Studies on nuclear fine structure

Nuclear

497

fine sfructure

14. 15. 16. 17.

MARTIN, R. G. and AMES, B. N., Proc. Natl Acad. Sci. U.S. 48, 2171 (1962). MCCOY, T. A., MAXWELL, M. and NEWMAN, R. E., Cancer Res. 16, 979 (1956). RAIN&A., Acta Physiol. &and. 60 Suppl. 218 (1963). RAZIN. S. and ROZANSKY. R., Arch. Biochem. Biophys. 81, 36 (1959). 18. SCHNEiDER, w. c., in C~LO~ICK, R. S. and KAPLAN, N..O. (eds.), Methods in Enzymology, Vol. 3, p. 680. Acad. Press, New York, 1957. 19. SPIEGELMAX, S., in TUNEVALL, G. (ed.), Recent Progress in Microbiology, p. 81. Almqvist and Wiksell, Stockholm, Sweden, 1959. 20. TABOR, H., TABOR, C. W. and ROSENTAAL, S. M., Ann. Rev. Biochem. 30, 579 (1961).

STUDIES

ON NUCLEAR

THREE

d’Histologie,

STRUCTURE

PHASES OF THE HELA CELL CYCLE’

B. BLONDEL Institut

FINE

&ole Received

and L. J. TOLMACHa de MJdecine, October

Geneva,

Switzerland

21, 1964

THEmethod of selective synchronization

described by Terasima and Tolmach [12] for cultured cells yields populations of cells 75-90 per cent of which are in mitosis, without the introduction of any inhibiting agents. We have used this method to investigate two aspects of the cell division cycle: (a) morphological manifestations of the transition from mitotic to interphase chromosomes, and (b) possible morphological differences between nuclei in G, and in S. The results reported here indicate that the M to G, transition is progressive and hence, can be morphologically characterized on an ultrastructural level. However, we observed no morphological differences between nuclei in G, and in S. Material

and methods

Synchronous cultures of HeLa S3 cells were initiated with mitotic cells harvested from random cultures and the degree of synchronization controlled by cytological observations [12]. In addition, the S phase was traced by exposing aliquots of the synchronized cells to 3H or 1% thymidine for 30 min periods at intervals, throughout the duration of the experiment. Following variable periods of growth cells were prepared for observation with the electron microscopeby a procedure of fixation derived from Ryter and Kellenberger’s technique, which stabilizes DNA filaments [6, 91: 1 This investigation was supported from the National Cancer Institute 2 Permanent address: Department St. Louis, MO., U.S.A.

by U.S. Public Health Service Research Grant No. CA-04483 and the Swiss National Foundation for Scientific Research. of Radiology, Washington University School of Medicine,

