Chromosome studies of Mustela putorius in tissue culture

Transcription

1 Hereditas 70: (1972) Chromosome studies of Mustela putorius in tissue culture INGRID FRYKMAN Institute o,f Genetics, University of Lund, Sweden (Received September 15, 1971) Tissue cultures from skin, lung, and heart were made from a male polecat (Musteluputorius putorius L.) and at a later time from a female ferret (Mustela putorius furo L.). In both cases the cultures from heart and lung died after a few passages and the skin cultures were the only ones that could be used for further studies. An idiogram was calculated from measurements of 10 karyotypes of each subspecies. No morphological differences between the karyotypes of the two subspecies were found. The chromosome number was 2n = 0 with 38 autosomes and XXjXY sex chromosomes. The m-group included 5 pairs of autosomes, the X and the small Y-chromosome. A secondary constriction was regularly found in one of the homologues of the second smallest pair of the t-group. The chromosome number in the skin cultures was studied with regular intervals during 25 passages. After passage 21, soon before the cultures died, there was an increase in the proportion of tetraploid cells to some ". The chromosome number of the ferret (Musteku polecat and the ferret and to investigate the putorius juro L.), here indicated by M. p. J, was behaviour of cells in tissue culture from these two first determined to 2n = 3 by KOLLER (1936). subspecies. Later, the chromosome number 2n = 0 was * found by several workers (LANDE 1957; BASRUR 1966; Hsu and ARRIGH~ 1966; FREDGA 1966, 1967a). The chromosome number of the polecat Material and methods (Mustela putorius putorius L.), here indicated by M. p. p. is also 2n = 0 (OMODEO and RENZONI One adult male polecat (M. p. p.) and one new- 1966; FREDGA 1966,1967a). ldiograms were made by BASRUR (1966) and by OMODEO and RENZONI ( 1966) for M. p. f. and M. p. p., respectively. Preliminary results show that the wild form (M. p. p.) has chromosomes identical with those of the fer- ret (FREDGA 1966, 1967a). None of the previous workers have compared measurements of the chromosomes of the two subspecies and no reports on their karyotype variation in vitro exist. The ferret is considered to have developed from the European polecat. There are differences in colour and skull form between the two subspecies. The ferret has been bred since the fourth century as an albino mutant especially suitable for hunt- ing (MORRIS 1965). The purpose of the present study is to compare the chromosomes of the born female ferret (M. p. $) were examined. The tissue culture and chromosome preparation techniques were those routinely applied in the Cancer Chromosome Laboratory (FREDGA 1967b). The cells were grown in Hank's medium. Tissue cultures were made from skin, lung and heart tissues. The polecat was examined 8 months before the ferret. Chromosome number and morphology were studied in mitotic metaphases from the cultures. Chromosome preparations from passage 1 (P,) were used for the analysis of the normal karyotype. To study environmental adaptations during the serial carriage of the cell cultures, preparations were made from, on an average, every fifth passage. On each occasion chromosome numbers were determined in about Hereditas 70, 1972

2 60 INGRID FRYKMAN m #a X Y sm I;t 5 st t b Fig. I. Chromosomes of male Mustela putorius putorius L. a: chromosomes in situ; b: karyotype with chromosomes arranged in four groups m, sm, st and t. The chromosomes within each group are arranged in decreasing length order, except the two sex chromosomes which are placed to the right in their group. - x 1700.

3 x CHROMOSOMES OF MUSTELA IN TISSUE CULTURE x x 1 i 2 i 5 I * Fig. 2. Chromosomes of female Mustela puforius furo L. a: chromosomes in situ; b: karyotype with chromosomes arranged in the same way as in Fig. 1. ~~ Hereditas

