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Arrangement of spindle apparatus in mitoses of different ploidy
Printed in Sweden Copyright 8 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental Cell Research 92 (1975) 419-427
ARRANGEMENT
OF SPINDLE APPARATUS IN MITOSES OF DIFFERENT PLOIDY F. PERA
Anatomisches Institut der Universikit Bonn, 53 Bonn, BRD
SUMMARY In non-hypotonically treated mitoses from tissue cultures of Micro&s agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, trlploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99 % of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45 % werebipolar,29.5 %tripolar, 7.5 % quadripolar and 18 % formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.
The methods of staining the constitutive heterochromatin [l, 10, 151produce in nonhypotonically treated cells not only a preferential staining of the constitutive heterochromatin but also a staining of the spindle apparatus [ll]. As in Mcrotus agrestis the constitutive heterochromatin is located mainly in the sex chromosomes, the number of darkly stained heterochromatic chromosomes can be used, particularly in this species, for ploidy determination of the densely packed mitotic figures. In this paper we report on the number of spindle poles and the mitotic behaviour of cells of different ploidy and on the frequency of multipolar mitoses in mono- and binucleate cells. MATERIALS AND METHODS The field-voles (Microtus ugrestis) were caught in woodland areas near Bonn. For the present study, 28-751808
tissue cultures of a female and two male M. agrestis
wereused.One of the male cultures,originallydiploid, became oredominantly trioloid during culture time [13]; the investigations for this paper were carried out on a cloned triploid cell line with the karyotype 3n, XXY. The other two cultures comprised diploid and some tetraploid and binucleate cells. Tissue culture. For fibroblast cultures, lung tissue was used. Culture medium consisted of Eagle’s MEM with 20 parts fetal calf serum, antibiotics and phenol red. Subcultures of the fibroblasts were set up in Pasteur tubes with coverslips. The cells were fixed with methanol-acetic acid 3 : 1 for 30 min and airdried. Other fixation methods (ethanol, glutaraldehyde, formalin, osmium tetroxyde) and abrupt drying in an exsiccator were found to produce worse staining results after Giemsa. Lubelling with *H-thymidine. Coverslips with fibroblasts were exposed to [*H]thymidine-methyl (1 @i/ml culture medium, spec. act. 10 Ci/mmole; NEN Dreieichenhain) for 4 h. After fixation and airdrying, the coverslips were mounted on slides, stained according to Feulgen and covered with stripping film (Kodak AR 10). The exposure time was about 10 days. Staining of spindle apparatus and constitutive heterochromatin. The staining procedures are described in detail elsewhere 1111.The preparations were treated in a mixture of sodium chloride and sodium citrate Exptl Cell Res 92 (1975)
420
F. Pera
(SSC), first in 0.1 x SSC (5 parts of 2 ,xSSC and 95 parts distilled water; 2 z SSC: 0.3 M sodium chloride and 0.03 M sodium citrate) for 3 min at 92’C, transferred to 70 and 96 “<, ethanol, air-dried and then treated in 2 x SSC at 65°C overnight. They were then stained with Giemsa (Merck, Darmstadt; IO parts of the alcoholic stem solution and 90 parts phosphate buffer pH 6.8) for 5 to 60 min; the staining effect was continuously microscopically controlled. After rinsing in tap water the preparations were air-dried and mounted on slides with DePeX.
terphase cells and in early prophase when the nuclear membrane has not yet disappeared, since they cannot be differentiated from other nuclear structures, especially nucleoli and heterochromatic particles. In prophases where the euchromatic chromosomes had been rendered unstainable by prolonged heat treatment (more than 3 min at 92°C) and only appeared as ghosts, the centrioles with their spindle fibers were loRESULTS cated between the chromosomes (figs 5, 8: Staining effects of the G’iemsa method another example of staining of only the In non-hypotonically treated cells from tis- spindle fibers is shown in fig. 25). Since sue cultures the constitutive heterochromatin centrioles are structures of the cytoplasm as well as the spindle apparatus can be de- and not of the nucleus, we must presume monstrated by heat treatment and Giemsa that they lie above and below the nucleus in staining. Both in interphase nuclei and in cells grown on cover glasses. Only in 7 out prophase cells the nucleoli become stained as of 146 diploid prophases the centrioles were well (fig. 5). The centrioles appear as small found beside the nucleus (fig. 4). granules beside the nucleus, but it is difficult In preparations of a male M. agrestk, 162 to distinguish them from other cytoplasmatic unselected prophase cells were examined. structures; in many cells they are not vis- Their ploidy was determined by counting the ible at all. number of heterochromatic sex chromosomes (figs l-3). The results in respect of Localization and number of the centrioles number and localization of centrioles are in interphase, prophase and given in table 1. In diploid prophases and prometaphase ceils prometaphases in which centrioles were visIn