Chromosome Numbers in Bone Marrow Erythroid Cells of the

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1 CELL STRUCTURE AND FUNCTION 3, 8993 (1978) C by Japan Society for Cell Biology Chromosome Numbers in Bone Marrow Erythroid Cells of the Mouse, Rat and Rabbit Syoichi Yamashita, Shigeru Okada, Keiki Hayashi(Fang), Yuji Baba and Satimaru Seno Department of Pathology, Okayama University Medical School, Okayama 700, Japan ABSTRACT. The chromosomes were counted in cells of erythroid bone marrow and spleen of anemic mice, rats and rabbits. Diploid chromosome numbers were evident in most observed cells; however in a few cells, a hypoploid state was observed. The G-band karyotype did not revealed a regular absence of chromosomes. This suggests that the hypoploid number is an artifact resulting from inadequate chromosome preparation. The data indicate that in the mouse, rat and rabbit, the number of erythroblast chromosomes is maintained at diploid level through the nucleated period and is not reduced in differentiation. Genetic material is expected to be constant in quantity in all the somatic cells of a given species (1), i.e. complete gene duplication occurs in each cell division and the chromosome number is maintained at the diploid level. However, erythroblasts of the Chinese hamster, which loose their nuclei at maturation, have been reported to reduce their chromosome number during differentiation (2). A reduction in chromosome number is also known to be the common phenomenon in hybrid somatic cells formed by cell fusion in culture (3). In mammalian erythroid cells the rate of DNA synthesis seems to decrease with the advance of maturation, as observed in rabbit erythroblasts incubated with 3H-Tdr (4). The DNA contents of the cells may also decrease with differentiation as revealed by the microspectrophotometry of cells stained by the Feulgen reaction (4). The phenomena may fit the reduction in chromosome number in erythroid cells, which suggest defective gene replification during differentiation. However, the rabbit erythroblast may maintain its DNA at the diploid level throughout differentiation division, because Feulgen-DNA levels have been shown to be constant in nuclei at metaphase (5), i.e. a reduction in DNA content was observed solely in cells with picnotic nuclei at the last stage of maturation. The reduction in the incorporation rate of 3H-Tdr in the advanced stage of differentiation may be due to elongation of the s-phase, not to the defective replication of DNA. We, thus, reinvestigated the chromosome number of erythroid cells, and here report that the erythroblasts of mice, rats and rabbits keep diploid chromosomes throughout the differentiation divisions and do no lose any chromosomes. MATERIALS AND METHODS Six male mice of ddn strain weighing 20 to 25 g, 3 male rats of Wister strain weighing about 200 g, and adult white rabbits weighing about 3 kg were used. 89

2 90 S. Yamashita et al. Phenylhydrazine 0.05 mg/10 g daily was subcutaneously injected to three mice for 3 succesive days. The other 3 mice and the 3 rats were bled from the retrobulbar venous plexus ; 0.6 ml daily per mouse and 5 ml daily per rat for 3 days. Three rabbits were made anemic by being bled from the ear vein ; about 40 ml per day per animal for 4 succesive days. All the animals were sacrificed by being bled from the heart 1-2 days after the last phenylhydrazine injection or the last blood-letting. Two hours before sacrifice all the animals were injected subcutaneously with 1 mg/kg colchicine. Immediately after death the spleens and femurs were removed. In the mice and the rats both femoral epiphyses were severed. One end of the diaphysis was connected to a syringe by a vinyl tube and bone marrow tissue was forced into a Petri dish containing homologous blood serum. With the rabbits, the bone marrow was taken out by breaking the bone with scissors. The bone marrow tissue was put in a glass homogenizer and crushed gently adding a small amount of blood serum by which hemopoietic cells were freed from connective tissue. The hematopoietic cell suspension was obtained by sieving the cluster of connective tissue through nylon filter mesh. The bone marrow cells were then sedimented by centrifuging the suspension at 1,000 rpm for 10 minutes. Spleen tissues were taken in a Petri dish, added small amount of blood serum, chopped up with scissors and the cells were freed from connective tissue by using glass homogenizer and nylon filter mesh. Then the cells were sedimented by centrifugation. Sedimented cells were smeared and stained with May-Grilwald- Giemsa for the morphologic observation of mitotic cells. With the remaining sample the chromosome preparation was made according to the method of Omura (6); one sample for each organ and more than 100 well-dispersed chromosome sets were analysed per sample. Each chromosome was identified by the G-band technique of Caspersson (7). RESULTS Classification of bone marrow cells on the smears revealed that erythroblasts made up about 50 % of bucleated cells from blood-depleted animals and % of cells from phenylhydrazine-injected mice. Chromosome counts of mouse bone marrow cells revealed that the chromosome number was 40 in most cells, but a few cells were hyperploid or hypoploid within the range of chromosomes (Fig. la). Spleen cells had a chromosome number similar to that of bone marrow cell (Fig. lb.) In rats, the chromosome number ranged from 40 to 44[42 in most cells (Fig. 2) ], and in rabbits from 41 to 44[44 in most cells (Fig. 3)]. Identification of lost chromosomes was made in the hypoploid chromosome sets of each animal by the G-band technique. No evidence was obtained to show that the lost chromosomes were specific ones. DISCUSSION Some hypodiploid chromosome numbers were found in cells from erythroid bone marrow and from the spleen; in the mouse, in the rat and in the rabbit. No cells with extremely small chromosome numbers of nearly haploid level, as reported by Weiker in erythroid cells of the Chinese hamster, were found in these animals (2). As mitotic erythroblasts were more than 80 % of the total number of mitotic cells in the bone marrow of these anemic animals, about 80 % of the chromosome sets analysed should be erythroblasts at least. In the spleen of the rat and the rabbit, whose mitotic cells should not be erythroblasts, hypodiploid chromosomes

