J. Cell Sd. 74, 239-256 (1985)
239
Printed in Great Britain © Company of Biologists Limited 1985
SIMULTANEOUS ULTRASTRUCTURAL
LOCALIZATION OF Ag-NOR (NUCLEOLAR
ORGANIZER REGION) PROTEINS AND
RIBONUCLEOPROTEINS DURING MITOSIS, IN
HUMAN BREAST CANCEROUS TISSUES
D. PLOTON, M. MENAGER AND J. J. ADNET
Laboratoire d'Histologie, Faculte de Medecine de Reims, 51, rue Cognacq-Jay,
51100 Reims, France
SUMMARY
The simultaneous ultrastructural localization of the Ag-NOR proteins and ribonucleoproteins
(RNP) was carried out during mitosis in human breast cancerous tissues using a one-step silver
staining method followed by acetylation.
Owing to the good preservation of the cells and to the high precision of the Ag staining we were able
to follow the behaviour of the argyrophilic nucleolar components from prophase to late telophase.
During all the phases of mitosis the Ag-NOR proteins were associated with fibrillar RNP component (without visible fibrillar centre), which originated, during prophase, from the dense fibrillar
component of the nucleolus and then integrated within the periphery of some metaphasic
chromosomes as irregular structures. The Ag staining of these structures was homogeneous but
lower compared to those of nucleoli during prophase.
The RNP sheath that covered the chromosomes was never silver stained.
From anaphase to early telophase the argyrophilic RNP structures disposed within the periphery
of the chromatin mass formed roundish and then spherical structures in which the Ag-NOR proteins
were clustered as centrally localized spheres.
These findings showed the usefulness of our method for the precise study of the spatial relationship between the Ag-NOR proteins and the chromosomes. Thus we were able to show striking
segregation of both the argyrophilic structures at the periphery of the chromatin mass and of the AgNOR proteins within these structures. This suggests that a high degree of the structural organization
of the future nucleus is already present during anaphase. This could be related to the rapid resumption of ribosomal RNA synthesis.
INTRODUCTION
The argyrophily of the nucleolar organizer regions (NORs) is due to non-histone
proteins, the Ag-NOR proteins, which are localized in the vicinity of the ribosomal
DNA genes (Hubbell, Rothblum & Hsu, 1979; Angelier, Hernandez-Verdun &
Bouteille, 1982; Hernandez-Verdun, Derenzini & Bouteille, 1982; Howell, 1982;
Hernandez-Verdun & Derenzini, 1983).
The exact biochemical nature and role of the Ag-NOR proteins are still under
discussion: they could correspond to the larger subunit of RNA polymerase I
(Williams, Kleinschmidt, Krohne & Franke, 1982), to 'Ag-NOR proteins' (Hubbell
Key words: mitosis, nucleoli, nucleolar organizer region, Ag-NOR proteins, RNP, human breast
cancerous tissues.
240
D. Ploton, M. Menager andjf. jf. Adnet
et al. 1979) or to B23 and C23 proteins (Lischwe, Richards, Busch & Busch, 1981;
Busch et al. 1982; Ochs, Lischwe, O'Leary & Busch, 1983).
Their structural function in the condensing state of rDNA has been also suggested
(Hernandez-Verdun & Derenzini, 1983; Medina, Risueno, Sanchez-Pina &
Fernandez-Gomez, 1983).
It is now well recognized that the silver stainability of the Ag-NOR proteins may
be related to the activity of the NORs (Howell, 1982). Moreover their use as a good
ultrastructural marker for the rDNA genes has been recently proposed (HernandezVerdun & Derenzini, 1983).
Thus, in the nucleolus, the Ag-NOR proteins are localized within the fibrillar
centres and the dense fibrillar component (Hernandez-Verdun, Hubert, Bourgeois &
Bouteille, 1980; Ploton, Bobichon & Adnet, 1982; Vio-Cigna, Pebusque & Seite,
1982; Ploton, Bendayan & Adnet, 1983; Goessens, 1984), where rDNA has been
demonstrated (Hernandez-Verdun et al. 1982). These two components both constitute the interphase counterpart of the mitotic NORs (Hernandez-Verdun, 1983).
