J. Cell Sci. 25, 103-110 (1977) 103

Printed in Great Britain

THE NUCLEOLAR CYCLE IN MAN

MARLEN ANASTASSOVA-KRISTEVAInstitute of Morphology, Bulgarian Academy of Sciences,Sofia 1113, Bulgaria

SUMMARY

Tissue cultures of human embryonal kidney and ovary were examined. In the nuclei of bothtissues, one to ten nucleoli have been found. The maximum number of nucleoli is connectedwith the gene expression of rDNA of the 10 nucleolus organizers of chromosome pairs Nos. 13,14, 15, 21 and 22, which have secondary constrictions and are the satellite chromosomes in man.The small percentage of cells with 10, 9 and 8 nucleoli is attributed to the rapid association of 3of the homologous acrocentrics (perhaps of group D). Two of the satellite (SAT) pairs probablyassociate later after mitosis. The process of fusion is dynamic, resulting in one interphasenucleolus - a manifestation of the association of all SAT chromosomes. Dissociation of thenucleolus occurs upon entering prophase, due to the condensation of the chromosomes andretreat of rDNA to the respective secondary constrictions. As a result, the nucleolar numberincreases again. The pattern of the nucleolar kinetics within the course of one mitotic division isdescribed.

INTRODUCTION

The possibility of plant chromosomes being identified in histological sections resul-ted in more rapid progress in plant cytogenetics in the 1930's, in comparison with thatof animals and man. Studying the chromosomes of various plant species, it was ob-served that during the mitotic cycle the nucleolus disappears, changing in size, shapeand number. The role of the nucleolus was entirely unknown at that time, but never-theless the data obtained using the classical histological techniques are frequentlyremarkably precise. In his work Heitz (1931) defends in an extremely convincingmanner the hypothesis that, ' the nucleoli are formed on the SAT chromosomes(Nukleolenchromosomen) below the satellite', hence ' the number of telophase nucleoli(Primarzahl) is constant as well as the number of the SAT chromosomes for a givenkaryotype, e.g. 2 SAT chromosomes - 2 nucleoli (Vicia faba, Crepis virens, Crepissibirica); 4 SAT chromosomes - 4 nucleoli (Vicia lutea, Viciapanonica, Crepispulchra)'.

The improvement of karyological methods has contributed to the final determina-tion of the human karyotype (Tjio & Levan, 1956). The findings of Heitz in plantshave not been taken fully into account in the studies of nucleolar origin in animals andman. Dearing (1934), Kaufmann (1938) and later Wallace (i960), Barr (1966) andothers pointed out that the secondary constrictions in the SAT chromosomes ofanimals also play the role of nucleolar organizers (NORs).

The karyotype of man contains 5 SAT chromosome pairs with secondary constric-tions. These are the acrocentric chromosomes Nos. 13, 14, 15, 21 and 22. Therefore,the genome of the human cell contains 10 NORs. There are, however, scanty data

104 M. Anastassova-Kristeva

concerning the nucleolar kinetics and morphology during the human mitotic cycle. It isstated that the cells of a given tissue possess one or two nucleoli and no connexion withthe number of NORs in the corresponding karyotype has been sought. According toHsu, Spirito & Pardue (1975) the maximum number of 10 nucleoli is seldom reachedin highly differentiated cells, where a single nucleolus per nucleus is the predominantfeature.

In some previous studies (Nicoloff, Anastassova-Kristeva & Kiinzel, 1976) themode of association between the 4 NORs in Hordeum was described. A similar processin man has not, however, been proved.

The present paper represents an attempt to follow the morphological manifestationof gene expression of the 10 human NORs during the mitotic cell cycle.

MATERIAL AND METHODSThe nucleoli were investigated in embryonic ovarian and kidney cells cultivated in vitro.

Under these conditions the number of mitotic dividing cells increases and the probability offinding nuclei which contain the primary number of nucleoli is greater.

Pieces of ovaries of 4- to 5-month old human embryos were cultivated on coverslides inmedium Parker 199 and 20% calf serum. After cultivation for 8 days the cells were fixed inCarnoy's fluid and stained with haematoxylin-eosin and methyl green-pyronin.

Primary suspension cultures of embryonal kidney were provided by the Virology Depart-ment of Research Institute of Infectious and Parasitic Diseases-Sofia by Associate ProfessorDr Sv. Atzev and by the Department of Microbiology and Virology at the Medical Faculty inVarna by Dr V. Gurdevsca, to whom the author's gratitude is due.