Experimental

Cell Research

37

498

B. Blonde1

and L. J. Tolmach

Fixative.-1 per cent OsO,, 0.01 M CaCl,, 0.68 per cent NaCl in Michaelis buffer pH 7.25. Fixation.-Cells prepared soon after seeding were centrifugedwith dilute(lOper cent) fixative, while attached cells were first trypsinized and then similarly collected. The supernatants were removed and the cells treated with fixative containing added tryptone (0.01 per cent final concentration). Following over-night fixation at room temperature, the cells were treated for 14-2 hr with 0.5 per cent uranyl acetate, dehydrated with acetone and embedded in Vestopal. Results Mitotic cells.-Harvested mitotic cells constitute a mixture representing all the stages of mitosis, but the majority are in metaphase [12]. The chromosomes appear as masses of concentrated granular material (Fig. 1). Careful observations reveal a mixture of granules 100 to 150 A in diameter, and of what appears to be a network of filaments of 45 to 100 A diameter. A certain amount of variation in composition is noticeable, some cells having chromosomes more filamentous or more granular than the average. Similar variation is found in any given cell, one part of the chromosomal mass sometimes being more filamentous than the remainder. Furthermore, the chromosomal masses do not display homogeneous density. We found no preferential orientation of the chromosomal elements, except for occasional short regions of filaments oriented in parallel. Passage from M to G,.-At telophase the chromosomes are united in a rather comThe nuclear membrane surrounds closely this pact bulk and show anastomosis. bulky mass. By fixing cells between harvesting and 14 hr, we could observe the decondensation of the chromosomal mass which, then, takes place. The condensed material no longer appears as a single unit, but is divided into several patches separated by areas of much less dense material (Fig. 2). This phenomenon is progressive as we observed at each step nuclei in different degree of decondensation (i.e. 50 per cent more or less, of the nucleoplasm still condensed). G, cells.-From the morphological and chemical tests it was concluded that cells fixed between 16 and 6 hr after harvesting were in G,. Their nucleoplasm (Fig. 3) shows two kinds of areas. One is composed of small patches of compact material distributed mostly along the nuclear membrane but also scattered throughout the nucleus and sometimes around the nucleolus. They have the same appearance as condensed chromosomes. The second area is composed of a much less concentrated mixture of filaments and granules, again without defined structure or organization. These granular and filamentous components have the same dimension as those of the chromosomes. No areas containing either granules or filaments alone were seen. One

Fig.

l.-HeLa

Cell in metaphase.

x 24,000.

ch,

Condensed

chromosome;

cy, cytoplasm.

Fig. 2.-HeLa cell 38 min post-synchronization. The chromosomal mass is in the process of decondensing. About 50 per cent of the chromosomal material is still condensed (c). Areas of much less dense material are visible. x 24,000. cy, Cytoplasm, m, nuclear membrane. Fig. 3.-HeLa cell in G, phase-2f the maximum of decondensation. Cytoplasm; m, nuclear membrane.

Experimental

Cell Resenrch 37

hr post-synchronization. The chromosomal mass has achieved Some patches of dense material remain (arrows). x 24,000. cv.

Nuclear

33 ~

651810

fine structure

499

Experimenlal

Cell Research

37

500

B. Blonde1

and

L. J. Tolmach

or more nucleoli were observed, often located close to or even in contact with the nuclear membrane. S cells.-The foregoing description of the nuclei of G, cells applies to the nuclei of cells undergoing DNA synthesis also. That is, with the method of specimenpreparation used, we have observed no conspicuousdifferences between the G, and S phase. Discussion

According to our observation the condensed chromosomesof HeLa cells have a fine structure difficult to characterize. They appear to be both filamentous and granular and we cannot claim as Ris does that the chromosomesare composed of filaments [7]. These chromosomesare similar to those of the cancer cells described by Bernhard and Granboulan [l] and resemble the micrographs of those of HeLa published by Robbins and Gonatas [8]. We suggestthat the presenceof granules in these chromosomescompared to their absencein chromosomesof Amphidinium and spermatozoa [2, 41 cannot be explained only on the basis of the coiling of nucleohistones [I], but might also indicate a chromosomericher in protein. In addition, the lack of homogeneousdensity of the condensed chromosome, detected with this method of fixation, suggeststhe existence of a superstructure. The progressive dispersion or decondensation of the chromosomal massfollowing telophase appears to be incomplete, as condensedmaterial is still observed during G, and S. Two hypotheses may be advanced: (I) Penetration of the nucleus by extranuclear substances might scatter the chromosomal mass. The remaining condensates observed in G, and S would then represent the totality of the chromosomes. This seemsto be difficult to conceive when considering the total volume occupied by chromosomes. (2) The chromosomal material might undergo decondensation, this processextending to only some of the chromosomesor to only a part of each. The result would be an expansion of much of the chromosomal material, which would appear as the less dense regions described above and denoted as interchromatinic areas by some authors [I, IO]. We favor this hypothesis which is an agreement with Hay and Revel’s interpretation of their observations on Amblystoma larvae, although we have not observed in G, and S areaswhich are distinctly either granular or filamentous as described in Amblystoma [5]. The decondensedregions have a fine structure as difficult to characterize as that of the condensedchromosomes.Comparison of our observations with those reported for expanded nucleoplasmsof a number of different organisms[3, 4, 5, 61, all showing a fibrous structure, suggeststhat in these regions, an underlying fibrous structure might be present but masked by an additional component. We have found that the nucleus of a cell in G, cannot be distinguished from that of a cell which is actively synthesizing DNA. Although it might have been expected that ultrastructural changeswould be found, arising from the presenceof fully extended DNA molecules undergoing replication, our failure to identify such changes does not rule out their occurrence in restricted regions of the nucleus. Replication of DNA does not proceed simultaneously among all of the chromosomesof a cell, nor ever within a given chromosome [ill. Consequently, the number or extent of replicating Experimenfnl

