Section 6 — Cytology 



rather than interphase chromosomes, preser- 

 vation of chromosome morphology can be used 

 as a significant criterion in the evaluation of the 

 techniques used. Chinese hamster cells grown 

 in vitro provide an excellent source of material 

 for chromosome isolation since 30 to 50 per cent 

 of the cells can be accumulated at metaphase 

 with a 12-hr colcemid treatment. Treatment 

 of metaphase cells for 3 min with 0.1 

 m sucrose containing 7 x 10~ 4 m CaCl-2 and 

 3 x 10 _4 m MgCb serves to hydrate cells sufficient- 

 ly for chromosome spreading while maintaining 

 a minimal degree of chromosomal hydration. 

 Following this hypotonic treatment, cells can be 

 disrupted in 0.4 m sucrose containing 5 x 10~ 4 m 

 each of CaCl2 and MgClo by drawing the cell 

 suspension into a 30 ml syringe and expelling it 

 through a 15 gauge hypodermic needle. Divalent 

 ions are required to preserve chromosome 

 morphology when chromosomes are isolated in 

 sucrose. In order to test the effects of ions on 

 metaphase chromosomes, a drop of 0.4 m 

 sucrose (5 x 10 _4 m CaCb and MgCl2) containing 

 isolated chromosomes was placed on a slide and 

 covered with a cover slip. Chromosomes were 

 perfused with 0.4 m sucrose containing desired 

 test ions. Changes in chromosome morphology 

 were observed with phase contrast optics. 

 Chromosomes will swell or uncoil if divalent 

 ions are replaced by monovalent ions, e.g. NaCl. 

 Uncoiling can be reversed by subsequent addition 

 of divalent ions. If extensive swelling or uncoiling 

 has occurred, the metaphase configuration is 

 destroyed, and the divalent ions promote a 

 contraction to the chromonema level only. 



6.13. Chromosomes of Normal and Tumour Epithelioid 

 Derivatives (2n=22) of the Chinese Hamster. 



George Yerganian (Boston, U.S.A.). 



Normal adult epithelia derived from various 

 organs of the Chinese hamster, Cricetulus 

 giiseus, fail to proliferate in vitro beyond several 

 passages. In contrast, "spontaneous" carcino- 

 matous derivatives proliferate extensively and 

 have presently exceeded the 45th passage while 

 averaging 18 hr generation times. In addition to 

 the retention of classic diploidy, tumor de- 

 rivatives lack contact inhibition, display a 

 metaphase: anaphase ratio of 10 : 1, and an 

 average single cell plating efficiency of 20 per 

 cent. 



Eighty per cent or more of the metaphases 

 are classic diploid, and some 5 per cent each are 

 hypodiploid, hyperdiploid, and tetraploid. Equal- 

 ly stable are clonal derivatives (XiO quasidiploid 

 4- trans I: III, and In + Iso LAIII). Each clonal 



subline exhibits varying degrees of cytopathic 

 effect (CPE) caused by an adenovirus (type 

 undetermined) isolated from one of five original 

 primary sublines. 



Of possible significance is the slight morpho- 

 logical variation in the short arm of the hetero- 

 chromatic X in a good portion of normal and 

 tumor epithelioid forms. In the majority of 

 fibroblast-like derivatives, this heterochromatic 

 segment or X2 is slenderer and undergoes late 

 DNA synthesis. In epithelioids, this portion ap- 

 pears intermediate in dimensions or X 1-2. If 

 this morphological feature is substantiated by a 

 corresponding intermediate type of tritiated 

 thymidine uptake pattern, epithelioid derivatives 

 would then have an X1X1-2 status, whereas 

 fibroblast-like cells feature the moie differenti- 

 ated or X1X2 status. 



These observations suggest that some chromo- 

 somal segments may reflect patterns of nuclear 

 and cellular differentiation, possess intermediate 

 levels of heterochromatization (gene expression), 

 and govern rates of CPE caused by viruses. 



6.14. The Chromosomes of A- and B-spermatogonia 

 of the Mouse. Eva Hansen-Melander (Lund, 

 Sweden). 



In the tubules of the mouse two types of 

 spermatogonial cells are found, the A- and 

 B-spermatogonia, which differ considerably in 

 their chromosomal behaviour. The chromosomes 

 of the A-spermatogonia resemble from prophase 

 to telophase the chromosomes of any other 

 dividing somatic cell. The A-spermatogonia give 

 rise to new-A-spermatogonia or to B-spermato- 

 gonia. 



From the onset of prophase the chromosomes 

 of the B-spermatogonia behave differently from 

 those of the A-spermatogonia. The differences 

 are visible already during prophase and become 

 striking during full metaphase. When the division 

 of the B-spermatogonia is over the cells enter 

 meiosis. 



The heteropycnosis of the sex chromosomes, 

 particularly of the Y, is conspicuous during 

 prophase in both kinds of spermatogonia. 



6.15. Origin of Sex Chromatin and Related Phe- 

 nomena. Yngve Melander (Lund, Sweden). 



The sex chromatin of the female rabbit 

 originates during early embryogenesis. The long 

 arm of one X chromosome becomes positively 

 heteiopycnotic during interphase as a result of a 



105 