RESULTS AND DISCUSSION

Some kidney tissue cultures reveal 'organoid' reassociation of epitheloid cellssurrounded by connective tissue cells. It should be noted that the centrally locatedepitheloid cells have predominantly one or two nucleoli, while the surrounding cellsexhibit a higher number of nucleoli (Fig. 1). In diploid human fibroblast culturesSchnedl & Schnedl (1972) have counted up to 7 nucleoli per nucleus. Unlike theseauthors we have found cells with 8, 9 and 10 nucleoli in both ovarian and kidneycultures (Fig. 2). These cells, which had just undergone division, were usually foundsituated in pairs in the culture (Fig. 1). One of the kidney cultures proved to be par-ticularly rich in cells with 9 or 10 nucleoli (Fig. 2B).

The chromatin of the tissue culture cells is very fine and evenly distributed, withoutdeeply stained chromocentres typical of the interphase nuclei in histological sections.Only the sex chromatin in some of the cultivated cells of the ovary and kidney can beseen as a heterochromatic cluster attached to the nuclear membrane. Against thebackground of the rather homogenous and pale nucleoplasm, the nucleoli are veryprominent. According to their morphology the nucleoli can be subdivided into 2types: the first type are well rounded, quite dense and with a regular outline, while theothers are less dense and irregular in shape. Multiple nucleoli with irregular outlineappear in telophase and their maximum number - 10 - corresponds to the number ofNORs in the human karyotype. It is known that the ultrastructure of secondary con-

Nucleolar cycle in man 1 0 ;

; * ,

m[MS

V

* • • • • • . 0

1 A

• • » .

Fig. i. Tissue cultures of human embryonic kidney; A, epitheloid cells containingpredominantly 1-2 nucleoli and connective tissue cells with more nucleoli; H-E,daughter cells immediately after mitosis, containing 9 and 10 nucleoli. A, X 160; B, c,x 320; D, E, x 800.

strictions and of the nucleolar light fibrillar centres is similar (Lafontaine, 1968). Thelow electron density is probably due to different types of histones and nonhistoneproteins bound to rDNA, which perhaps determine the lower specificity of rDNAderepression. Experimental injuries to one of the nucleoli in a given nucleus result in acompensatory enlargement of the other. This is an indication of the identity of all

io6 M. Anastassova-Kristeva

10

8 , $ & ' • ' •

i«

6

Fig. 2. Nuclei containing I - IO nucleoli. A, from human embryonic ovary; B, C, fromhuman embryonic kidney, x 8oo.

Nucleolar cycle in man 107

rDNA cistrons localized in NORs of various homologous chromosome pairs. It mightbe the reason for the simultaneous expression of rDNA in all NORs of a nucleus.Presumably the inductor for all rDNA cistrons is the same, while the duration andintensity of the synthesis are influenced by some additional factors.

It should be pointed out that fusion of the nucleoli is a very active and dynamicprocess. Very often the nucleoli are found fixed at what appears to be the moment offusion, or connected by thin or wide bridges. This creates difficulties in the precise de-termination of the boundary between the individual nucleoli and their counting. Whilethe primary number of the nucleoli is under genetical control, the order of their con-secutive association does not seem to be exactly determined. In spite of that, in thehuman ovarian and kidney cells some regular features have been observed. For ex-ample in the case of 2 nucleoli per nucleus one is larger than the other. A comparison ofkidney and ovarian nuclei with 3, 4 and 5 nucleoli shows great similarity in the size ofthe nucleoli of the 2 different tissues (Fig. 2).

The small percentage of cells with 8, 9 and 10 nucleoli shows that 6 of the acrocen-tric chromosomes in man associate very quickly after mitosis and 4 of them associatelater. It can be supposed that the longer homologues of group D will associate earlier.The 3 nucleoli obtained, together with the 4 nucleoli of group G, which will associ-ate later, will produce nuclei with 7 nucleoli, which occur more frequently. The gra-dual reduction of the nucleolar number shows that the process of association is notsynchronous. As a result of the association the nucleoli became larger and denser.In our opinion the single interphase nucleolus is a manifestation of complete associa-tion between the acrocentric chromosomes (Fig. 3). One or two nucleoli are obviouslytypical of this interphase period, during which the functions specific for the cellulartype are realized.