coverslip preparations the cells can be ible, always two centrioles occurred (figs 4-6), studied in the position they had attained in tetraploid prophases and prometaphases when growing in vitro at the time of fixa- four centrioles (figs 8, 9). Likewise, in bition. They can only be seen from one angle, nucleate cells with two diploid nuclei, four namely from above. The cells are so flat centrioles were found (fig. 10). In earlier that all cell structures are in one optical prophase stages of binucleate cells it was plane even at the highest magnification. Thus, noticed that always two centrioles were adjasuperimposed cell structures appear to lie cent to each nucleus; at later stagesthe chromosomesof both nuclei were not distinguishside by side. The centrioles are not visible in most in- able so that a co-ordination of centrioles to a Figs I-II. Prophase and pro-metaphase stages of cells of Microrus agrestis after heat treatment and Giemsa staining. The centrioles are marked by arrows. (I) I)iploid; (2) triploid; and (3) tetraploid prophase with staining of the constitutive hetercchromatin of the sex chromosomes and the centromeres; (4) prophase with two centrioles beside the nucleus; (5) prophase with two oentrioles apparently within the nucleus (nu, nucleolus); (6) diploid pro-metaphase with two &nrrioles; (7) triploid pro-metaphase with two centrioles; (8). (9) tetraploid pro-metaphases with four centtioles within (&and beside (9) the bulk of chromosomes; (IO) binucleate cell with two diploid nuclei in.prophase with four ce+i6les; (II) binucleate cell with two tetraploid nuclei in prophase with eight centrioles. Exptl Celf Res 92 (1975)
Spindle apparatus in mitoses of different ploidy
421
Exptl Cell Res 92 (1975)
422 F. Pera
Table 1. Number and apparent position of the centrioles in prophase cells of different ploidy Centrioles No. of nuclei/cell 1 1 1 1 2 2 2 Total
Ploidy/nucleus
No.
Diploid Diploid Diploid Tetraploid Diploid Diploid Tetraploid
Not noticeable Within the nucleus ii Beside the nucleus 4 Within the nucleus Not noticeable 4 Beside the nucleus 8 Beside the nucleus
Apparent position
Fl
%
70 69 7 6
43.2 42.6 4.3 3.7
: 1 162
2.5 3.1 0.6 100.0
certain nucleus could not be carried out. A binucleate cell with two tetraploid nuclei contained 8 centrioles (fig. 11). In all of the examined triploid prophases and prometaphases, two centrioles were found (fig. 7).
(seefig. 30). In 29.5 % a tripolar (fig. 15) and in 7.5 % a quadripolar spindle was observed (fig. 16). The chromosomes of 18 % of the tetraploid cells, indeed, had formed a normal bipolar metaphase plate, but always two separate centrioles with their spindle fibers Arrangement of spindle apparatus were arranged at each side and formed two in metaphase cells parallel bipolar spindle figures (fig. 18). Ploidy and arrangement of the spindle ap- Sometimes there was one spindle pole at the paratus were determined in 560 metaphases one side and two poles at the other. On the from a predominantly diploid fibroblast cul- other hand, some mitoses with four chromoture of female A4. agrestis. 462 mitoses were some branches were found where only two diploid, 78 tetraploid. Like studies were car- or three spindle poles were visible (fig. 17). These mitoses were reckoned among bipolar ried out on 435 metaphases from a triploid cell line of male 44. agrestis. The results are and tripolar ones respectively. In a few cases two diploid bipolar metaphases were found summarized in table 2. (1) Diploid and triploid cells: In most of in one cell (fig. 19). Fig. 29 shows a cell with the diploid (figs 12, 13) and triploid meta- 4 nuclei of which 2 in each caseare connected phases (fig. 14) a bipolar spindle arrange- by thin chromatin bridges; this cell possibly ment was found. Three or four spindle poles contains the 4 daughter nuclei of a parallel mitosis or a double mitosis. occurred in less than 2 %. In tetraploid metaphasesit cannot be dis(2) Tetraploid cells: 45% of the tetraploid tinguished in general whether it is a mitosis mitoses showed a bipolar spindle apparatus
Figs 12-20: 22-23. Metaphase stages; (21) prophase stage. (12-19) Giemsa staining; (12), (13) diploid bipolar metaphases with staining of preponderantly the heterochromatin (12) and preponderantly the spindle fibers (13); (14) bipolar triploid metaphase; (15) tripolar tetraploid metaphase; (16) quadripolar tetraploid metaphase; (17) pseudoquadripolar metaphase with only two spindle poles; (18) tetraploid parahel metaphase; (19) double metaphase: two separated metaphase figures within a cell; (20-23) Feulgen stained mitoses after labelling with *H-thymidine: (20) quadripofar metaphase with one late labelled chromosome in each branch; (22) asynchronously labelled binucleate cell in prophase; (22-23) half-labelled tripolar (22) and quadripolar (23) metaphase. Exptl Cell Res 92 (1975)
Spindle apparatus in mitoses of different ploidy
423
Exptl Cell Res 92 (1975)
424
F. Pera
Table 2. Variations
of
the spindle arrangemem
in metaphase cells Spindle arrangement
Diploid n
Triploid %
n
Table 3. Labelling pattern of multipolar tetraploid metaphases 4 h after pulse-labelling with 3H-thymidine
Tetraploid %
Bipolar 456 98.7 428 98.4 Tripolar 2 0.4 5 1.1 Quadripolar 4 0.9 1 0.2 Parallel 0 0.0 1 0.2 Total 462 100.0 435 100.0
