J. Cell Sci. 4, 3-15 (1969)
Printed in Great Britain
SOME OBSERVATIONS ON THE NUCLEOLUS
IN SPIROGYRA
E.G.JORDAN* AND M. B. E. GODWARD
Department of Botany, Queen Mary College, Mile End Road, London, E. 1, England
SUMMARY
Several species of Spirogyra have been collected and fixed for electron microscopy in either
2 % osmium tetroxide or 5 % glutaraldehyde. Mitotic cells were selected with the light microscope from filaments of Spirogyra embedded in Epon.
Evidence is given for an interphase cycle in the nucleolus of Spirogyra. A clear association
between nucleolar material and chromosomes occurs at mitosis. The breakdown of the nucleolus
is shown to occur in two stages, one as a consequence of the withdrawal of the nucleolar
chromosomes, and the other after telophase in the new nucleus. The significance of these
findings is discussed.
INTRODUCTION
The nucleolus is usually the only component which can be seen easily in the interphase nucleus by light microscopy.
For some time little was known about it apart from its association with a particular
chromosome region (Heitz & Bauer, 1933; McClintock, 1934). It is only recently that its
major role in ribosome production has been clearly demonstrated (Brown & Gurdon,
1964; Perry, 1966; Wallace & Birnstiel, 1966; Birnstiel, Wallace, Sirlin & Fischberg,
1966). Now that this is known it is more meaningful to attempt explanations of its behaviour at mitosis. It is commonly taught that the nucleolus disappears at nuclear division to reappear again in the new daughter nuclei. Studies of this phenomenon with the
electron microscope have been presented (Swift, 1959; Lafontaine & Chouinard, 1963).
There are cases in which the nucleolus persists at mitosis (Brown & Emery, 1957;
Hsu, Humphrey & Somers, 1964). It would seem better in the light of some studies
(Hsu, Arrighi, Klevecz & BrinkJey, 1965) to refer to degrees of persistence or disappearance rather than to speak in absolute terms.
Spirogyra, although having specific variation, shows a very high degree of persistence
ofthenucleoli(Geitler, 19350,6; Godward, 1950,1953; Godward & Newnham, 1965).
The nucleoli do not persist throughout mitosis as spherical bodies, however, but show a
remarkable redistribution of their material around the chromosomes. It is this feature
which has been studied here by electron microscopy.
• Present address: Department of Biology, Queen Elizabeth College, Campden Hill, London,
W. 8.
4
E. G. Jordan and M. B. E. Godward
MATERIALS AND METHODS
Spirogyra was collected from the field and grown in Godward's solution (Godward,
1942) enriched with 10% soil extract. The mitotic cells were obtained by growing the
Spirogyra in water-cooled culture chambers with a light/dark cycle of 18 h light and
6 h dark. The cells were grown for 3-5 days in these conditions and fixed at a time
when the mitotic index was highest. This time varied, since the material was never very
well synchronized, but about 2 h after the beginning of the dark period was generally
found to be best. Preparations for the light microscope using the iron alum acetocarmine technique (Godward, 1948) were used to check the mitotic index and find the
most suitable time for fixation.
Fixations were made in 2% osmium tetroxide in veronal acetate buffer at pH 7-0
or in 5 % glutaraldehyde with 5 % sucrose in cacodylate buffer at pH 7-0. The
glutaraldehyde solution was first neutralized with 1 N sodium hydroxide. The fixation
was carried out at 0-4 °C. The material fixed in glutaraldehyde was washed in buffer
overnight and then treated with 2% osmium tetroxide. Alcohol dehydration was
followed by Epon embedding.
The resin was polymerized in shallow plastic dishes to give a thin disk which could
be viewed subsequently with the light microscope. Cells in mitosis were identified and
selected in this way. The microtomes used in this study were the Cambridge Huxley
and the LKB Ultratome. The cylindrical cells were oriented vertically to the knife
edge for cutting. This ensured that there was no sudden change in texture due to
cutting the longitudinal cell walls because they were always in contact with the knife
throughout the cutting process. Sections were mounted on carbon-Formvar films or
directly on to copper grids; 2% aqueous uranyl acetate was used routinely as a stain.