During mitosis the clustering of the rDNA genes in the mitotic NORs is
concomitant with the cessation of ribosomal RNA synthesis and with the disaggregation of the nucleolus.
Morphological studies have shown this phenomenon and the association of various
nucleolar remnants with some chromosomes during prophase and the fusion of
prenucleolar bodies, which form the interphase nucleolus, during telophase (Stevens,
1965; Hsu, Arrighi, Klevecz & Brinkley, 1965; Brinkley, 1965; Schwarzacher, Mikelsaar & Schnedl, 1978; Ploton & Gontcharoff, 1979).
The Ag-NOR proteins appear to be a good tracer, which may facilitate the study
of the behaviour of the NOR (fibrillar centre and fibrillar component) during all the
phases of mitosis particularly when nucleolar remnants are difficult to identify.
Ultrastructural studies of mitosis with Ag-NOR staining are rare (HernandezVerdun et al. 1980; Paweletz & Risueno, 1982) and to our knowledge have never been
carried out on human cancerous tissues.
The aim of the present work is to describe a detailed study of the simultaneous localization of the Ag-NOR proteins and ribonucleoproteins (RNP) during all the phases of
mitosis in human breast cancerous tissues using a recent improvement (Ploton, Menager
& Adnet, 1984a) of the one-step silver staining method (Ploton et al. 1982, 1983).
We were able to demonstrate the presence of Ag-NOR proteins within the dense
fibrillar component that becomes partially integrated into the prophase chromosomes.
At anaphase the Ag-NOR proteins are segregated as roundish structures localized on
RNP fibrillar component within the surface of the chromosomes. They then become
integrated within the early telophase chromosomes and fuse to form the prenucleolar
bodies of late telophase.
MATERIALS
AND
METHODS
Tissues
Three human breast cancers (infiltrating duct carcinomas) in which mitosis was relatively
Ag-NOR proteins during mitosis
241
numerous were U9ed for this study. After surgical excision the tissues were cut into slices about
0-5—1 mm thick and were processed as follows.
(1) In the first group the slices were fixed with 1-6% glutaraldehyde in 0 - l M-phosphate buffer
(pH 7 - 2) for lOmin at4°C, rinsed in the same buffer and fixed for 5 min at 4 °C in Carnoy's solution
( 3 : 1 , v/v, ethanol/acetic acid), rehydrated in graded ethanol solutions, rinsed in distilled water and
then silver stained as described previously (Plotonet al. 1982, 1983, 1984a). For the silver staining
two solutions were prepared; the first consisted of 2 % (w/v) gelatin in 1 % formic acid; the second
consisted of 50% (w/v) silver nitrate in water.
The staining solution was obtained by mixing one part of gelatin solution with two parts of the
silver nitrate solution. The slices were stained in this mixture for 10min at 70°C, then thoroughly
rinsed in distilled water, immersed in a 5 % (w/v) thiosulphate solution for 10 min and then rinsed
in water.
The slices were then processed for acetylation as follows. They were dehydrated in graded acetone
solutions (5 min each in 25, 50, 75 and 90%, v/v) and then in three changes of pure acetone for
10 min each. The slices were passed through mixtures of pyridine/acetone ( 1 : 1 , 2 : 1 , 3 : 1 , 4 : 1 ,
v/v) for 5 min each. They were then rinsed in three changes of pure pyridine for 10 min each and
finally acetylated for 4h at 45°C in a mixture of pyridine and acetic anhydride (60 :40, v/v).
The slices of tissues were rinsed three times in pure pyridine for 10 min each, then for 5 min in
pyridine/acetone (50 :50, v/v) and finally three times in pure acetone for 10 min each.