n
%
35 45.0 23 29.5 6 7.5 14 18.0 78 100.0
of a mononucleate tetraploid cell or whether polyploidy has arisen from a confluence of the chromosomes of two diploid nuclei (fig. 20). The origin from a binucleate cell is only demonstrable by labelling with 3H-thymidine, if both nuclei of a binucleate cell have not synchronously carried out their DNA synthesis, but start mitosis at the same time (fig. 21). The chromosomes of the two nuclei can be differentiated in the common metaphase plate by means of their different labelling. Bipolar tetraploid metaphases with unequal labelling of two parts of the mitotic figure were not found hitherto, all half-labelled tetraploid mitoses were multipolar. As shown in table 3, about 6% of the tripolar mitoses and more than half of the quadripolar mitoses showed a labelling of only half of the mitotic figure. In the tripolar mitoses, one branch was half, the second full and the third not labelled (fig. 22). In quadripolar metaphases two consecutive branches were labelled, the other two were unlabelled (fig. 23). In these mitoses the origin from an asynchronously replicated binucleate cell was presumed.
Tripolar Labelling pattern
n
Half-labelled Full-labelled Total
5 82 87
Quadripolar %
5.7 94.3 100.0
n
%
4 3 7
57 43 100
The spindle apparatus at anaphase and telophase
By means of the retarded chromatid separation of the heterochromatic sections of the sex chromosomes in anaphase, the number of these chromosomes and the ploidy of the mitosis can be determined also in mitoses without specific staining of heterochromatin (fig. 27). Fundamental differences between the behaviour of the spindle apparatus of diploid, triploid and tetraploid cells were not found (figs 24, 26, 27). The spindle fibers surrounding the centrioles disappear in telophase but the fibers between the daughter nuclei remain for a long time and are visible after cytokinesis as thin interzonal region between the daughter cells (figs 28, 30). Then, they can be helpful indications for determining a run-down multipolar mitosis (figs 31, 32). DISCUSSION In non-hypotonically treated cells of Microtus agrestis, both constitutive heterochromatin and spindle apparatus become stained
Figs 24-32. Anaphase and telophase stages. (24) Bipolar diploid anaphase; (25) bipolar anaphase with exclu-
sive staining of the spindle apparatus; arrows mark the position of the chromosomes. (26), (27)Tripioid bipolar anaphases showing the retarded chromatid separation of the heterochromatib chromosomes; (28) diploid telophase with staining of the interzonal region; (29) reconstruction phase of a parallel or a double mitosis with chromatin bridges between the daughter nuclei; (30) telophase of a tetraploid bipolar mitosis; (31) teiophaae of a quadripolar mitosis with staining of the interzonal region; (32) tripolar telophase with interzonal region. (24-32) Giemsa staining. Exptl
Cell Res 92 (1975)
Spindle apparatus in mitoses of different ploidy
425
Exptl Cd Res 92 (1975)
426 F. Pera
with Giemsa if the cells are pretreated with 0.1 x SSC at 92°C for a few minutes and 2 x SSC at 65°C for about 18 h [ll, 121. The mechanism of staining the spindle fibers is unknown. Mitoses which are stained with Giemsa for a long time without any pretreatment show the spindle apparatus in some casestoo, but there is little contrast with the cytoplasm and the chromosomes. The same long staining times are necessaryin pretreated mitoses in order to produce a clear coloration of the spindle fibers. From the fact that the staining procedures used for a preferential staining of constitutive heterochromatin also render visible the spindle fibers, we conclude that the spindle fibers are more resistant to heat treatment than other cell structures, even more so than constitutive heterochromatin itself. We observed staining of the spindle fibers only when using elongated boiling times. The question as to whether the microtubules of the spindle apparatus themselves, or other cell structures in their place, are stained cannot be decided by the light microscope. By heat treatment, either the microtubules could be separated one from another by swollen vesicles of the endoplasmic reticulum [2], or the microtubules could be concentrated to clusters of fibrils which in this state are stainable [6]. In the case of M. agrestis there are marker structures, namely the large sex chromosomes which for the most part consist of constitutive heterochromatin. The number of the sex chromosomes is proportional to the number of chromosome sets, i.e. to the cell ploidy. In tissue cultures of M. agrestis, however, especially in older cultures, there are anomalies of the karyotype in which the number of marker structures is no longer proportional to the ploidy. Mitotic non-disjunction of the sex chromosomes, for instance, leads to an additional or absent heterochromatic chromosome in the daughter nucleus. Both daughter Exptl Cell Res 92 (1975)