4 ~ 62 INGRID FRYKMAN 50 metaphases and representative cells were photographed. All measurements were made on photographs enlarged 3200 times. Relative length, absolute length and arm ratio were determined for each chromosome and the data were treated statistically. The chromosomes were arranged into groups according to the location of the centromere, as suggested by LEVAN et al. (196). Within each group, the chromosomes were arranged in order of falling length. To test whether there existed a possible difference in karyotype between the polecat and the ferret, a classical t-analysis was undertaken. The idiogram was based on measurements of representative metaphases from 10 male and 10 female cells of skin cultures at PI of the polecat and the ferret, respectively. Observations 1. Karyotype and idiogram The chromosome number of both subspecies was 2n = 0. A metaphase plate and a karyotype of each of the two subspecies are presented in Fig. 1 and 2. The relative lengths and arm ratios of the different chromosomes of the polecat and the ferret karyotypes are presented in Table 1. A difference with a level of significance of p = 0.05 was found only in case of chromosomes ml, m5 and sml as regards length and m2 as regards arm ratio. As there were no great differences between the karyotypes of the polecat and the ferret, it was possible to combine the measurements made on the male and female materials for making the idiogram (Table 2 and Fig. 3). In the m group there are five pairs of autosomes and the X and Y chromosomes, all distinguishable by means of length and arm ratio. The X shows great resemblance to m3 and m but is intermediate between these two chromosomes both in total length and arm ratio. According to FREDCA (unpubl.) the late replicating X chromosome of the female falls between m3 and m in size. The relative length of the X chromosome is.7 % of (na + X). The Y is the smallest chromosome of the karyotype, 0.83p, and thus easy to identify. The sm group includes five pairs. The largest pair of the whole complement, sml has a Table 1. Relative chromosome length and arm ratio in the female (Mustelaputoriusfuro L.) compared by t-analysis with the male (Mustela putoriirsputorius L.) The unit of relative length is thousands of the haploid chromosome set of the female (na + X). Means and standard errors are calculated on 10 cells from each subspecies. Chromosome Relative length Arm ratio No. M.p.p. M.p,f: t M.p.p. M.p.j: t ml 80.6 f f f i m f I.20 1.I2 It 0.01 I.OO f m t f f f X 6. f f f f m 0.9 f f I 1.52 f f m5 Y sm I sm2 sm 3 sm sm5 St I st2 st3 st tl t2 t3 t t5 Hereditas 70, f f f f f f f f f f f f I. I7 f I 5 f f f f I.9l f f f f f f f f f f fo f f f 0.07 I.23 f f f 0.0 I.85 f f f f f f f f f f f f f m m 2.7 f f m m 26.1 f f m m f f m m

5 CHROMOSOMES OF MUSTELA IN TISSUE CULTURE 63 Table 2. Absolute length, relative length and arm ratio of the chromosomes of Mustelaputorius based on measurements from 10 male and 10 female cells The unit of relative length is thousands of the total length of the haploid chromosome set of the female (na + X). Chromo- Absolute Relative length Arm ratio some length No. Mean Mean Standard Mean Standard error error ml m m X m m5 I Y sm sm sm sm sm stl st st3 2.7 I st ti t m t m t m t m I length of.5~. Chromosome sm, being of about the same length as m3 and X, may be difficult to distinguish from these chromosomes. The difference in arm ratio, however, is striking. The st group consists of four pairs of chromosomes. It may be difficult sometimes to separate st3 and sm3 visually, but it can be done by determining the arm ratio. Within the two groups st and sm, st3 and sm3 are easily distinguishable by their length values. Among the five pairs of the t group, the two smallest ones, t and t5, are of the same length; t is recognizable by a secondary constriction near the centromere. This can be seen in all cells and is as a rule more evident in one of the homologues. In both subspecies the tl chromosome has a distinct measurable short arm. The chromosome pairs t2-t5, on the other hand, have no clear short arms. What in some cells appears as very short arms in these chromosomes (Fig. 1 and 2) may represent parts of the centromeric region. 2. Chromosome variation in tissue culture Chromosome numbers were determined in PI cells from heart, lung and skin cultures. The cell culture; from heart and lung tissues died early (passage 2-) and therefore the long-term chromosome analysis was made only on skin cells. Usually every fifth passage was studied. The data obtained are presented in Table 3. In both subspecies of Mustela putorius, PI had the normal chromosome number 2n = 0 in the majority of the cells. A few cells, however, had a chromosome number of 39 or 38. The analysis of these cells showed that the missing chromosomes were of different types indicating that the losses were probably artificial due to the squashing. Up to passages only single tetraploid cells were seen but after that their frequency rapidly increased. At P,, and PZ6 of the female and the male, 85 and 88 "/o of the cells, respectively, were in the tetraploid region. Also in the tetraploid cells Hereditas 70, 1972

