J. Cell Sd. 74, 239-256 (1985) 239Printed in Great Britain © Company of Biologists Limited 1985

SIMULTANEOUS ULTRASTRUCTURALLOCALIZATION OF Ag-NOR (NUCLEOLARORGANIZER REGION) PROTEINS ANDRIBONUCLEOPROTEINS DURING MITOSIS, INHUMAN BREAST CANCEROUS TISSUES

D. PLOTON, M. MENAGER AND J. J. ADNETLaboratoire 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 silverstaining method followed by acetylation.

Owing to the good preservation of the cells and to the high precision of the Ag staining we were ableto 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 com-ponent (without visible fibrillar centre), which originated, during prophase, from the dense fibrillarcomponent of the nucleolus and then integrated within the periphery of some metaphasicchromosomes as irregular structures. The Ag staining of these structures was homogeneous butlower 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 proteinswere clustered as centrally localized spheres.

These findings showed the usefulness of our method for the precise study of the spatial relation-ship between the Ag-NOR proteins and the chromosomes. Thus we were able to show strikingsegregation of both the argyrophilic structures at the periphery of the chromatin mass and of the Ag-NOR proteins within these structures. This suggests that a high degree of the structural organizationof the future nucleus is already present during anaphase. This could be related to the rapid resump-tion of ribosomal RNA synthesis.

INTRODUCTION

The argyrophily of the nucleolar organizer regions (NORs) is due to non-histoneproteins, the Ag-NOR proteins, which are localized in the vicinity of the ribosomalDNA 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 underdiscussion: 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 breastcancerous 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 maybe related to the activity of the NORs (Howell, 1982). Moreover their use as a goodultrastructural marker for the rDNA genes has been recently proposed (Hernandez-Verdun & Derenzini, 1983).

Thus, in the nucleolus, the Ag-NOR proteins are localized within the fibrillarcentres 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 beendemonstrated (Hernandez-Verdun et al. 1982). These two components both con-stitute the interphase counterpart of the mitotic NORs (Hernandez-Verdun, 1983).

During mitosis the clustering of the rDNA genes in the mitotic NORs isconcomitant with the cessation of ribosomal RNA synthesis and with the disaggrega-tion of the nucleolus.

Morphological studies have shown this phenomenon and the association of variousnucleolar remnants with some chromosomes during prophase and the fusion ofprenucleolar bodies, which form the interphase nucleolus, during telophase (Stevens,1965; Hsu, Arrighi, Klevecz & Brinkley, 1965; Brinkley, 1965; Schwarzacher, Mikel-saar & Schnedl, 1978; Ploton & Gontcharoff, 1979).

The Ag-NOR proteins appear to be a good tracer, which may facilitate the studyof the behaviour of the NOR (fibrillar centre and fibrillar component) during all thephases of mitosis particularly when nucleolar remnants are difficult to identify.

Ultrastructural studies of mitosis with Ag-NOR staining are rare (Hernandez-Verdun et al. 1980; Paweletz & Risueno, 1982) and to our knowledge have never beencarried out on human cancerous tissues.

The aim of the present work is to describe a detailed study of the simultaneous localiza-tion of the Ag-NOR proteins and ribonucleoproteins (RNP) during all the phases ofmitosis 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 densefibrillar component that becomes partially integrated into the prophase chromosomes.At anaphase the Ag-NOR proteins are segregated as roundish structures localized onRNP fibrillar component within the surface of the chromosomes. They then becomeintegrated within the early telophase chromosomes and fuse to form the prenucleolarbodies 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 about0-5—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 andthen silver stained as described previously (Plotonet al. 1982, 1983, 1984a). For the silver stainingtwo solutions were prepared; the first consisted of 2 % (w/v) gelatin in 1 % formic acid; the secondconsisted of 50% (w/v) silver nitrate in water.

The staining solution was obtained by mixing one part of gelatin solution with two parts of thesilver nitrate solution. The slices were stained in this mixture for 10min at 70°C, then thoroughlyrinsed in distilled water, immersed in a 5 % (w/v) thiosulphate solution for 10 min and then rinsedin water.