nuclei may be viable, but ploidy determination is no longer possible only by means of counting the heterochromatic chromosomes. Fortunately, however, such karyotype aberrations in a given culture are either so infrequent that they can be ignored in a random sample, or so frequent that practically all the cells are affected [14]. From these observations we must draw the conclusion that evaluation of cell ploidy by the number of heterochromatic chromosomes is permissible only if the euploidy of the chromosome set in hypotonically treated parallel preparations is ensured. This is particularly important in Giemsa stained preparations, since here an additional cytophotometric ploidy determination cannot be carried out. Whether a cell is divided by a normal bipolar or by a multipolar mitosis seemsto be dependent on the number of centrioles in it. In tetraploid mitoses that have originated from a binucleate cell with two diploid nuclei, four centrioles are visible. As already presumed by Matthews et al. [5], each diploid nucleus of a multinucleate cell enters its own pair of centrioles into the common mitosis. We were able to verify this assumption by direct cytological evidence of the centrioles in binucleate cells. But not every centriole will later form its own spindle pole; not each tetraploid mitosis is four-poled and will divide into four daughter nuclei. About half the tetraploid mitoses possessonly two spindle poles. It is possible that these tetraploid mitoses have resulted from mononucleate tetraploid cells; the tetraploid multipolar mitoses, on the other hand, from binucleate cells. Not every binucleate cell, indeed, must be divided by a multipolar mitosis [4, 91, but at least in the case of binucleate cells with nuclei which have had an asynchronous DNA synthesis, only tri- or quadripolar mitoses were observed.
Spindle apparatus in mitoses of different ploidy If we postulate that every tetraploid mitosis contains four centrioles, then in tripolar mitoses two of the four centrioles must form a common spindle pole and in bipolar mitoses two pairs of centrioles in each case ought not to be divided. Unfortunately, by means of light microscope and the applied Giemsa technique it is not possible to optically resolve a paired centriole as consisting of two individual centrioles. More than half of all tetraploid mitoses in our cultures are multipolar, i.e. either triand quadripolar or in the form of parallel and double mitoses. The daughter nuclei of such mitoses possessa lower ploidy than the mother cell. In other words, the probability that a tetraploid cell will establish a stemline with stable tetraploidy is only 45 %. The chances of tetraploid cells establishing themselves in a preferentially diploid culture are further diminished by the fact that their cell cycle is longer than that of diploid cells [3, 8, 13). The situation is different with triploid cells. They represent the most frequent ploidy class resulting from tripolar mitoses of tetraploid cells. In the most frequent instance two triploid and one diploid daughter nuclei result [12]. As mentioned above, in tripolar mitoses two of the four centrioles probably form one spindle pole. If a diploid chromosome set goes to this pole, the two triploid daughter nuclei will receive one centriole in each case; the diploid nucleus two. After division of the centriole the triploid nuclei will carry out a
427
normal bipolar mitosis, whereas the diploid cell with its originally two centrioles will probably be divided by a multipolar mitosis. In our material most of the triploid mitoses are bipolar.The few triploid multipolar mitoses could result either from a separation of centrioles [2, 71, or from such triploid cells that have received the paired centrioles from their multipolar mother cell. This work was supported by the Bundesministerium fiir Forschung und Technologie of the Federal Republic of Germany.
REFERENCES 1. Arrighi, F E, Hsu, T C, Saunders, P & Saunders, G F. Chromosoma 32 (1970) 224. 2. Friehllnder, M & Wahrman, J, J cell sci 7 (1970) 65. 3. Gimenez-Martin, G, Lopez-Sbz, J F, Moreno,. P & Gonzales-Fernandez, Chromosoma 25 (1968) 282. 4. Heneen, W K, Hereditas 67 (1971) 221. 5. Matthews, J L, Martin, J H & Race, G J, Science 155 (1967) 1423. 6. McIntosh, J R &Landis, S C, J cell biol40 (1971) 468. 7. Mazia, D, The cell (ed J Brachet & A E Mirsky) vol. 3, p. 77. Academic Press, New York (1961). 8. Oftebro, R, Rep fourth Stand congr cell res, p. 74 (1965). 9. Oftebro, R & Wolf, I, Exptl cell res 48 (1957) 39. 10. Pardue, M L & Gall, J G, Science 168 (1970} 1356. 11. Pera, F, Stain technol 49 (1974) 335. 12. Pera, F & Rainer, B, Chromosoma 42 (1973) 71. 13. Pera, F & Scholz, P, Human genet 21 (1974) 17. 14. Pera, F, Firsching, V & Scholz, P, Verh Anat Ges 69 (1974). In Dress. 15. Y&s, J J, Roldan, L, Yasmineh, W G & Lee J C, Nature 231 (1971) 532. Received October 21, 1974 Revised version received November 25, 1974
Exptl Cell Res 92 (1975)
Experimental Cell Research 92 (1975) 419-427
ARRANGEMENT
OF SPINDLE APPARATUS IN MITOSES OF DIFFERENT PLOIDY F. PERA
Anatomisches Institut der Universikit Bonn, 53 Bonn, BRD
SUMMARY In non-hypotonically treated mitoses from tissue cultures of Micro&s agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, trlploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99 % of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45 % werebipolar,29.5 %tripolar, 7.5 % quadripolar and 18 % formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.