Upon entering prophase and with increasing degree of spiralization of the chromo-somes, the association of nucleoli is reversed and dissociation begins (Fig. 3). Now thenucleolus loses its sharp contours and its density decreases. This is presumablycorrelated with the lower synthetic activity of rDNA. The stronger the spiralization ofthe chromosomes, the greater the number of nucleoli. Schnedl & Schnedl (1972) alsoreport an increase in the number of nucleoli in cells after replication of DNA. It seemsthat rDNA of the NOR is contracted to the respective chromosomal secondary con-striction and parallel with this process it entrains part of the granular component of thenucleolus, as a result of which more and smaller nucleoli are obtained. Some pre-liminary data in our studies on the nucleolus in human lymphocyte cultures (Anastas-sova-Kristeva, 1973 a, b) indicate that the number of nucleoli here may again reachten. On the other hand, it should be established whether in the case of some humanacrocentrics the dissociation does not occur later in prophase, as happens in some plantSAT chromosomes (Nicoloff et al. 1976). In such cases the primary number of nucleolicannot be observed in prophase. In the tissue cultures studied, cells with various num-bers of nucleoli were observed. However, further investigations are necessary in orderto reconstruct all stages of dissociation until the complete disappearance of the nucle-olus in metaphase. It should be pointed out here that by the term 'complete disap-pearance' we refer to the absence of a nucleolus visible in the light microscope. In the

io8 M. Anastassova-Kristeva

electron microscope this corresponds to destruction oi pars granulosa nucleoli, parts ofwhich may persist scattered among the chromosomes. The persistence of lightfibrillar centres of the nucleoli in the structure of the metaphase chromosomes at theultrastructural level has been established on many occasions (Brinkley, 1965; Hsu,Arrighi, Klevecz & Brinkley, 1965; Hsu, Brinkley & Arrighi, 1967; Goessens &Lepoint, 1974; Goessens, 1975). This supports the view that nucleolar material is

Fig. 3. Pattern of the human nucleolar kinetics within the framework of one mitoticdivision. The dotted, discontinuous and continuous circles express the nucleolar, mitoticand synthetic cycles respectively.

transferred from mother to daughter cells. It also suggests that the primary nucleolioccur as a result of the expression of rDNA cistrons of the NORs. The appearance ofthe primary nucleoli also coincides with the beginning of rRNA synthesis in them,which has been proved autoradiographically by Woods & Taylor (1959) and by La-fontaine (1968). This is in contradiction to the assertion that NORs are passive organ-izers, mechanically collecting previously produced nucleolar material from the mothercell. A 'repopulation' (Phillips, 1972; Chentsov & Poliakov, 1974) of primary nucleoliwith such material is possible, but it is a consequence and not the cause of their forma-tion.

Nucleolar cycle in man 109

On the basis of the results obtained, a model of the nucleolar cycle in human cells ispresented (Fig. 3). It reflects the kinetics of the nucleolus within the framework of onemitotic division, starting with the appearance of the primary nucleoli in telophase,their gradual association into a single interphase nucleolus and some stages of the dis-sociation of the nucleolus until its disappearance in metaphase. The duration of theindividual phases is not given, since they may vary depending on the level of differentia-tion and functional state of the different cells. In the case of intensive proliferation,interphase may be reduced to a minimum, while in highly differentiated cells it mayalready be extremely prolonged at the stage of 1-2 nucleoli. The study of the nucleolarkinetics does not give reason to assume one Go period preceding Gx (Epifanova &Terskikh, 1969). Obviously the cell cannot pass into a resting period immediatelyafter telophase. This is evidenced by the active process of association of the primarynucleoli, leading the cell to a specific functional state. Evidently the Gx period includesthe specific cell functions, which are an essential part of the cell life cycle.

In our opinion, the number of nucleoli can be used as an indication of the degree ofassociation of the SAT chromosomes in the interphase nucleus.

The present study reveals that the processes of formation, fusion and dissociationof the nucleolus, described for some plant species (Nicoloff et al. 1976; Anastassova-Kristeva & Nicoloff, 1976) take place in the human cells too. Knowledge of nucleolarkinetics in the mitotic cycle may be useful in determining the different functionalstages of both normal and pathological cells.

The author is grateful to Associate Professors Dr Georgi Markov and Emanuel Tchakarovfor their critical reading of the manuscript and some useful suggestions.

REFERENCES

ANASTASSOVA-KRISTEVA, M. (1971). The nucleolar cycle at mitosis and meiosis. IVth int. Congr.CytoL, London, May 1971. Summary of papers No. 84, 97.