Photographs were taken with Siemens Elmiskop 1 A, Zeiss EM 9, Jeol Jem 7, or AEI
EM 6 electron microscopes.
EXPERIMENTAL RESULTS
The structure of the nucleolus is clearly related to the chromosome material which
has been shown to be the organizing region of the nucleolar organizing chromosome
(Godward & Jordan, 1965). The terms used to describe the two zones of the nucleolus
have been well defined in the work of Bernhard (1966). The nucleolar material adjacent
to the chromosome is the fibrillar region shown to be the first region labelled by isotopic RNA precursors (Granboulan & Granboulan, 1965; Bernhard, 1966). The rest
of the spherical nucleolus is composed of the granular region characterized by 150-A
granules.
In Spirogyra the distinction between the fibrillar and the granular material of the
nucleolus is much clearer in some nucleoli than others. There seems to be a correlation
between smaller early interphase nucleoli and this sharper distinction between the two
zones shown in Fig. 1. There is another feature which varies with the age of the
nucleolus and that is the distribution of the chromosome within it. The two extremes of
this are shown in Figs. 1 and 2. In the first, which is the smaller early interphase
Nucleolus of Spirogyra
5
nucleolus, the chromosome is restricted to more or less two regions and these are also
the only places where the fibrillar material is seen. In Fig. 2, which is much larger and
at a later stage of interphase, the chromosome material is distributed as smaller units
throughout the nucleolus and the fibrillar regions adjacent to it cannot be so clearly
defined as in early interphase. The chromosome region at this later stage would appear
to be a thread of uniform thickness around 0-25 fi in diameter which meanders through
the nucleolus (see Fig. 2, where both transverse and longitudinal sections of this
chromosome region can be seen).
The fibrillar region which can be so clearly demonstrated at early interphase and
which can still be defined, though not so easily, at the end of interphase, by prophase
has completely disappeared, at least as a separate definable zone.
After prophase and before metaphase all the chromosomes move into the nucleolus.
This association of the material of the nucleolus with the chromosomes persists until
telophase.
The nucleolar material around the chromosomes in mitosis is not demonstrably
different from the granular material of interphase (Figs. 2, 8). It has an overall reticular
structure with a thickness around o-i fi (Fig. 3). The reticular nature of the nucleolar
material becomes very clear as the initial chromosome component of the nucleolus,
the organizing chromosomes, is withdrawn from the interstices (Fig. 3). This reticular
structure would be called the nucleolonema by some workers (Estable, 1966). The
organizing chromosome can be identified in the prophase picture (Fig. 3) but in later
stages of mitosis it is not possible to distinguish it from the other chromosomes. At
prometaphase (Fig. 4) and all later stages all the chromosomes are included in the
nucleolar material. This indicates that although the organizing chromosomes had a
region which was within the nucleolus, this is withdrawn when all the chromosomes
condense, only to return to the nucleolus again with the other chromosomes later.
There does not appear to be any structural continuity between the chromosomes and
the nucleolar material in this mitotic association. There is, however, a closer association
between them in anaphase than in metaphase (compare Figs. 6, 5).
The precise stage at which the anaphase separation starts has not been seen, but the
early anaphase picture (Fig. 6) shows the arrangement immediately after separation.
The chromosomes are still surrounded by nucleolar material and it is not possible to
say which surfaces of the chromosomes were once in contact as chromatids because all
the surfaces of the chromosomes are covered with the nucleolar material and they are
completely embedded in it.
In the telophase stage (Fig. 7) no chromosome can be seen. This may be due to the
fact that they were missed by the plane of the sections. No pictures have yet been
obtained showing the condition at the time when the chromosomes leave the nucleolar
material. The nature of the nucleolar material of telophase is clearly the same as that
of the granular zone of interphase (Fig. 9). The new nuclear membranes are formed
by the coalescence of vesicles around these two groups of nucleolar material (Figs. 7, 9).