The slices were then processed for acetylation as follows. They were dehydrated in graded acetonesolutions (5 min each in 25, 50, 75 and 90%, v/v) and then in three changes of pure acetone for10 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 andfinally 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 inpyridine/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-phosphatebuffer (pH 7-2) for 1 h at 4°C, rinsed in the game buffer and processed for acetylation as describedabove, for 15 h (Wassef, Burglen & Bernhard, 1979).

EmbeddingThe 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 sectionsUltrathin 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 forribonucleoproteins (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 allowsfor 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 thechromatin is high whereas that of RNP structures (nucleolus, peri- and inter-chromatin fibrils and granules) is low.

The regular silver dots (50-100 A in diameter) were specifically localized withinfibrillar centres and part of the dense fibrillar component. The granular component,which is easily localized, is never silver stained. When sections were preferentiallycontrasted for RNP by EDTA regressive staining the chromatin clumps were wellbleached whereas RNP components showed a high contrast. Silver dots were notdisplaced by the EDTA treatment. The bleaching of the chromosomes greatly en-hanced the precision of the localization of the Ag-NOR proteins and allows their studyparticularly during nucleolar disintegration and reformation. In order to gain a moreprecise view of the fine structure of some components we also studied sections ofacetylated blocks only in which the general definition of the nuclear structures isgreatly enhanced (Wassef et al. 1979; Ploton et al. 1983, 1984a).

242 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 andthe preferential localization of the condensing chromosomes near the nuclear en-velope.

The nucleoli, which frequently linked several chromosomes, had irregular shapesas they were beginning to disaggregate. This consisted of a segregation of the nucleo-lar components, which eventually led to their partial or complete separation.

We noticed frequent extrusions of the fibrillar component that was directly incontact with the chromosomes and seemed to become integrated within them.

The Ag-NOR proteins were localized within part of the fibrillar component of thenucleolus and within the nucleolar extrusions. As seen on a tangential section of ajunction of a chromosome with Ag-NOR proteins the latter never formed regularstructures but always contorted zones extending within or on the surface of thechromosome.

Fig. 1. Localization of the Ag-NOR proteins during prophase. A. The bleached con-densing 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 nucleolusand chromosomes, B. The Ag-NOR proteins are localized within the RNP fibrillar com-ponent of the nucleolus (arrowhead), or extruded from the nucleolus and integrated withinthe chromosomes (arrow). EDTA regressive staining. X 46000. c. On this tangentialsection the Ag-NOR proteins are seen within contorted zones that extend on or into thechromosome. EDTA regressive staining. X 46 000. D. The Ag-NOR proteins are localizedwithin 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 anddisorganization (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 proteinsare localized within the periphery of some chromosomes (arrows). Uranyl and leadstaining. X 16 000. B. Higher magnification of one of the argyrophilic structures. The Ag-NOR 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, whichmight 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 withina fibrillar RNP component of irregular shape and size; the Ag dots are few but arehomogeneously distributed within the fibrillar components (large arrows). Thechromosomal RNP sheath was not silver stained (small arrows). EDTA regressivestaining. 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 theargyrophilic RNP structures (large arrows) (0-3-0-4 fini) are localized. The clusters of Ag-NOR proteins (0-1-0-2 /*m) are centrally localized within these structures and never seenwithin the chromatin mass. No silver dots are seen in the chromosomal RNP sheath (smallarrows). 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 andreniform shape. The clusters of Ag-NOR proteins are centrally localized within RNPfibrillar 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. EDTAregressive staining, c, X 23000; D, X 66000.

244 D. Ploton, M. Menager andjf. jf. Adnet

2A

BFig. 2. For legend see p. 243

Ag-NOR proteins during mitosis 245

- f

Fig. 3. For legend see p. 243

246 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 RNPsheath, which was never silver stained. The Ag-NOR proteins were localized only onstructures composed of fibrillar RNP component that had an irregular size and shape.They were systematically integrated partly within the periphery of somechromosomes and showed an homogeneous but poor silver staining, which was verylow in comparison to that seen in prophasic nuclei.

Anaphase (Fig. 3). During early anaphase (Fig. 3A,B) the chromosomes fused andformed a compact chromatin mass in which spindle fibres were seen.

The Ag-NOR proteins formed roundish aggregates (of about 0-1-0-2/im indiameter) centrally localized within fibrillar RNP components. The latter werelocated 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 orpockets. The RNP sheath that covered the chromosomes had an irregular thicknessand was composed of both fibrillar and granular components.