(2) In the second group the slices were fixed with 1-6% glutaraldehyde in O'l M-phosphate
buffer (pH 7-2) for 1 h at 4°C, rinsed in the game buffer and processed for acetylation as described
above, for 15 h (Wassef, Burglen & Bernhard, 1979).
Embedding
The slices of tissues were passed through acetone-Epon mixtures and then through pure Epon
at room temperature. Polymerization was carried out at 60°C for 3 days.
Ultrathin sections
Ultrathin sections (70 nm thick) were cut with a diamond knife and stained with uranyl acetate
and lead citrate or with the Bernhard's regressive preferential staining method for
ribonucleoproteins (Bernhard, 1969) and viewed in a Hitachi H.300 microscope at 75 kV.
RESULTS
The use of the specific one-step Ag-NOR staining followed by acetylation allows
for the precise localization of the Ag-NOR proteins during all the phases of mitosis.
When sections are contrasted with uranyl acetate and lead citrate the contrast of the
chromatin is high whereas that of RNP structures (nucleolus, peri- and interchromatin fibrils and granules) is low.
The regular silver dots (50-100 A in diameter) were specifically localized within
fibrillar centres and part of the dense fibrillar component. The granular component,
which is easily localized, is never silver stained. When sections were preferentially
contrasted for RNP by EDTA regressive staining the chromatin clumps were well
bleached whereas RNP components showed a high contrast. Silver dots were not
displaced by the EDTA treatment. The bleaching of the chromosomes greatly enhanced the precision of the localization of the Ag-NOR proteins and allows their study
particularly during nucleolar disintegration and reformation. In order to gain a more
precise view of the fine structure of some components we also studied sections of
acetylated blocks only in which the general definition of the nuclear structures is
greatly enhanced (Wassef et al. 1979; Ploton et al. 1983, 1984a).
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D. Ploton, M. Menager andj. J. Adnet
Fig. 1
Ag-NOR proteins during mitosis
243
Ag-NOR staining followed by acetylation
Prophase (Fig. I). Prophase was recognized by the great volume of the nucleus and
the preferential localization of the condensing chromosomes near the nuclear envelope.
The nucleoli, which frequently linked several chromosomes, had irregular shapes
as they were beginning to disaggregate. This consisted of a segregation of the nucleolar components, which eventually led to their partial or complete separation.
We noticed frequent extrusions of the fibrillar component that was directly in
contact with the chromosomes and seemed to become integrated within them.
The Ag-NOR proteins were localized within part of the fibrillar component of the
nucleolus and within the nucleolar extrusions. As seen on a tangential section of a
junction of a chromosome with Ag-NOR proteins the latter never formed regular
structures but always contorted zones extending within or on the surface of the
chromosome.
Fig. 1. Localization of the Ag-NOR proteins during prophase. A. The bleached condensing chromosomes (c) are preferentially localized along the nuclear envelope. Nucleoli
(«) linked several chromosomes. The Ag-NOR proteins are seen within the nucleolus
(arrowhead) or in contact with the chromosomes (arrows). EDTA regressive staining.
X 16000. B and c. Two serial but not consecutive sections of contact between a nucleolus
and chromosomes, B. The Ag-NOR proteins are localized within the RNP fibrillar component of the nucleolus (arrowhead), or extruded from the nucleolus and integrated within
the chromosomes (arrow). EDTA regressive staining. X 46000. c. On this tangential
section the Ag-NOR proteins are seen within contorted zones that extend on or into the
chromosome. EDTA regressive staining. X 46 000. D. The Ag-NOR proteins are localized
within irregular extrusions of the fibrillar component in contact with the chromosomes
(arrows). The rest of the nucleolus was not silver stained and shows micro-segregation and
disorganization (appearance of a central vacuole) (v). Uranyl and lead staining. X 46000.