ANASTASSOVA-KRISTEVA, M. (1973 a). The nucleolus as an ingredient of the cellular geneticapparatus. 2nd A. Meet. eur. Environ. Mutagen. Soc, Czechoslovakia, May 1972. Summaryin: Mutation Res. 21, 21-22.

ANASTASSOVA-KRISTEVA, M. & KANCHEVA, L. (19736). On the autoradiography of human lym-phocytes activated by PHA. C. r. Bulg. Acad. Sci. 26, 549-552.

ANASTASSOVA-KRISTEVA, M. & NICOLOFF, H. (1976). Nucleolus organizing regions andnucleoli formation in Allium odorum L. (in Press).

BARR, H. J. (1966). Problems in the development and cytogenetics of nucleoli in Xenopus.Natn. Cancer Inst. Monogr. 23, 411-424.

BRINKLEY, B. R. (1965). The fine structure of the nucleolus in mitotic division of Chinesehamster cells in vitro. J. Cell Biol. 27, 411-422.

CHENTSOV, J. S. & POLIAKOV, V. J. (1974). Ultrastructure of cell nucleus. Publ. Nauka, Moscow,pp. 1-175. (In Russian.)

DEARING, N. H. (1934). The material continuity and individuality of the somatic chromosomesof Amblystoma tigrinum, with special reference to the nucleolus as a chromosomal component.J. Morph. 56, 157-159-

EPIFANOVA, O. J. & TERSKIKH, V. V. (1969). The Autoradiographic Method in Cell Cycle Re-search. Moscow. (In Russian.)

GOESSENS, G. & LEPOINT, A. (1974). The fine structure of the nucleolus during interphase andmitosis in Ehrlich tumour cells cultivated in vitro. Expl Cell Res. 87, 63-72.

8 CEL 25

n o M. Anastassova-Kristeva

GOESSENS, G. (1975). Etude Ultrastructurale des Nucleoles au cours du Cycle Cellulaire. DoctoratSci. Biomed. Univ. Liege, 1-173.

HEITZ, E. (1931). DieUrsache der gesatzmassigen Zahl, Lage und Form pflanzlicher Nukleolen.Planta 12, 775-844.

Hsu, T. C, ARRICHI, F. E., KLEVECZ, R. R. & BRINKLEY, B. R. (1965). The nucleoli in mitoticdivisions of mammalian cells in vitro. J. Cell Biol. 26, 539-553.

Hsu, T. C, BRINKLEY, B. R. & ARRIGHI, F. E. (1967). The structure and behaviour of thenucleolus organizers in mammalian cells. Chromosoma 23, 137-153.

Hsu, T. C., SPIRITO, S. E. & PARDUE, M. L. (1975). Distribution of 18 + 28 s ribosomal genesin mammalian genomes. Chromosoma 53, 26-36.

KAUFMANN, B. P. (1938). Nucleolus-organizing regions in salivary gland chromosomes ofDrosophila melanogaster. Z. Zellforsch. mikrosk. Anat. 28, 1-11.

LAFONTAINE, J. G. (1968). Structural components of the nucleus in mitotic plant cells. InThe Nucleus (ed. A. J. Dalton & F. Haguenau), pp. 151-196. New York: Academic Press.

NICOLOFF, H., ANASTASSOVA-KRISTEVA, M. & KUNZEL, G. (1976). Changes in nucleolarorganizer activity due to segmental interchanges between satellite chromosomes in barley.Biol. Zbl. (in Press).

PHILLIPS, S. G. (1972). Repopulation of the postmitotic nucleolus by preformed RNA. jf. CellBiol. 53, 611-623.

SCHNEDL, W. & SCHNEDL, M. (1972). Nucleoluszahl und -grosse wahrend des Zellzyclus. Z.Zellforsch. mikrosk. Anat. 126, 374-382.

TJIO, J. & LEVAN, A. (1956). The chromosome number of man. Hereditas 42, 1-6.WALLACE, H. (i960). The development of anucleolate embryos of Xenopus laevis. J. Embryol.

exp. Morph. 8, 405-413.WOODS, P. S. & TAYLOR, J. H. (1959). Studies of ribonucleic acid metabolism with tritium-

labeled cytidine. Lab. Invest. 8, 309-318.

{Received 14 July 1976 - Revised 19 October 1976)