3 Chromosomes of Mammalian Erythroblasts 91 Fig. 1. Distribution pattern of the chromosome number of bone marrow cells (Fig. 1 a) and of spleen cells (Fig. 1 b) in blood-depleted, anemic mice. Each column shows the mean value obtained for 3 mice. Fig. 2. Distribution pattern of the chromosome number of blood-depleted rats. Each column shows the mean value obtained for 3 animals.

4 92 S. Yamashita et al, Fig. 3. Distribution pattern of the chromosome number of bone marrow cells from 3 anemic rabbits. Fig. 4. Micrograph of rat chromosomes. The photo shows the cells from etryhroid bone marrow and 3 metaphase. chromosomes. were also found. This may mean that hypodiploid chromosomes are not specific to erythroblasts. A check of the bands of hypodiploid chromosomes showed that the type of lost chromosome(s) differed from cell to cell. This indicates that the hypoploid number observed in some chromosomes is an artifact probably formed during chromosome preparation. The present observations failed to show the hypoploidy in the chromosomes of cells from the erythroid marrow and the spleen of anemic animals, mice, rats and rabbits. In an earlier work Seno reported that in erythroid cells the rate of DNA synthesis and DNA contents decrease with maturation, as seen in the incorporation of 3H-thymidine into DNA, which shows a reduction of both the labelling index and the grain count, and in microspectrophotometry in Feulgen-stained cells (4). The reduction in the labelling index is explained by the elongated generation time in the

5 Chromosomes of Mammalian Erythroblasts 93 more mature cells; about 7 to 11 hours in proerythroblasts and basophilic erythroblasts, and 48 hours in orthchromatic erythroblasts (8, 9). The reduction in grain count and DNA level is due to the elongated s-phase in relatively mature erythroblasts; 2.5 hours in immature cells and 6.5 hours in mature ones. REFERENCES 1.SWIFT, H. Quantitative aspects of nuclear nucleoprotein. Internat. Rev. Cytol. 2, 1-76, WEICKER, H. and K.H. TERWAY. Die Chromosomenzahl der Erythroblasten. Klin. WoSchft 36, , YOSHIDA, M.C. Recent advances in somatic cell hybrization. Symposium for cell biology, Vol. 26, SENO, S. Studies on the differentiation of erythroblasts using radioisotopes. Acta Haemat. Jap. 27, , INouE, M. Studies on the DNA metabolism of erythroid cells. Acta Med. Okayama, 24, , OMURA, T. A method of chromosome preparation for fresh bone marrow cells of the mouse. Biol. Okayama Univ. 16, 29-34, SEABRIGHT, M. A rapid banding technique for human chromosomes. Lancet, , LAJTHA, L.G. Recent studies in erythroid differentiation and proliferation. Medicine 43, , FRINDEL, E., M. TUBIANA and R. VASSORT. Generation cycle of mouse bone marrow, Nature 214, , 1964 (Received for publication, February 9, 1978)