Fig. 2. Localization of the Ag-NOR proteins during metaphase. A. The Ag-NOR proteins
are localized within the periphery of some chromosomes (arrows). Uranyl and lead
staining. X 16 000. B. Higher magnification of one of the argyrophilic structures. The AgNOR proteins are localized within a fibrillar structure at the periphery of the chromosomes
(arrow). One can note the absence of silver staining of a fibrillo-granular structure, which
might represent a cluster of interchromatin granules or nucleolar remnants (arrowhead).
Uranyl and lead staining. X 46000. c and D. The Ag-NOR proteins are localized within
a fibrillar RNP component of irregular shape and size; the Ag dots are few but are
homogeneously distributed within the fibrillar components (large arrows). The
chromosomal RNP sheath was not silver stained (small arrows). EDTA regressive
staining. X 46000.
Fig. 3. Localization of the Ag-NOR proteins during anaphase. A. and B. Early anaphase.
The chromosomes partly fused and formed a compact mass, at the periphery of which the
argyrophilic RNP structures (large arrows) (0-3-0-4 fini) are localized. The clusters of AgNOR proteins (0-1-0-2 /*m) are centrally localized within these structures and never seen
within the chromatin mass. No silver dots are seen in the chromosomal RNP sheath (small
arrows). Note the presence of some spindle fibres (arrowheads). Uranyl and lead staining.
A, X 23 000; B, X 66000. candD. Late anaphase. The chromatin mass has an irregular and
reniform shape. The clusters of Ag-NOR proteins are centrally localized within RNP
fibrillar structures located within the periphery of the chromatin in depressions or pockets
(arrows). The forming nuclear envelope is absent at the level of these pockets. EDTA
regressive staining, c, X 23000; D, X 66000.
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D. Ploton, M. Menager andjf. jf. Adnet
2A
B
Fig. 2. For legend see p. 243
Ag-NOR proteins during mitosis
- f
Fig. 3. For legend see p. 243
245
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D. Ploton, M. Menager andjf. J. Adnet
Metaphase (Fig. 2). The observation of well-orientated metaphase plates was rare.
Metaphase chromosomes had a regular shape and were limited by a continuous RNP
sheath, which was never silver stained. The Ag-NOR proteins were localized only on
structures composed of fibrillar RNP component that had an irregular size and shape.
They were systematically integrated partly within the periphery of some
chromosomes and showed an homogeneous but poor silver staining, which was very
low in comparison to that seen in prophasic nuclei.
Anaphase (Fig. 3). During early anaphase (Fig. 3A,B) the chromosomes fused and
formed a compact chromatin mass in which spindle fibres were seen.
The Ag-NOR proteins formed roundish aggregates (of about 0-1-0-2/im in
diameter) centrally localized within fibrillar RNP components. The latter were
located at the periphery of the chromatin mass and constituted irregular masses
(0-3-0-4 fim in diameter), which were disposed within either slight depressions or
pockets. The RNP sheath that covered the chromosomes had an irregular thickness
and was composed of both fibrillar and granular components.
At the end of anaphase (Fig. 3c,D) the mass of chromatin had an irregular and
kidney-shaped cross-section incompletely lined with the new nuclear envelope. The
Ag-NOR proteins formed roundish aggregates centrally localized within RNP
fibrillar components systematically located in pockets within the chromatin. It was
noted that the nuclear envelope was always interrupted at the level of these pockets.
Telophase (Fig. 4). The chromosomes were completely fused and formed a mass
with a characteristic kidney-shaped cross-section regularly limited by the nuclear
envelope. The aggregates of the Ag-NOR proteins constituted spherical masses (0 • 1 ^m
in diameter) centrally localized within spherical fibrillar RNP structures (0-2 /Am in
diameter) scattered within the chromatin.
Although they were frequently located at the border of the nuclei these RNP
structures were always separated from the nuclear envelope by some chromatin.
Moreover some of them were in contact with decondensing chromatin.
Late telophase (Fig. 5). The chromatin of late telophase nuclei showed typical
decondensation characterized by the greater volume of the nucleoplasm.