The few pictures that have been obtained of post-telophase nuclei show that the
nucleolar material breaks down completely prior to being reorganized into a nucleolus.
It is clear that the granular material persists at least in part through mitosis and the
6
E. G. Jordan and M. B. E. Godward
granules of this appear not to be destroyed at the time of the breakdown of nuclear
material which occurs in telophase (Fig. io). The nature of the organizing process has
not yet been elucidated but the evidence of Fig. io points to the preliminary formation
of a dense body resembling in structure the fibrillar zone of the interphase nucleolus.
Figure 11 is a newly formed nucleolus of the same species as Fig. io and a chromosome
body, possibly a chromocentre, adjacent to the organizing region of the organizing
chromosome can be seen. Fibrillar regions appear in the region of the organizing
chromosome.
DISCUSSION
From the increase in size and change in distribution of components, particularly
the chromosome, there would appear to be an interphase cycle in the nucleolus of
Spirogyra. At the beginning of the cycle the nucleolus is smaller and the chromosome
regions are clumped together; later the chromosome material is more evenly distributed
and the nucleolus is larger in size. The appearance of the fibrillar region is also associated with this cycle; at the start it is more distinct from the granular material than
at the end.
Among the most interesting aspects of this nucleolar behaviour are the two steps in
the breakdown process, the first at prophase consequent on the withdrawal of the
nucleolar organizing chromosome; and the second at telophase, where a complete
dispersal precedes the formation of the new nucleolus. After the prophase withdrawal
of chromosome the nucleolar material still has a definite structure showing clearly that
the first breakdown process is not a complete one.
It is tempting to think that inside the sheets and strands of the nucleolar material
remaining after the first step in the breakdown process there are threads of DNA
giving them their structural continuity. The peripheral nucleoli of the newt oocyte
have been shown to have a ring of DNA within them (Miller, 1964). Chromosome
models have been proposed from considerations of lampbrush chromosome structure
implicating the production of detached copies of the genie DNA (Callan, 1967;
Whitehouse, 1967). Such proposals fit well with our suggested interpretation of persistent nucleolar structure. The idea that there is a possible DNA backbone to the
nucleolonema has been suggested by Lettre, Siebs & Poivelity (1966).
The second stage in the breakdown, the complete dispersal which takes place after
telophase, presents two questions. First, why is it that the structure which clearly
exists throughout mitosis in the nucleolar material breaks down; and secondly, why
is it necessary that this material is reorganized into a nucleolus rather than remaining
dispersed in the nucleus? This latter question is based on the assumption, which is
difficult to prove from photographs, that the material which is dispersed collects again
at the site of the nucleolus. Whether this is a false assumption or not, it is clear that the
nucleolus represents a large accumulation of material, some being RNA which was
produced earlier at this particular site on the chromosome. It is possible that in interphase some control over the release of this material and, therefore, over protein
synthesis, is exercised by virtue of the fact that it is held as an aggregate in the nucleolus.
Nucleolus of Spirogyra
7
An answer to the first question relating to the breakdown of the persistent nucleolus
material must await a clearer understanding of the basis of the structure which persists
initially.
The remarkable appearance of the mitotic figures with all the chromosomes associated
with the nucleolar material does not suggest any simple explanation. It is clear from
other organisms that this behaviour of the nucleolus is in no way necessary for mitosis
and even here a normal spindle seems to be operating. It may be viewed as a mechanism
for the distribution of nucleolar material to the two new cells. Whatever the explanation
it is difficult to envisage the nature of the forces which bring the chromosomes into the
nucleolar material and which maintain this relationship through mitosis.
The nucleolar material which persists through mitosis is the granular zone material
of the nucleolus. The fate of the fibrillar zone is indicated by its changes in interphase,
becoming progressively more indistinct from the granular zone and at prophase ceasing
to exist as a separate definable region.
The prophase reticular structure, or nucleolonema (Estable, 1966), consists entirely
of granular zone material. But this not to say that the nucleolonema is always composed
only of granular material, since the structure which would most merit this name in
early interphase nuclei because of its reticular appearance is the fibrillar material.