Cell

Research

37

Culture

of preimplantation

mammalian

501

embryos

regions present at any instant might be too small to be detected. Alternatively, the changes might occur at a level of organization beyond the resolving power of the techniques employed. We thank Miss N. Kempers for excellent technical assistance. REFERENCES 1. BERNHARD, W. and GRANBOULAN, N., Exp:pt[ Cell Res. Sup@. 9, 19 (1963). 2. GRASS& P. P., CARASSO, N. and FAVARD, P., Compt. Rend. Acad. Sci. 242, 1395 (1956). 3. GRELL, K. G. and WOHLFARTH-BOTTERMANN, K. E., Z. Zellforsch. 47, 7 (1957). 4. DE HALLER, G., KELLENBERGER, E. and ROUILLER, C., J. Microscop. In press. 5. HAY, E. D. and REVEL, J. P., J. Cell Biol. 16, 29 (1963). 6. KELLENBERCER, E., in Interpretation of Ultrastructure (Symp. Intern. Sot. Cell Biol., N. p. 233. Academic Press, New York 1962. 7. RIS, H., Can. J. Genet. Cytol. 3, 95 (1961). 8. ROBBINS, E. and GONATAS, N., J. Cell BioZ. 21, 429 (1964). 9. RYTER, A. and KELLENBERGER E., Z. Naturforsch. 13 b, 597 (1958). 10. SWIFT, H., in Structure and Function of Genetic Elements, p. 134. Brookhaven Symposium in Biology No 12, Brookhaven National Laboratory, Upton New York, 1959. 11. TAYLOR, J. H., J. Biophys. Biochem. CytoI. 7, 455 (1960). 12. TERASIMA, T. and TOLMACH, L. G., Exptl Cell Res. 30, 344 (1962).

CYTODIFFERENTIATION AND

CELL

STRAINS

IN

DERIVED

BLASTOCYSTS

CELL

FROM OF THE

of Biochemistry, Received

The November

COLONIES

CLEAVING

OVA

AND

RABBIT1

R. J. COLE, R. G. EDWARDS’ Department

1)

and

University,

J. PAUL

Glasgow,

Scotland

9, 1964

Smmms on cytodifferentiation in mammals might be greatly assisted if undifferentiated cells capable of profound differentiation were available in culture. A possible source of such cells is the earliest stages of mammalian embryogenesis, especially the preimplantation stages. This is a report of successful attempts to culture tissue from rabbit embryos aged between the fertilised egg and the g-day blastocyst and to initiate cell strains from the blastocysts. Embryos at appropriate stages were obtained by flushing out the oviducts or uteri. The albumin layer and zona pellucida were removed either by treatment with a 0.1 to 1 per cent (W/V) solution of pronase [2] or by dissection. When pronase was used 1 This research was supported by Public Health Service. 2 Present address: Marshall Laboratory, England.

grant

CA-05855 Physiological

from

the

Laboratory,

National

Cancer

Downing

Experimenfal

Institute, Street,

U.S.

Cambridge,

Cell Research

37