A continuous sheath of condensed chromatin was seen along the nuclear envelope.
In the nucleoplasm the chromatin clumps were lined with perichromatin fibrils;
numerous interchromatin fibrils and granules were also seen. The Ag-NOR proteins
were grouped as spherical clusters (0-2 /im in diameter) localized within spherical
aggregates of fibrillar RNP component (0-3 pan in diameter), which were located in
Fig. 4. Localization of the Ag-NOR proteins during early telophase. A and B. The mass
of chromatin has a reniform shape.
The spherical clusters of Ag-NOR proteins (0-1 /an in diameter) are centrally localized
within spherical structures of RNP fibrillar component (0-2/an in diameter) surrounded
by chromatin (arrows). Some of them are in contact with nucleoplasmic vacuoles
(arrowhead). EDTA regressive staining, A, X 23 000; B, X 66000. c. The spherical
clusters of Ag-NOR proteins are centrally disposed in the spherical RNP structure
(arrow). Note the presence of numerous nucleoplasmic vacuoles, which correspond with
the decondensation of the chromatin. Uranyl and lead staining. X 66000.
Ag-NOR proteins during mitosis
B
Fig. 4
248
D. Ploton, M. Menager andj. J. Adnet
Ag-NOR proteins during mitosis
249
the nucleoplasm in contact with chromatin. These structures may be interpreted as
prenucleolar bodies.
In early interphasic nuclei, prenucleolar bodies were bigger, irregular, less
numerous and incompletely silver stained in comparison to interphase nucleoli.
Acetylation (Fig. 6). The observation of sections of acetylated blocks showed details
of the fine structure during disaggregation and reconstruction of the nucleolus.
During interphase some nucleoli showed typical fibrillar centres, fibrillar and
granular components.
During prophase the segregation of the granular and fibrillar components of the
nucleolus was followed by the incorporation of the latter within the chromosomes.
During metaphase this study confirmed the presence, within the periphery of some
chromosomes, of RNP clusters composed of both fibrillar and granular components
in which roundish structures of fibrillar dense component were centrally localized.
During anaphase and telophase the progressive incorporation of spherical RNP
structures composed of both fibrillar and granular components within the chromatin
was noticed. These structures showed a centrally localized sphere (about 0-1 fim)
composed of fibrillar dense component.
Finally, during late telophase both the fusion of the prenucleolar bodies and the
appearance of preribosomal granules were noticed.
This study also confirmed the absence of visible fibrillar centre within the nucleolar
remnants and within prenucleolar bodies during telophase.
Fig. 5. Localization of the Ag-NOR proteins during late telophase and early interphase.
A and B. Late telophase. At this stage chromatin decondensation leads to the extension of
the nucleoplasm. The clusters of Ag-NOR proteins are bigger (0-2/an in diameter) and
fewer (large arrows). They are localized at the periphery of some chromatin clumps within
RNP fibrillar structures, which they completely cover (prenucleolar bodies of 0-3 /un in
diameter). Note the presence of abundant peri- and inter-chromatin fibrils and granules
(small arrows). EDTA regressive staining, A, X 23 000; B, X 66000; c. Early interphase.
During early interphase some bigger prenucleolar bodies may be seen (0 - 4/im in
diameter). They are characterized by an irregular shape. The Ag-NOR proteins are
irregularly disposed and not present in all the fibrillar component. EDTA regressive
staining. X 66000.
Fig. 6. Behaviour of the nucleolar components during mitosis as seen after acetylation
only, and uranyl and lead staining, A. Interphase. When nucleoli with fibrillar centres are
visible they show the following typical distribution of their components: a spherical
fibrillar centre (0-3 fJm in diameter), which appears as a low-stained structure (fc); a ring
of fibrillar component (/) and a granular area (g). Some chromatin clumps can also be
seen. X 66000. B. Prophase. During prophase a segregation of the nucleolar components
can be noticed. The fibrillar component is integrated within the periphery of the condensing chromosomes (arrow). X 66000. c. Metaphase. Some fibrillo-granular RNP structures were seen within the periphery of some chromosomes. The fibrillar component
constitutes some roundish structures in contact with the chromatin (arrow). X 66000.