The nucleolar material which persists through mitosis shows no sign of a distinct
fibrillar region. The explanation for this absence of fibrillar material in mitosis could
quite easily be the inactivity of the nucleolus organizing region during this time. It has
been shown that this zone is the first to show any activity in labelling experiments
with RNA precursors (Granboulan & Granboulan, 1965; Bernhard, 1966), and this
would seem to confirm that the fibrillar material is the first-formed material, and is
visible only if fixation coincides with its synthesis.
REFERENCES
W. (1966). Ultrastructural aspects of normal and pathological nucleolus in mammalian cells. Natn. Cancer Inst. Monogr. 23, 13-38.
BIRNSTIEL, M. L., WALLACE, H., SIRLIN, J. L. & FISCHBERC, M. (1966). Localization of the
ribosomal DNA complement in the nucleolar organizer region of Xenopus laevis. Natn.
Cancer Inst. Monogr. 23, 431-447.
BROWN, D. D. & GURDON, J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolate
mutant of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 51, 139—146.
BROWN, W. V. & EMERY, H. P. (1957). Persistent nucleoli and grass systematics. Am.J. Bot. 44,
585-59°CALLAN, H. G. (1967). The organization of genetic units in chromosomes. J. Cell Sci. 2, 1—7.
ESTABLE, C. (1966). Morphology, structure and dynamics of the nucleolonema. Natn. Cancer
Inst. Monogr. 23, 91-105.
GEITLER, L. (1935a). Neue Untersuchungen iiber die Mitosen von Spirogyra. Arch. Protistenk.
85, 10-19.
GEITLER, L. (19356). Untersuchungen iiber den Kernbau von Spirogyra mirtels feulgens
Nuklealfarbung. Ber. dt. bot. Ges. 53, 270-276.
GODWARD, M. B.E. (1942). Life cycle of Stigeoclonium amoenum Kutz. New Phytol.41, 293-301.
GODWARD, M. B. E. (1948). The iron alum acetocarmine method for algae. Nature, Lond. 161,
BERNHARD,
203.
M. B. E. (1950). On the nucleolus and the nucleolar-organising chromosomes of
Spirogyra. Ann. Bot. 14, 39-53.
GODWARD,
8
E. G. Jordan and M. B. E. Godward
GODWARD, M. B. E. (1953). Geitler's nucleolar substance in Spirogyra. Ann. Bot. 17, 403-416.
GODWARD, M. B. E. & JORDAN, E. G. (1965). Electron microscopy of the nucleolus of Spirogyra
britannica and Spirogyra ellipsospora. Jl R. microsc. Soc. 84, 347—360.
GODWARD, M. B. E. & NEWNHAM, R. E. (1965). Cytotaxonomy of Spirogyra. II. 5. neglecta
(Hass) Kutz., S. punctulata Jao, S. majuscular (Kutz.) Czurda emend., 5 . ellipsospora
Transeau, S. porticalis (Muller) Cleve. J. Linn. Soc. (Bot.) 59, 99-110.
GRANBOULAN, N. & GRANBOULAN, P. (1965). Cytochimie ultra-structurale du nucleole. II.
Etudes des sites de synthese du RNA dans le nucleole et la noyau. Expl Cell Res. 38, 604—619.
HEITZ, E. & BAUER, H. (1933). Beweise filr die chromosomennatur der kernschleifen in den
knauelkernen von Bibio hortulanus L. Z. Zellforsch mikrosk. Anat. 17, 67-82.
Hsu, T . C , ARRIGHI, F. E., KLEVECZ, R. R. & BRINKLEY, B. R. (1965). The nucleoli in mitotic
divisions of mammalian cells in vitro. J. Cell Biol. 26, 539-555.
Hsu, T . C , HUMPHREY, R. M. & SOMERS, C. E. (1964). Persistent nucleoli in animal cells
following treatment with fluorodeoxyuridine and thymidine. Expl Cell Res. 33, 74-77.
