CHROMOSOME NUMBER OF MAN

While staying last summer at the Sloan-Kettering Institute, New York, one of us tried out some modifications of Hsu’s technique (1952) on various human tissue cultures carried in serial in vitro cultivation at that institute. The results were promising inasmuch as some fairly satisfactory chromosome analyses were obtained in cultures both of tissues of normal origin and of tumours (Levan, 1956).

In our opinion the hypotonic pre-treatment introduced by Hsu, although a very significant improvement especially for spreading the chromosomes, has a tendency to make the chromosome outlines somewhat blurred and vague. We consequently tried to abbreviate the hypotonic treatment to a minimum, hoping to induce the scattering of the chromosonies without unfavourable effects on the chromosome surface. Pre-treatment with hypotonic solution for only one or two minutes gave good results. In addition, we gave a colchicine dose to the culture medium 12-20 hours before fixation, making the medium 50X lo-" mol/l for the drug. The colchicine effected a considerable accumulation of mitoses and :I varying degree of chromosome contraction. Fixation followed in 60 "/o acetic acid, twice exchanged in order to wash out the salts left from the culture medium and from the hypotonic solution that would otherwise have caused precipitation with the orcein. Ordinary squash preparations were made in 1 "/o acetic orcein. For chromosome counts the squashing was made very mild in order to keep the chromosomes in the metaphase groups. For idiogram studies a more thorough squashing was preferable. In many cases single cells were squashed under the microscope by a slight pressure of a needle. In such cases it was directly observed that no chromosomes escaped.

THE CHROMOSOME NUMBER
With the technique used exact counts could be made in a great number of cells. Figs. 1 N and b represent typical samples of the appearance of the chromosomes at early nietaphase ((1) and full inetaphase ( h ) , showing the ease with which the counting could be made. In Table 1 the numbers of counts made from the four embryos studied are recorded. We were surprised to find that the chroinosome number 46 predominated in the tissue cultures froin all four embryos, only single caws deviating froni this number. Lower numbers were frequent, of course, but always in cells that seemed damaged. These were consequently disregarded just :is the solitary chromosomes and the groups with but a few chromosomes, which were frequent. In some doubtful cases the numbers 47 and 48 were counted (in four cases not included in thc table). This may be due to one or two solitary chromosonies having been pressed into a 46-chroniosome plate at the squashing. It is also possible that deviating numbers may originate through non-dis,junction, thus representing a real chromosome number variation in the living tissue. This kind of variation will probably increase as a consequence of the change in environment for the tissue involved in the in uitro esplantation. HSU (1952) reports a certain degree of such variation in his primary cultures. LEVAN (195G), studying long-carried serial subcultures, found hypotriploid stemline numbers in two of them, and a near-diploid number in a third culture. In this culture one cell with 48 chromosomes was analysed. Naturally, at that time, this was thought to represent the normal diploid number.

CHROMOSOME MORPHOLOGY
Some data on the chromosome morphology of the 46 human chroinosonies will be communicated here. The detailed idiogram analysis will be postponed, however, until we are able to study individuals of known sex, the sex of ihe present embryos being unknown. The coinparative study of germline chromosomes in spermatogonial mitoses coilstitutes an urgent supplement to the present work.
In Fig. 2 four cells are analysed ranging from late prophase ( ( I ) to late c-metaphase i d ) . The chromosomes of metaphases with moderate colchicine contraction vary in length between 1 and 8 p (Fig. 2 b), but the entire range of variation of Fig. 2 is from 1 to 11 EL. The chromosome morphology is roughly concordant with the observations of earlier workers, as, for instance, the idiogram of HSU (1952). The chromosomes may be divided into three groups: hl chromosomes (median-submedian centroniere; index long arm : short arm 1-1,9), S chromosomes (subterminal centromere; arm index 2-4,9), and T chromosomes (nearly terminal centromere; arm index 5 or more).
The h l and S chromosomes are present in about equal numbers (twenty of each), while six T chroniosonies are found. The classification of the three groups is arbitrary, of course, since gradual transitions of arm indices occur between the three groups. Certain submedian M chromosomes are hard to distinguish from some of the S chromosomes, and the most asymmetric S chromosomes approach the T group.
The chroniosomes are easily arranged in pairs, but only certain of these pairs are individually distinguishable. Thus, the P I 1 chromosomes include the three longest pairs, which can always be identified. The two longest pairs are different: the second having a decidedly more asymmetric localion of its centromere. The two or three smallest M p?' '11s are also recognizable. lktween the three longest and the three shortest pairs there are four intermediate pairs that cannot be individually recognized.
The S chromosomes are hardly identifiable, since they form a series of gradually decreasing length. The largest pair, however, is characteristic. Certain chromosomes were seen to have a small satellite on their short arms. Secondary constrictions, too, have been observed now and then, so that it may be hoped that the detailed morphologic study will lead to the identification of more chromosome pairs. The T chromosomes are recognizable; they constitute three pairs of middle-sized chromosomes. Unlike the mouse chromosomes, the human T chroniosomes evidently have a small shorter arm.

CONCLUSION
The almost exclusive occurrence of the chromosome number 46 in one somatic tissue derived from four individual human embryos is a very unexpected finding. To assume a regular mechanism for the esclusion of two chromosomes from the idiogram at the formation of a certain tissue is unlikely, even if this assumption cannot be entirely dismissed at this stage of inquiry. Our experience from one somatic tissue in mice and rats, viz., regenerating liver, speaks against this assumption. The exact diploid chromosome set was always found in regenerating liver.
After the conclusion had been drawn that the tissue studied by us had 46 as chromosome number, Dr. EVA HANSEN-MELANDER kindly informed us that during last spring she had studied, in cooperation with Drs. YNGVE MELANDER and STIG KULLANDER, the chromosomes of liver mitoses in aborted human embryos. This study, however, was temporarily discontinued because the workers were unable to find all the 48 human chromosomes in their material; as a matter of fact, the number 46 was repeatedly counted in their slides. We have seen photomicrographs of liver pcophases from this study, clearly showing 46 chromosomes. These findings suggest that 46 may be the correct chromosome number for human liver tissue, too.
With previously used technique it has been extremely difficult to make counts in human material. Even with the great progress involved in Hsu's method exact counts seem difficult, judging from the photoniicrographs published (Hsu, 1952 and elsewhere). For instance, we think that the excellent photomicrograph of Hsu published in DARLING-TON'S book (1953, facing p. 288) is more in agreement with the chromosome number 46 than 48, and the same is true of many of the photomicrographs of human chromosomes previously published.
Refore a renewed, careful control has been made of the chromosome number in spermatogonial mitoses of man we do not wish to generalize our present findings into a statement that the chromosome number of inan is 2n=46, but it is hard to avoid the conclusion that this would be the most natural explanation of our observations.