At the end of anaphase (Fig. 3c,D) the mass of chromatin had an irregular andkidney-shaped cross-section incompletely lined with the new nuclear envelope. TheAg-NOR proteins formed roundish aggregates centrally localized within RNPfibrillar components systematically located in pockets within the chromatin. It wasnoted that the nuclear envelope was always interrupted at the level of these pockets.

Telophase (Fig. 4). The chromosomes were completely fused and formed a masswith a characteristic kidney-shaped cross-section regularly limited by the nuclearenvelope. The aggregates of the Ag-NOR proteins constituted spherical masses (0 • 1 ^min diameter) centrally localized within spherical fibrillar RNP structures (0-2 /Am indiameter) scattered within the chromatin.

Although they were frequently located at the border of the nuclei these RNPstructures 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 typicaldecondensation 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 proteinswere grouped as spherical clusters (0-2 /im in diameter) localized within sphericalaggregates 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 massof chromatin has a reniform shape.

The spherical clusters of Ag-NOR proteins (0-1 /an in diameter) are centrally localizedwithin spherical structures of RNP fibrillar component (0-2/an in diameter) surroundedby chromatin (arrows). Some of them are in contact with nucleoplasmic vacuoles(arrowhead). EDTA regressive staining, A, X 23 000; B, X 66000. c. The sphericalclusters of Ag-NOR proteins are centrally disposed in the spherical RNP structure(arrow). Note the presence of numerous nucleoplasmic vacuoles, which correspond withthe decondensation of the chromatin. Uranyl and lead staining. X 66000.

Ag-NOR proteins during mitosis

BFig. 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 asprenucleolar bodies.

In early interphasic nuclei, prenucleolar bodies were bigger, irregular, lessnumerous and incompletely silver stained in comparison to interphase nucleoli.

Acetylation (Fig. 6). The observation of sections of acetylated blocks showed detailsof the fine structure during disaggregation and reconstruction of the nucleolus.

During interphase some nucleoli showed typical fibrillar centres, fibrillar andgranular components.

During prophase the segregation of the granular and fibrillar components of thenucleolus was followed by the incorporation of the latter within the chromosomes.

During metaphase this study confirmed the presence, within the periphery of somechromosomes, of RNP clusters composed of both fibrillar and granular componentsin which roundish structures of fibrillar dense component were centrally localized.

During anaphase and telophase the progressive incorporation of spherical RNPstructures composed of both fibrillar and granular components within the chromatinwas 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 theappearance of preribosomal granules were noticed.

This study also confirmed the absence of visible fibrillar centre within the nucleolarremnants 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 ofthe nucleoplasm. The clusters of Ag-NOR proteins are bigger (0-2/an in diameter) andfewer (large arrows). They are localized at the periphery of some chromatin clumps withinRNP fibrillar structures, which they completely cover (prenucleolar bodies of 0-3 /un indiameter). 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 indiameter). They are characterized by an irregular shape. The Ag-NOR proteins areirregularly disposed and not present in all the fibrillar component. EDTA regressivestaining. X 66000.

Fig. 6. Behaviour of the nucleolar components during mitosis as seen after acetylationonly, and uranyl and lead staining, A. Interphase. When nucleoli with fibrillar centres arevisible they show the following typical distribution of their components: a sphericalfibrillar centre (0-3 fJm in diameter), which appears as a low-stained structure (fc); a ringof fibrillar component (/) and a granular area (g). Some chromatin clumps can also beseen. X 66000. B. Prophase. During prophase a segregation of the nucleolar componentscan be noticed. The fibrillar component is integrated within the periphery of the condens-ing chromosomes (arrow). X 66000. c. Metaphase. Some fibrillo-granular RNP struc-tures were seen within the periphery of some chromosomes. The fibrillar componentconstitutes some roundish structures in contact with the chromatin (arrow). X 66000.D. Anaphase. The fibrillar RNP structures are localized within depressions or pocketswithin the periphery of the chromosomes (arrow). X 66000. E. Telophase. The fibrillarRNP structures become spherical (0-1 jan in diameter) and are incorporated within thechromatin 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. Thechromatin is decondensed. X 66000.