LAFONTAINE, J. G. & CHOUINARD, L. A. (1963). A correlated light and electron microscope
study of the nucleolar material during mitosis in Vicia faba. J. Cell Biol. 17, 167-201.
LETTRE, R., SIEBS, W. & POIVELETY, N. (1966). Morphological observations on the nucleolus of
cells in tissue culture, with special regard to its composition. Natn. Cancer Inst. Monogr. 23,
107-123.
MCCLINTOCK, B. (1934). The relation of a particular chromosome element to the development
of the nucleoli in Zea mays. Z. Zellforsch. mikrosk. Anat. 21, 294-328.
MILLER, O. L. JR. (1964). Extrachromosomal nucleolar DNA in amphibian oocytes. J. Cell
Biol. 23, 60A.
PERRY, R. P. (1966). On ribosome biogenesis. Natn. Cancer Inst. Monogr. 23, 527-545.
SWIFT, H. H. (1959). Nucleolar function. In Symposium on Molecular Biology (ed. R. E. Zirkle),
pp. 266—303. Chicago: University of Chicago Press.
WALLACE, H. & BIRNSTIEL, M. I. (1966). Ribosomal cistrons and the nucleolar organizer.
Biochim. biophys. Acta 114, 296—310.
WHITEHOUSE, H. L. K. (1967). A cycloid model for the chromosome. J. Cell Set. 2, 9-22.
{Received 19 August 1967—Revised 5 June 1968)
Fig. 1. Spirogyra neglecta x 25000. Nucleolus at the start of interphase. c, chromos o m e ; / , fibrillar material; g, granular material.
Fig. 2. Spirogyra neglecta x 18500. Nucleolus at the end of interphase. The chromosome, c, is distributed in the form of a meandering thread which has been cut in both
transverse, t, and longitudinal, /, section. The fibrillar material, / , is not so strikingly
different from the granular, g, at this stage.
Nucleolus of Spirogyra
io
E.G. Jordan and M. B. E. Godward
Fig. 3. Spirogyra britannica x 14000. Nucleolus at prophase. The chromosome region,
c, has withdrawn and condensed from the nucleolus leaving it as a reticulum.
Fig. 4. Large unidentified species x 6000. Prometaphase condition. The chromosomes,
c, have migrated into the nucleolar material, nm. For high power see Fig. 8.
Fig. 5. Spirogyra neglecta x 11000. Metaphase. The chromosomes, c, are aligned at the
equator together with the nucleolar material, nm.
Fig. 6. Spirogyra neglecta x 8500. Anaphase. Compacting of the nucleolar material
occurs as the two groups of chromosomes, c, and nucleolar material, nm, separate.
Fig. 7. Large unidentified species x 3000. The telophase nuclei are masses of nucleolar
material surrounded by the nuclear envelopes. Chromosomes do not appear in this
section. For high power see Fig. 9.
Nucleolus of Spirogyra
. V.
12
E.G. Jordan and M. B. E. God-ward
Fig. 8. Large unidentified species x 35000. Prometaphase. The association between
nucleolar material, nm, and chromosomes, c, does not interfere with normal spindle
production, m, spindle microtubules.
Fig. 9. Large unidentified species x 30000. Telophase. The nuclear envelope, ne, is
the result of the coalescence of vesicles which collect on the surface of the nucleolar
material, nm.
Nucleolus of Spirogyra
14
E. G. Jordan and M. B. E. Godward
Fig. 10. Spirogyra submargaritata x 30000. Telophase nucleus. The nucleolar material
which filled the whole nucleus earlier has been largely dispersed into ribosome-like
granules throughout the nucleus. The two arrowed regions are probably chromatin,
and the darker structure could be a developing nucleolus.
Fig. 11. Spirogyra submargaritata x 24000. A young nucleolus in an early interphase
cell. The nucleolus has fibrillar regions, /, associated with a chromosome region, c,
which is adjacent to a chromocentre, cc.
Nucleolus of Spirogyra