The methods of staining the constitutive heterochromatin [l, 10, 151produce in nonhypotonically treated cells not only a preferential staining of the constitutive heterochromatin but also a staining of the spindle apparatus [ll]. As in Mcrotus agrestis the constitutive heterochromatin is located mainly in the sex chromosomes, the number of darkly stained heterochromatic chromosomes can be used, particularly in this species, for ploidy determination of the densely packed mitotic figures. In this paper we report on the number of spindle poles and the mitotic behaviour of cells of different ploidy and on the frequency of multipolar mitoses in mono- and binucleate cells. MATERIALS AND METHODS The field-voles (Microtus ugrestis) were caught in woodland areas near Bonn. For the present study, 28-751808
tissue cultures of a female and two male M. agrestis
wereused.One of the male cultures,originallydiploid, became oredominantly trioloid during culture time [13]; the investigations for this paper were carried out on a cloned triploid cell line with the karyotype 3n, XXY. The other two cultures comprised diploid and some tetraploid and binucleate cells. Tissue culture. For fibroblast cultures, lung tissue was used. Culture medium consisted of Eagle’s MEM with 20 parts fetal calf serum, antibiotics and phenol red. Subcultures of the fibroblasts were set up in Pasteur tubes with coverslips. The cells were fixed with methanol-acetic acid 3 : 1 for 30 min and airdried. Other fixation methods (ethanol, glutaraldehyde, formalin, osmium tetroxyde) and abrupt drying in an exsiccator were found to produce worse staining results after Giemsa. Lubelling with *H-thymidine. Coverslips with fibroblasts were exposed to [*H]thymidine-methyl (1 @i/ml culture medium, spec. act. 10 Ci/mmole; NEN Dreieichenhain) for 4 h. After fixation and airdrying, the coverslips were mounted on slides, stained according to Feulgen and covered with stripping film (Kodak AR 10). The exposure time was about 10 days. Staining of spindle apparatus and constitutive heterochromatin. The staining procedures are described in detail elsewhere 1111.The preparations were treated in a mixture of sodium chloride and sodium citrate Exptl Cell Res 92 (1975)
420
F. Pera
(SSC), first in 0.1 x SSC (5 parts of 2 ,xSSC and 95 parts distilled water; 2 z SSC: 0.3 M sodium chloride and 0.03 M sodium citrate) for 3 min at 92’C, transferred to 70 and 96 “<, ethanol, air-dried and then treated in 2 x SSC at 65°C overnight. They were then stained with Giemsa (Merck, Darmstadt; IO parts of the alcoholic stem solution and 90 parts phosphate buffer pH 6.8) for 5 to 60 min; the staining effect was continuously microscopically controlled. After rinsing in tap water the preparations were air-dried and mounted on slides with DePeX.
terphase cells and in early prophase when the nuclear membrane has not yet disappeared, since they cannot be differentiated from other nuclear structures, especially nucleoli and heterochromatic particles. In prophases where the euchromatic chromosomes had been rendered unstainable by prolonged heat treatment (more than 3 min at 92°C) and only appeared as ghosts, the centrioles with their spindle fibers were loRESULTS cated between the chromosomes (figs 5, 8: Staining effects of the G’iemsa method another example of staining of only the In non-hypotonically treated cells from tis- spindle fibers is shown in fig. 25). Since sue cultures the constitutive heterochromatin centrioles are structures of the cytoplasm as well as the spindle apparatus can be de- and not of the nucleus, we must presume monstrated by heat treatment and Giemsa that they lie above and below the nucleus in staining. Both in interphase nuclei and in cells grown on cover glasses. Only in 7 out prophase cells the nucleoli become stained as of 146 diploid prophases the centrioles were well (fig. 5). The centrioles appear as small found beside the nucleus (fig. 4). granules beside the nucleus, but it is difficult In preparations of a male M. agrestk, 162 to distinguish them from other cytoplasmatic unselected prophase cells were examined. structures; in many cells they are not vis- Their ploidy was determined by counting the ible at all. number of heterochromatic sex chromosomes (figs l-3). The results in respect of Localization and number of the centrioles number and localization of centrioles are in interphase, prophase and given in table 1. In diploid prophases and prometaphase ceils prometaphases in which centrioles were visIn coverslip preparations the cells can be ible, always two centrioles occurred (figs 4-6), studied in the position they had attained in tetraploid prophases and prometaphases when growing in vitro at the time of fixa- four centrioles (figs 8, 9). Likewise, in bition. They can only be seen from one angle, nucleate cells with two diploid nuclei, four namely from above. The cells are so flat centrioles were found (fig. 10). In earlier that all cell structures