D. Anaphase. The fibrillar RNP structures are localized within depressions or pockets
within the periphery of the chromosomes (arrow). X 66000. E. Telophase. The fibrillar
RNP structures become spherical (0-1 jan in diameter) and are incorporated within the
chromatin mass (arrows). X 66000. F. Early interphase. The prenucleolar bodies (arTOw)
have a roundish shape in which fibrillar (/) and granular (g) components can be seen. The
chromatin is decondensed. X 66000.
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D. Ploton. M. Menaser andjf. J. Adnet
9
r*
Fig. 6. For legend see p. 249
Ag-NOR proteins during mitosis
251
DISCUSSION
The redistribution of the Ag-NOR proteins throughout mitosis of human breast
cancerous cells in vivo has been studied using a one-step silver staining method
followed by acetylation.
The use of this technique resulted in the localization of small-sized Ag dots on
structures whose morphology and cytochemical nature may be established by the use
of the acetylation procedure and EDTA regressive staining (Ploton et al. 1984a).
Moreover, the bleaching of the chromatin after EDTA staining facilitated the study
of the spatial relationships between the argyrophilic structures and the chromosomes.
In the human cancerous tissues examined in this study, it appeared that during all
the phases of mitosis the Ag-NOR proteins were never localized within condensed
chromatin itself but only within fibrillar clumps that possessed a high contrast after
EDTA regressive staining and were associated with condensed chromatin.
As confirmed by examination of sections of blocks that had been acetylated only the
supposed Ag-stained structures were mainly composed of fibrillar components in
which fibrillar centres were no longer visible.
It also appeared that the RNP sheath that covered the chromosomes, whose nucleolar origin had been supposed (Moyne & Garrido, 1976), was never silver stained. This
confirms results of Hernandez-Verdun et al. (1980) and our preliminary results
(Ploton et al. 1984a) but is the opposite of those of Paweletz & Risueno (1982).
The fact that this RNP sheath is not argyrophilic may be explained by the use, in
our work, of an ethanol/acetic acid fixation step, which is necessary to obtain 'specific'
Ag-NOR staining. This interpretation was corroborated by the absence of any background in the nucleoplasm or the cytoplasm.
Our study has followed the behaviour of the clusters of argyrophilic RNP fibrillar
component seen on metaphase chromosomes from their origin. There is no doubt that
these clusters originated at prophase from the Ag-staining fibrillar component that
extruded from the nucleolus and thus represent what is generally called nucleolar
remnants.
This phenomenon is concomitant with the disaggregation of the nucleolus, which
follows a kind of micro-segregation of the granular and fibrillar components.
The ability of these clusters to fuse during telophase to form prenucleolar bodies
suggests that they could contain the NORs.
The fact that no fibrillar centre was found in these clusters is in contrast with
previous results of Goessens & Lepoint (1974), who showed that in Ehrlich cells the
fibrillar centre is the only nucleolar component integrated within the chromosomes.
This follows the disappearance of the fibrillar and granular components, respectively.
Moreover, Goessens (1984) recently showed that the fibrillar centre is the only
argyrophilic structure present during mitosis.
The differences concerning the localization of the Ag-NOR proteins within the
chromosomes in Ehrlich cells and in the human material used by us may be due to
variations of the size of the fibrillar centres within the nucleoli (big in Ehrlich cells and
small, rare or absent in our material) and to variations in the size and location of the
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D. Ploton, M. Menager andj. J. Adnet
secondary constrictions (conspicuous on telocentric chromosomes of Ehrlich cells and
inconspicuous on the short arm of the human acrocentrics; Howell, 1982).