250 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 breastcancerous cells in vivo has been studied using a one-step silver staining methodfollowed by acetylation.

The use of this technique resulted in the localization of small-sized Ag dots onstructures whose morphology and cytochemical nature may be established by the useof the acetylation procedure and EDTA regressive staining (Ploton et al. 1984a).

Moreover, the bleaching of the chromatin after EDTA staining facilitated the studyof the spatial relationships between the argyrophilic structures and the chromosomes.

In the human cancerous tissues examined in this study, it appeared that during allthe phases of mitosis the Ag-NOR proteins were never localized within condensedchromatin itself but only within fibrillar clumps that possessed a high contrast afterEDTA regressive staining and were associated with condensed chromatin.

As confirmed by examination of sections of blocks that had been acetylated only thesupposed Ag-stained structures were mainly composed of fibrillar components inwhich fibrillar centres were no longer visible.

It also appeared that the RNP sheath that covered the chromosomes, whose nucleo-lar origin had been supposed (Moyne & Garrido, 1976), was never silver stained. Thisconfirms 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, inour 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 back-ground in the nucleoplasm or the cytoplasm.

Our study has followed the behaviour of the clusters of argyrophilic RNP fibrillarcomponent seen on metaphase chromosomes from their origin. There is no doubt thatthese clusters originated at prophase from the Ag-staining fibrillar component thatextruded from the nucleolus and thus represent what is generally called nucleolarremnants.

This phenomenon is concomitant with the disaggregation of the nucleolus, whichfollows a kind of micro-segregation of the granular and fibrillar components.

The ability of these clusters to fuse during telophase to form prenucleolar bodiessuggests that they could contain the NORs.

The fact that no fibrillar centre was found in these clusters is in contrast withprevious results of Goessens & Lepoint (1974), who showed that in Ehrlich cells thefibrillar 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 onlyargyrophilic structure present during mitosis.

The differences concerning the localization of the Ag-NOR proteins within thechromosomes in Ehrlich cells and in the human material used by us may be due tovariations of the size of the fibrillar centres within the nucleoli (big in Ehrlich cells andsmall, rare or absent in our material) and to variations in the size and location of the

252 D. Ploton, M. Menager andj. J. Adnet

secondary constrictions (conspicuous on telocentric chromosomes of Ehrlich cells andinconspicuous on the short arm of the human acrocentrics; Howell, 1982).

However, it must be kept in mind that the dense fibrillar component and thefibrillar centres are the sites where the NOR chromatin is located during interphase(Hernandez-Verdun, 1983) and that both were always stained in our material. Theabsence of visible fibrillar centres during mitosis could be due to a kind of maskingby fibrillar component, which has been evoked to explain the absence of fibrillarcentres in some nucleoli (Knibiehler, Mirre & Rosset, 1982; Williams et al. 1982;Knibiehler & Mirre, 1983; Boloukhere, 1984; Goessens, 1984), or in secondaryconstriction of chromosomes where rDNA is, however, localized (Hsu, Spirito &Pardue, 1975).

Moreover, our finding of the NORs within the periphery of some chromosomespartly confirms previous work of Schwarzacher et al. (1978), who found that theNORs of isolated human chromosomes seemed to be in an extrachromosomalposition.

Our study also demonstrates important variations during mitosis in the localizationof the Ag-NOR proteins within clusters of RNP fibrillar components. Thus a segrega-tion of the Ag-NOR proteins within these structures characterized the second part ofmitosis following the disruption of the chromosomes. This segregation is alwaysfollowed by the acquisition by RNP clusters of a spherical shape and by their integra-tion within the telophasic mass of chromatin. It also appears from our study that theAg-NOR proteins are not localized at random within the early telophase nuclei butare preferentially localized at the periphery of the nuclei, in direct contact with thenuclear envelope, and are never localized within the chromatin mass. This couldindicate that the telophase nuclei are highly ordered structures at least as regards thefuture nucleolar bodies and their Ag-NOR proteins. In particular, a preferentialdisposition of the future prenucleolar bodies in contact with the new nuclear envelopemay be compared with the frequent junctions of the interphasic nucleolus with thenuclear envelope (Bourgeois, Bard & Bouteille, 1983).