are in one optical prophase stages of binucleate cells it was plane even at the highest magnification. Thus, noticed that always two centrioles were adjasuperimposed cell structures appear to lie cent to each nucleus; at later stagesthe chromosomesof both nuclei were not distinguishside by side. The centrioles are not visible in most in- able so that a co-ordination of centrioles to a Figs I-II. Prophase and pro-metaphase stages of cells of Microrus agrestis after heat treatment and Giemsa staining. The centrioles are marked by arrows. (I) I)iploid; (2) triploid; and (3) tetraploid prophase with staining of the constitutive hetercchromatin of the sex chromosomes and the centromeres; (4) prophase with two centrioles beside the nucleus; (5) prophase with two oentrioles apparently within the nucleus (nu, nucleolus); (6) diploid pro-metaphase with two &nrrioles; (7) triploid pro-metaphase with two centrioles; (8). (9) tetraploid pro-metaphases with four centtioles within (&and beside (9) the bulk of chromosomes; (IO) binucleate cell with two diploid nuclei in.prophase with four ce+i6les; (II) binucleate cell with two tetraploid nuclei in prophase with eight centrioles. Exptl Celf Res 92 (1975)
Spindle apparatus in mitoses of different ploidy
421
Exptl Cell Res 92 (1975)
422 F. Pera
Table 1. Number and apparent position of the centrioles in prophase cells of different ploidy Centrioles No. of nuclei/cell 1 1 1 1 2 2 2 Total
Ploidy/nucleus
No.
Diploid Diploid Diploid Tetraploid Diploid Diploid Tetraploid
Not noticeable Within the nucleus ii Beside the nucleus 4 Within the nucleus Not noticeable 4 Beside the nucleus 8 Beside the nucleus
Apparent position
Fl
%
70 69 7 6
43.2 42.6 4.3 3.7
: 1 162
2.5 3.1 0.6 100.0
certain nucleus could not be carried out. A binucleate cell with two tetraploid nuclei contained 8 centrioles (fig. 11). In all of the examined triploid prophases and prometaphases, two centrioles were found (fig. 7).
(seefig. 30). In 29.5 % a tripolar (fig. 15) and in 7.5 % a quadripolar spindle was observed (fig. 16). The chromosomes of 18 % of the tetraploid cells, indeed, had formed a normal bipolar metaphase plate, but always two separate centrioles with their spindle fibers Arrangement of spindle apparatus were arranged at each side and formed two in metaphase cells parallel bipolar spindle figures (fig. 18). Ploidy and arrangement of the spindle ap- Sometimes there was one spindle pole at the paratus were determined in 560 metaphases one side and two poles at the other. On the from a predominantly diploid fibroblast cul- other hand, some mitoses with four chromoture of female A4. agrestis. 462 mitoses were some branches were found where only two diploid, 78 tetraploid. Like studies were car- or three spindle poles were visible (fig. 17). These mitoses were reckoned among bipolar ried out on 435 metaphases from a triploid cell line of male 44. agrestis. The results are and tripolar ones respectively. In a few cases two diploid bipolar metaphases were found summarized in table 2. (1) Diploid and triploid cells: In most of in one cell (fig. 19). Fig. 29 shows a cell with the diploid (figs 12, 13) and triploid meta- 4 nuclei of which 2 in each caseare connected phases (fig. 14) a bipolar spindle arrange- by thin chromatin bridges; this cell possibly ment was found. Three or four spindle poles contains the 4 daughter nuclei of a parallel mitosis or a double mitosis. occurred in less than 2 %. In tetraploid metaphasesit cannot be dis(2) Tetraploid cells: 45% of the tetraploid tinguished in general whether it is a mitosis mitoses showed a bipolar spindle apparatus
Figs 12-20: 22-23. Metaphase stages; (21) prophase stage. (12-19) Giemsa staining; (12), (13) diploid bipolar metaphases with staining of preponderantly the heterochromatin (12) and preponderantly the spindle fibers (13); (14) bipolar triploid metaphase; (15) tripolar tetraploid metaphase; (16) quadripolar tetraploid metaphase; (17) pseudoquadripolar metaphase with only two spindle poles; (18) tetraploid parahel metaphase; (19) double metaphase: two separated metaphase figures within a cell; (20-23) Feulgen stained mitoses after labelling with *H-thymidine: (20) quadripofar metaphase with one late labelled chromosome in each branch; (22) asynchronously labelled binucleate cell in prophase; (22-23) half-labelled tripolar (22) and quadripolar (23) metaphase. Exptl Cell Res 92 (1975)
Spindle apparatus in mitoses of different ploidy
423
Exptl Cell Res 92 (1975)
424
F. Pera
Table 2. Variations
of
the spindle arrangemem
in metaphase cells Spindle arrangement
Diploid n
Triploid %
n
Table 3. Labelling pattern of multipolar tetraploid metaphases 4 h after pulse-labelling with 3H-thymidine
Tetraploid %
Bipolar 456 98.7 428 98.4 Tripolar 2 0.4 5 1.1 Quadripolar 4 0.9 1 0.2 Parallel 0 0.0 1 0.2 Total 462 100.0 435 100.0
n
%
35 45.0 23 29.5 6 7.5 14 18.0 78 100.0