However, it must be kept in mind that the dense fibrillar component and the
fibrillar centres are the sites where the NOR chromatin is located during interphase
(Hernandez-Verdun, 1983) and that both were always stained in our material. The
absence of visible fibrillar centres during mitosis could be due to a kind of masking
by fibrillar component, which has been evoked to explain the absence of fibrillar
centres in some nucleoli (Knibiehler, Mirre & Rosset, 1982; Williams et al. 1982;
Knibiehler & Mirre, 1983; Boloukhere, 1984; Goessens, 1984), or in secondary
constriction of chromosomes where rDNA is, however, localized (Hsu, Spirito &
Pardue, 1975).
Moreover, our finding of the NORs within the periphery of some chromosomes
partly confirms previous work of Schwarzacher et al. (1978), who found that the
NORs of isolated human chromosomes seemed to be in an extrachromosomal
position.
Our study also demonstrates important variations during mitosis in the localization
of the Ag-NOR proteins within clusters of RNP fibrillar components. Thus a segregation of the Ag-NOR proteins within these structures characterized the second part of
mitosis following the disruption of the chromosomes. This segregation is always
followed by the acquisition by RNP clusters of a spherical shape and by their integration within the telophasic mass of chromatin. It also appears from our study that the
Ag-NOR proteins are not localized at random within the early telophase nuclei but
are preferentially localized at the periphery of the nuclei, in direct contact with the
nuclear envelope, and are never localized within the chromatin mass. This could
indicate that the telophase nuclei are highly ordered structures at least as regards the
future nucleolar bodies and their Ag-NOR proteins. In particular, a preferential
disposition of the future prenucleolar bodies in contact with the new nuclear envelope
may be compared with the frequent junctions of the interphasic nucleolus with the
nuclear envelope (Bourgeois, Bard & Bouteille, 1983).
The exact nature of the Ag-NOR proteins is still controversial. The more likely
candidates are the larger subunit of RNA polymerase I (Williams et al. 1982) and, as
more recently proposed, the C23 protein (Lischwe et al. 1981; Ochs et al. 1983),
which have both been immunolocalized within the secondary constrictions (Lischwe
et al. 1981; Scheer & Rose, 1984).
As demonstrated earlier (Hernandez-Verdun et al. 1982; Angelier et al. 1982;
Derenzini, Hernandez-Verdun, Pession & Novello, 1983; Hernandez-Verdun &
Derenzini, 1983; Ploton et al. 1983), the Ag-NOR proteins are associated with rDNA
genes during interphase and metaphase. Although Ag-NOR staining cannot be considered as really specific because Ag staining of non-nucleolar structures occurs in
some material (Seite, Pebusque & Vio-Cigna, 1982; Boloukhere, 1984), it has been
proposed as a good ultrastructural marker for rDNA (Hernandez- Verdun et al. 1982).
Can the behaviour of rDNA be partly deduced from that of the Ag-NOR proteins
during mitosis? If so we could then confirm the preferential segregation of rDNA on
some sites of the chromosomes (Heitz, 1931; Goodpasture & Bloom, 1975) and we
Ag-NOR proteins during mitosis
253
could also demonstrate the specific localization of the NOR-bearing chromosomes at
the periphery of the anaphase and early telophase masses of chromatin.
Such proximity of the NOR-bearing chromosomes has been previously proposed
by Bobrow & Heritage (1980) and has been evoked to explain the morphological
association of these chromosomes on karyotypes (Schwarzacher et al. 1978; Howell,
1982). This could also favour the rapid association of NORs for nucleolar formation
during telophase as noted by Anastassova-Kristeva (1977).
Our work confirms the strict association of the Ag-NOR proteins with structures
whose nucleolar origin and participation in late telophase reconstruction of the
nucleolus are undoubted.
However, it cannot be excluded that during some steps of mitosis (apart from
metaphase) some of the Ag-NOR proteins may leave the NOR and accumulate in
separate regions. Therefore, additional work is needed to demonstrate the eventual
relationships of rDNA and Ag-NOR proteins during all the phases of mitosis.