The exact nature of the Ag-NOR proteins is still controversial. The more likelycandidates are the larger subunit of RNA polymerase I (Williams et al. 1982) and, asmore recently proposed, the C23 protein (Lischwe et al. 1981; Ochs et al. 1983),which have both been immunolocalized within the secondary constrictions (Lischweet 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 rDNAgenes during interphase and metaphase. Although Ag-NOR staining cannot be con-sidered as really specific because Ag staining of non-nucleolar structures occurs insome material (Seite, Pebusque & Vio-Cigna, 1982; Boloukhere, 1984), it has beenproposed 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 proteinsduring mitosis? If so we could then confirm the preferential segregation of rDNA onsome 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 atthe periphery of the anaphase and early telophase masses of chromatin.

Such proximity of the NOR-bearing chromosomes has been previously proposedby Bobrow & Heritage (1980) and has been evoked to explain the morphologicalassociation of these chromosomes on karyotypes (Schwarzacher et al. 1978; Howell,1982). This could also favour the rapid association of NORs for nucleolar formationduring telophase as noted by Anastassova-Kristeva (1977).

Our work confirms the strict association of the Ag-NOR proteins with structureswhose nucleolar origin and participation in late telophase reconstruction of thenucleolus are undoubted.

However, it cannot be excluded that during some steps of mitosis (apart frommetaphase) some of the Ag-NOR proteins may leave the NOR and accumulate inseparate regions. Therefore, additional work is needed to demonstrate the eventualrelationships 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 NORsmay 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 Agstaining intensity and rDNA transcription as demonstrated after natural or artificialinhibition of rRNA synthesis (Boloukhere, 1984). Dimova et al. (1982) also putforward the hypothesis that Ag staining does not reflect actual transcription of rDNAgenes.

Another hypothesis has been recently presented, which suggests that Ag-NORstaining would detect rDNA in a decondensed state but not necessarily in an activeone (Hernandez-Verdun et al. 1983; Medina et al. 1983).

Similarly, during mitosis the Ag staining of the metaphase NORs, which is muchless than that of the interphase nucleolus (Hubbell et al. 1980), may be related to lowtranscription 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 tothose of anaphase and telophase.

We demonstrated a clustering of Ag dots on the argyrophilic structures duringanaphase and telophase and consequently a higher intensity of Ag staining, which maybe related to previous findings of Busch, Daskal, Gyorkey & Smetana (1979) and ofHubbell et al. (1980) at the optical level. Such a clustering would be related to thereactivation of rRNA transcription.

However, it is not evident from our study that the number of argyrophilic structuresis the same during early anaphase and early telophase. Such a problem could be solvedat 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 increasein Ag stainability during anaphase and telophase is due to a modification of theargyrophily of the Ag-NOR proteins or to an increase of their concentration. In the

254 D. Ploton, M. Menager andjf. J. Adnet

latter hypothesis the origin of the Ag-stainable material that accumulates around theNORs to form the prenucleolar bodies is still unclear. It may be formed by theaccumulation around the NOR of newly synthetized components or by the aggregationof chromosomal material. According to Paweletz & Risueno (1982), the argyrophilicRNP sheath surrounding the chromosomes originates from the nucleolus and formsthe prenucleolar bodies during telophase.

In our case a non-argyrophilic RNP sheath has been observed around thechromosomes. However, there is no argument against its nucleolar origin, its par-ticipation in nucleolar reconstruction and its silver stainability during telophase.Thus, as supposed by Ochs et al. (1983), the phosphorylated C23 protein, which isthe (or one of the) Ag-NOR protein(s), could be very sensitive to partial proteolysisor dephosphorylation during metaphase. This would explain its apparent loss (exceptin 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-NORproteins is still enigmatic (Goessens, 1984).

In conclusion, the simultaneous localization of Ag-NOR proteins andnucleoproteins allowed us to follow the behaviour of the Ag-NOR proteins during allthe phases of mitosis in human cancerous tissues.

Our study showed the peculiar localization of the Ag-NOR proteins within RNPfibrillar structures at the periphery of the metaphase and anaphase chromosomes.Segregation of the Ag-NOR proteins in these RNP structures during telophase hasbeen also demonstrated.

The authors are indebted to Dr E. G. Jordan for critical reading of the manuscript. We aregrateful 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 leCancer.

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{Received 6 September 1984 -Accepted 11 October 1984)