6 INGRID FRYKMAN 8-7, 6, 5-, 3, 2. I, 0, X S Y m sm rt Fig. 3. ldiogram of Mustela putorius. The chromosomes are divided into four classes: m, sm, st and t according to the position of the centrornere. Within each class the autosomes are numbered in order of decreasing length. A secondary constriction is present in the t pair. Scale unit = % of (na + X). t single chromosomes were lost, probably during the squashing, resulting in some cells with the chromosome number 79. Soon after the cultures had changed to tetraploid level, the growth stopped, the quality of chromosome preparations became inferior and no mitoses were found. Cells loosened from the glass were floating around freely, and the cultures rapidly declined and died. Discussion 1. Comparison between the chromosomes of the polecat and the ferret In the two investigations, by OMODEO and RENZONI (1966) and by BASRUR (1966), in which idiograms were presented of M. p. p. and M. p..f, respectively, data were also given of the arm ratios for all the individual chromosomes. According to these data the chromosomes were distributed on the abovementioned morphologic types in the following way: Group OMODEO and BASRUR Present work RENZONI m 6 10 I sm 9 5 st 2 2 t 5 5 A close analysis of the data indicated that the differences were mostly due to the fact that several chromosomes were near the borderlines be- Hereditas

7 3 1 CHROMOSOMES OF MUSTELA IN TISSUE CULTURE 65 Table 3. Chromosome counts in different tissues and passages Subspecies Sex Tissue Passage Chromosome numbers Total S: x No. counts level ~~ M.p.p. 8 Heart Lung I Skin 1 I ~ Total ~ M.p. f. 9 Heart ~- Lung Skin I ~ I Total 1--2 ~~ tween groups. It was possible with a few minor adjustments to bring both the earlier investigations into reasonable accordance with the present results. In the study of BASRUR the chromosomes were somewhat clearer and the secondary constriction in the t chromosome, not mentioned or illustrated by OMODEO and RENZONI, was pictured. As earlier mentioned, there were no distinct difference in karyotype between the two subspecies M. p. p. and M. p. f. The small differences indicated by the four t-values just below the 5 percent level of significance could be explained by random deviations, as the differences are rather small compared to the optical sources of error. Since the chromosomes are spread all over the view field, differences between those in the periphery and in the center will appear due to spherical aberration. This phenomenon might explain the differences in the present rather small material The conclusion is that there are no significant differences in the morphology of the chromosomes between the polecat and the ferret. 2. Chromosome variation in tissue culture The cell lines of normal diploid skin tissue from Mustelaputoriusp. and$ did not show any striking chromosomal variation during the first 21 passages. The growth of the normal diploid cells continued until passages 22-2, that is only a few passages before the cell population changed into the tetraploid level. At this stage very few mitoses were found and the cultures degenerated and died a few passages later (Table 3). The increase in the population of cells on the tetraploid level was evidently accompanied by loss in viability soon leading to the death of the population. It is a suggestive fact that the skin cultures from both subspecies behaved in exactly the same manner: declining at about the same age and showing the same pattern of chromosome changes. Since the two cultures were grown at different times, their concordant behaviour cannot be explained by some common disturbance, such as failure of the temperature regulation of the incubator, a mistake in the preparation of medium or trypsin or some similar laboratory mishap. Instead there is a strong suggestion that the cultures had an inherent tendency to respond at a certain stage of development with a change of ploidy level and loss of viability, and that this response is characteristic of the species Mustela putorius. As is well known, cells from different species behave in different ways when kept in long-term culture. Some of them undergo heteroploid transformation and develop new stemlines capable of permanent growth. This behaviour is 5 Hrrrditas 70, 1972