of a mononucleate tetraploid cell or whether polyploidy has arisen from a confluence of the chromosomes of two diploid nuclei (fig. 20). The origin from a binucleate cell is only demonstrable by labelling with 3H-thymidine, if both nuclei of a binucleate cell have not synchronously carried out their DNA synthesis, but start mitosis at the same time (fig. 21). The chromosomes of the two nuclei can be differentiated in the common metaphase plate by means of their different labelling. Bipolar tetraploid metaphases with unequal labelling of two parts of the mitotic figure were not found hitherto, all half-labelled tetraploid mitoses were multipolar. As shown in table 3, about 6% of the tripolar mitoses and more than half of the quadripolar mitoses showed a labelling of only half of the mitotic figure. In the tripolar mitoses, one branch was half, the second full and the third not labelled (fig. 22). In quadripolar metaphases two consecutive branches were labelled, the other two were unlabelled (fig. 23). In these mitoses the origin from an asynchronously replicated binucleate cell was presumed.
Tripolar Labelling pattern
n
Half-labelled Full-labelled Total
5 82 87
Quadripolar %
5.7 94.3 100.0
n
%
4 3 7
57 43 100
The spindle apparatus at anaphase and telophase
By means of the retarded chromatid separation of the heterochromatic sections of the sex chromosomes in anaphase, the number of these chromosomes and the ploidy of the mitosis can be determined also in mitoses without specific staining of heterochromatin (fig. 27). Fundamental differences between the behaviour of the spindle apparatus of diploid, triploid and tetraploid cells were not found (figs 24, 26, 27). The spindle fibers surrounding the centrioles disappear in telophase but the fibers between the daughter nuclei remain for a long time and are visible after cytokinesis as thin interzonal region between the daughter cells (figs 28, 30). Then, they can be helpful indications for determining a run-down multipolar mitosis (figs 31, 32). DISCUSSION In non-hypotonically treated cells of Microtus agrestis, both constitutive heterochromatin and spindle apparatus become stained
Figs 24-32. Anaphase and telophase stages. (24) Bipolar diploid anaphase; (25) bipolar anaphase with exclu-
sive staining of the spindle apparatus; arrows mark the position of the chromosomes. (26), (27)Tripioid bipolar anaphases showing the retarded chromatid separation of the heterochromatib chromosomes; (28) diploid telophase with staining of the interzonal region; (29) reconstruction phase of a parallel or a double mitosis with chromatin bridges between the daughter nuclei; (30) telophase of a tetraploid bipolar mitosis; (31) teiophaae of a quadripolar mitosis with staining of the interzonal region; (32) tripolar telophase with interzonal region. (24-32) Giemsa staining. Exptl
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Spindle apparatus in mitoses of different ploidy
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Exptl Cd Res 92 (1975)
426 F. Pera
with Giemsa if the cells are pretreated with 0.1 x SSC at 92°C for a few minutes and 2 x SSC at 65°C for about 18 h [ll, 121. The mechanism of staining the spindle fibers is unknown. Mitoses which are stained with Giemsa for a long time without any pretreatment show the spindle apparatus in some casestoo, but there is little contrast with the cytoplasm and the chromosomes. The same long staining times are necessaryin pretreated mitoses in order to produce a clear coloration of the spindle fibers. From the fact that the staining procedures used for a preferential staining of constitutive heterochromatin also render visible the spindle fibers, we conclude that the spindle fibers are more resistant to heat treatment than other cell structures, even more so than constitutive heterochromatin itself. We observed staining of the spindle fibers only when using elongated boiling times. The question as to whether the microtubules of the spindle apparatus themselves, or other cell structures in their place, are stained cannot be decided by the light microscope. By heat treatment, either the microtubules could be separated one from another by swollen vesicles of the endoplasmic reticulum [2], or the microtubules could be concentrated to clusters of fibrils which in this state are stainable [6]. In the case of M. agrestis there are marker structures, namely the large sex chromosomes which for the most part consist of constitutive heterochromatin. The number of the sex chromosomes is proportional to the number of chromosome sets, i.e. to the cell ploidy. In tissue cultures of M. agrestis, however, especially in older cultures, there are anomalies of the karyotype in which the number of marker structures is no longer proportional to the ploidy. Mitotic non-disjunction of the sex chromosomes, for instance, leads to an additional or absent heterochromatic chromosome in the daughter nucleus. Both daughter Exptl Cell Res 92 (1975)