It has been proposed that the Ag stainability of the mitotic and interphasic NORs
may directly reflect their activity (Smetana & Busch, 1979; Hubbell, Lau, Brown &
Hsu, 1980; Morton et al. 1983).
In contrast, other work suggests that there is no direct relationship between Ag
staining intensity and rDNA transcription as demonstrated after natural or artificial
inhibition of rRNA synthesis (Boloukhere, 1984). Dimova et al. (1982) also put
forward the hypothesis that Ag staining does not reflect actual transcription of rDNA
genes.
Another hypothesis has been recently presented, which suggests that Ag-NOR
staining would detect rDNA in a decondensed state but not necessarily in an active
one (Hernandez-Verdun et al. 1983; Medina et al. 1983).
Similarly, during mitosis the Ag staining of the metaphase NORs, which is much
less than that of the interphase nucleolus (Hubbell et al. 1980), may be related to low
transcription of rDNA genes (Schwarzacher et al. 1978; Howell, 1982; Ochs et al.
1983) but reflects the rRNA synthesis level of the preceding interphase nucleolus
(Morton et al. 1983).
We confirmed that during metaphase NORs are rare and less stained compared to
those of anaphase and telophase.
We demonstrated a clustering of Ag dots on the argyrophilic structures during
anaphase and telophase and consequently a higher intensity of Ag staining, which may
be related to previous findings of Busch, Daskal, Gyorkey & Smetana (1979) and of
Hubbell et al. (1980) at the optical level. Such a clustering would be related to the
reactivation of rRNA transcription.
However, it is not evident from our study that the number of argyrophilic structures
is the same during early anaphase and early telophase. Such a problem could be solved
at the ultrastructural level by the study of serial and thick sections (Ploton, Menager
&Adnet, 19846).
Moreover, as stated by Hubbell et al. (1980), it is not yet clear whether the increase
in Ag stainability during anaphase and telophase is due to a modification of the
argyrophily of the Ag-NOR proteins or to an increase of their concentration. In the
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D. Ploton, M. Menager andjf. J. Adnet
latter hypothesis the origin of the Ag-stainable material that accumulates around the
NORs to form the prenucleolar bodies is still unclear. It may be formed by the
accumulation around the NOR of newly synthetized components or by the aggregation
of chromosomal material. According to Paweletz & Risueno (1982), the argyrophilic
RNP sheath surrounding the chromosomes originates from the nucleolus and forms
the prenucleolar bodies during telophase.
In our case a non-argyrophilic RNP sheath has been observed around the
chromosomes. However, there is no argument against its nucleolar origin, its participation in nucleolar reconstruction and its silver stainability during telophase.
Thus, as supposed by Ochs et al. (1983), the phosphorylated C23 protein, which is
the (or one of the) Ag-NOR protein(s), could be very sensitive to partial proteolysis
or dephosphorylation during metaphase. This would explain its apparent loss (except
in the NORs) during metaphase as shown by immunostaining and Ag-NOR staining.
Finally, like the origin of the RNA in prenucleolar bodies the origin of the Ag-NOR
proteins is still enigmatic (Goessens, 1984).
In conclusion, the simultaneous localization of Ag-NOR proteins and
nucleoproteins allowed us to follow the behaviour of the Ag-NOR proteins during all
the phases of mitosis in human cancerous tissues.
Our study showed the peculiar localization of the Ag-NOR proteins within RNP
fibrillar structures at the periphery of the metaphase and anaphase chromosomes.
Segregation of the Ag-NOR proteins in these RNP structures during telophase has
been also demonstrated.
The authors are indebted to Dr E. G. Jordan for critical reading of the manuscript. We are
grateful for the technical assistance of Mrs G. Himber and for secretarial work of Miss N. Hubert.
This work was supported by grants from the Federation Nationale des Centres de Lutte Contre le
Cancer.
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{Received 6 September 1984 -Accepted 11 October 1984)