8 ~ J. J. Hereditas 66 INGRID FRYKMAN characteristic of the mouse, in which cultures from normal diploid tissue have a tendency to change at an early stage into tetraploidy, or rather hypotetraploidy. In such species permanent cell lines are readily established on the tetraploid level (Hsu et al. 1961). Other species form permanent cell lines without changing ploidy level. In experiments at our laboratory Chinese hamster cells of normal origin have remained diploid through nearly 00 passages, but during this time it was necessary to let them undergo repeated selective clonings to counteract their tendency to change into trisomy (KATO 1967; KRISTOFFERSSON and LEVAN unpubl.). Normal cells of the root vole remained diploid through 13 passages, but at passage 0-50 the stemline changed from 31 to 30 and at the same time the proportion of near-tetraploid cells increased; the fraction of tetraploid cells remained as a minority throughout the experiment (FERNO and MANDAHL 1970). Still other species are very difficult or impossible to transform into permanent cell lines just by growing them with best possible care. Man belongs to this group of species. Actively proliferating human fibroblasts enter a degenerative phase after 35-0 passages. During this phase chromosomal abnormalities increase in number, both structural changes and heteroploidy (SAK- SELA and MOORHEAD 1963). The present experiments with the two subspecies of Mustela putorius indicate that cells of this species behave in a similar manner to human cells, when maintained in long-term in vitro growth. In fact, the Mustela cells seem to have a somewhat shorter period before entering the degenerative phase. The reason for the varying behaviour of different species is unknown. A general feature is that sooner or later during the long-term culture the chromosomal stability becomes upset. In some species the chromosomal variation leads to so-called heteroploid transformation, by which the culture acquires the capacity for unlimited life. In other species the chromosome variation is a forerunner to degeneration and death. When a normal tissue is explanted in vitro, this involves a radical change in environment. The cells have been liberated from the control of the host organism, and variant cells that would normally be recognized and eliminated may survive. The foreign environment by itself may induce chromosomal disturbances and the population Hereditas 70, I972 becomes gradually more heterogeneous genetically with consequent capacity for selective changes. Whether these changes will result in the establishment of a permanent cell line seems to be determined by the inherent tendency of the species, in other words to which of the abovementioned types it belongs. It is highly interesting that infection of human cells in vitro with SV0 virus invariably leads to transformation, and that the spectrum of events involved in the transformation involves chromosomal variation including the induction of tetraploidy (MOORHEAD and SAKSELA 1965). It would be interesting to grow the Mustela cells during the critical final passages in varying environmental conditions, including infection with SV0 virus. Atknowledgement. ~ The present work was supported by a grant from the Swedish Cancer Society. Literature cited BASRUR, P. K The somatic chromosomes of the ferret. J. Herd. 57: FERNO, A. and MANDAHL, N In vitro chromosome studies in the root vole. ~ 66: FREDGA, K Chromosome studies in six species ol Mustelidae and one of Procyonidae. ~- M~mt?t~l. Chromos. Newslett. 21: a. Comparative chromosome studies of the family Mustelidae (Carnivora, Mammalia). ~- Hereditas 57: b. Chromosome studies in six different tissues of a male small Indian mongoose (Herpestes auropunctatus). - Ihitl. 57: Hsu, T. C. and ARRIGHI, F. E Karyotypes of 13 Carnivores. - - Mammal. Chronios. Newslett. 21: ~I 59. Hsu, T. C., BILLEN, D. and LEVAN, A Mammalian chromosomes in vitro. XV. Patterns of transformation. Nat. Cancer Inst. 27: KATO, R Localization of spontaneous and Rous sarcoma virus-induced breakage in specific regions of the chromosomes of the Chinese hamster. ~~~ Herediras 58: KOLLER, P. C Chromosome behaviour in the male ferret and mole during anoestrus. ~~ Proc. Roy. Soc. (Lonrf.) 121: KOPROWSKY, H., PONT~N, J. A., JENSEN, F., RAVDIN, R. G., MOORHEAD, P. and SAKSELA, E Trdnsformation of cultures of human tissue infected with Simian virus SV0. ~ Cell. Comp. Physiol. 59: LANDE, Chromosome number in the ferret (Putorius furo). - Nature 180: LEVAN, A., FREDGA, K. and SANDBERG, A. A Nomenclature for centromeric position on chromosomes. -- Hereditas 52: 201& 220. MOORHEAD, P. S. and SAKSELA, E The sequence of chromosome aberrations during SV0 transformation of a human diploid cell strain. - {bid. 52:

9 Proc. ~ Caryologia CHROMOSOMES OF MUSTELA IN TISSUE CULTURE 67 MORKIS, The Mammals. A guide to the living species. ~ Hodder and Stoughton, London, 8 pp. OMODEO, P. and RENZONI, A The karyotype of some Mustelidae. 19: 2 19~-226. SAKSELA, E. and MOORHEAD, P. S Aneuploidy in the degenerative phase of serial cultivation of human cell strains. P- Naf. Ac. Sri. 50: Ingrid Frykman Institute of Genetics S Lund, Sweden Hereditas 70, 1972