nuclei may be viable, but ploidy determination is no longer possible only by means of counting the heterochromatic chromosomes. Fortunately, however, such karyotype aberrations in a given culture are either so infrequent that they can be ignored in a random sample, or so frequent that practically all the cells are affected [14]. From these observations we must draw the conclusion that evaluation of cell ploidy by the number of heterochromatic chromosomes is permissible only if the euploidy of the chromosome set in hypotonically treated parallel preparations is ensured. This is particularly important in Giemsa stained preparations, since here an additional cytophotometric ploidy determination cannot be carried out. Whether a cell is divided by a normal bipolar or by a multipolar mitosis seemsto be dependent on the number of centrioles in it. In tetraploid mitoses that have originated from a binucleate cell with two diploid nuclei, four centrioles are visible. As already presumed by Matthews et al. [5], each diploid nucleus of a multinucleate cell enters its own pair of centrioles into the common mitosis. We were able to verify this assumption by direct cytological evidence of the centrioles in binucleate cells. But not every centriole will later form its own spindle pole; not each tetraploid mitosis is four-poled and will divide into four daughter nuclei. About half the tetraploid mitoses possessonly two spindle poles. It is possible that these tetraploid mitoses have resulted from mononucleate tetraploid cells; the tetraploid multipolar mitoses, on the other hand, from binucleate cells. Not every binucleate cell, indeed, must be divided by a multipolar mitosis [4, 91, but at least in the case of binucleate cells with nuclei which have had an asynchronous DNA synthesis, only tri- or quadripolar mitoses were observed.
Spindle apparatus in mitoses of different ploidy If we postulate that every tetraploid mitosis contains four centrioles, then in tripolar mitoses two of the four centrioles must form a common spindle pole and in bipolar mitoses two pairs of centrioles in each case ought not to be divided. Unfortunately, by means of light microscope and the applied Giemsa technique it is not possible to optically resolve a paired centriole as consisting of two individual centrioles. More than half of all tetraploid mitoses in our cultures are multipolar, i.e. either triand quadripolar or in the form of parallel and double mitoses. The daughter nuclei of such mitoses possessa lower ploidy than the mother cell. In other words, the probability that a tetraploid cell will establish a stemline with stable tetraploidy is only 45 %. The chances of tetraploid cells establishing themselves in a preferentially diploid culture are further diminished by the fact that their cell cycle is longer than that of diploid cells [3, 8, 13). The situation is different with triploid cells. They represent the most frequent ploidy class resulting from tripolar mitoses of tetraploid cells. In the most frequent instance two triploid and one diploid daughter nuclei result [12]. As mentioned above, in tripolar mitoses two of the four centrioles probably form one spindle pole. If a diploid chromosome set goes to this pole, the two triploid daughter nuclei will receive one centriole in each case; the diploid nucleus two. After division of the centriole the triploid nuclei will carry out a
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normal bipolar mitosis, whereas the diploid cell with its originally two centrioles will probably be divided by a multipolar mitosis. In our material most of the triploid mitoses are bipolar.The few triploid multipolar mitoses could result either from a separation of centrioles [2, 71, or from such triploid cells that have received the paired centrioles from their multipolar mother cell. This work was supported by the Bundesministerium fiir Forschung und Technologie of the Federal Republic of Germany.
REFERENCES 1. Arrighi, F E, Hsu, T C, Saunders, P & Saunders, G F. Chromosoma 32 (1970) 224. 2. Friehllnder, M & Wahrman, J, J cell sci 7 (1970) 65. 3. Gimenez-Martin, G, Lopez-Sbz, J F, Moreno,. P & Gonzales-Fernandez, Chromosoma 25 (1968) 282. 4. Heneen, W K, Hereditas 67 (1971) 221. 5. Matthews, J L, Martin, J H & Race, G J, Science 155 (1967) 1423. 6. McIntosh, J R &Landis, S C, J cell biol40 (1971) 468. 7. Mazia, D, The cell (ed J Brachet & A E Mirsky) vol. 3, p. 77. Academic Press, New York (1961). 8. Oftebro, R, Rep fourth Stand congr cell res, p. 74 (1965). 9. Oftebro, R & Wolf, I, Exptl cell res 48 (1957) 39. 10. Pardue, M L & Gall, J G, Science 168 (1970} 1356. 11. Pera, F, Stain technol 49 (1974) 335. 12. Pera, F & Rainer, B, Chromosoma 42 (1973) 71. 13. Pera, F & Scholz, P, Human genet 21 (1974) 17. 14. Pera, F, Firsching, V & Scholz, P, Verh Anat Ges 69 (1974). In Dress. 15. Y&s, J J, Roldan, L, Yasmineh, W G & Lee J C, Nature 231 (1971) 532. Received October 21, 1974 Revised version received November 25, 1974
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