Testicular preparations were obtained from 7 bulls, twins of freemartins, and 1 male marmoset, all proved XX/XY chimaeras. X and Y sex chromosomes were confidently identified in nearly all the 87 spermatogonia at mitotic metaphase and 1052 primary spermatocytes at diakinesis-metaphase examined: no cell was identified as containing two X chromosomes. The germ cell chimaerism previously reported in these species is therefore not confirmed. Cultures grown from presumptive somatic these species is therefore not confirmed. Cultures grown from presumptive somatic cells in the testes of two of the bulls yielded 248 identifiable mitotic spreads, all XY-type; cultures from the gonads of their freemartin twins yielded 442 mitotic spreads, all XX-type. Direct preparations from one freemartin gonad, however, yielded 3 XY mitotic spreads out of 18 examined. The conflicting evidence concerning germ cell chimaerism in cattle and marmosets is discussed, particularly in relation to reports of XX/XY bulls that have sired a great excess of daughters. The possibility that XX germ cells contributed to the functional spermatozoa of these bulls is not favoured by present information, but is not excluded.

Chimaeric mice were obtained by injecting embryonic (CBA/H-T6 X PDE)F1 cells into PDE blastocysts. Three of 15 young were overt chimaeras. One female and one male chimaera survived to adulthood and after completion of test breeding, which demonstrated chimaerism in the germ cells of both, they were killed for study when aged 32 and 33 weeks respectively. Chromosome spreads were scored for the presence or absence of the T6 marker chromosome in direct preparations from bone marrow, spleen, thymus, lymph nodes, Peyer's patches, corneas, gut epithelium and testes. Preparations from monolayer cultures of skin, kidney, ovary and gut from mitogen-stimulated blood cultures were scored in the same way. Both components of the chimaeras were identified in every one of 53 specimens studied, some of which, such as single lymph nodes, corneas, and segments of gut, may not have contained more than 10(5) proliferating cells. This result complements published evidence for fine-grained mixture obtained in morula-aggregation chimaeras by other methods and implies extensive cell movement during embryogenesis. Results obtained from the lymphomyeloid tissues show a clear partition into two groups in respect of the proportions of host-type to donor-type cells indentified. The one group consists of bone marrow, thymus and Peyer's patches, the other of spleen and lymph nodes. This result would be most simply explained in terms of two distinct stem cell pools and appears to conflict with the currently favoured hypothesis of a single stem cell pool for the whole lymphomyeloid complex located in bone marrow. Four groups of factors may, however, modify the relative representation of the two components in different lymphomyeloid sites: (1) The magnitude of embryonic founder populations. (2) Limited recruitment from the stem cell pool in post-natal life. (3) Variable size of clones produced by individual stem cells. (4) Differential cellular behaviour determined by genotypic differences.

Two fertile female chimaeras derived by aggregation of morulae from the inbred mouse strains AKR/J and CBA/H-T6 were found to be of mixed sex chromosome constitution, XX/XY. The XY component of both chimaeras was AKR (albino). The records showed that one of the chimaeras had two litters totalling seven young including an exceptional son with an albino coat. This implied that the maternal gamete had originated from the XY component. Other possible explanations are examined and effectively excluded. The exceptional son was sterile and was later found to have the rare 41, XXY (‘Klinefelter’) karyotype. The possibility of functional reversal of germ cells in mouse chimaeras had hitherto been dismissed on the basis of the apparently undisturbed sex ratio of their progeny and the great excess of single-phenotype progenies in test matings. This evidence is re-examined and shown to be indecisive for female chimaeras.

Marker chromosome analysis of 18 tetraparental AKR↔CBA/H-T6 chimaeras revealed a great excess of AKR mitoses over CBA mitoses in direct preparations from lymphomyeloid tissues, corneal epithelium, intestinal epithelium and skin (Table 1). The degree of AKR dominance was strongly influenced by anatomical site (Tables 2 and 3). The testes of three out of four XY/XY males contained a marked excess of AKR germ cells and produced a parallel excess of functional AKR gametes (Table 4). Mitotic spreads in mitogen-stimulated cultures of tail blood were overwhelmingly of AKR type in 1972, but less so in 1973 (Table 5). The coat phenotypes, and breeding results from known or presumptive XX/XX females, suggest that AKR and CBA cells were numerically balanced when melanoblasts and oocytes were formed during embryonic development, and therefore that the striking deviations from equality observed in mitotic populations of adult chimaeras arose later by differential proliferation or survival of AKR cells, or both. The low frequency of lymphomas, compared with normal AKR mice, previously reported in these chimaeras cannot therefore be accounted for by insufficiency of AKR cells in the thymus or elsewhere in the lymphomyeloid tissues. One of three lymphomas studied was CBA type. This suggests that the high risk of lymphomatous transformation is not an autonomous property of AKR cells.

Adult male mice were given gonadal doses of 0–1200 rad acute X-irradiation and mated the same day. 531 sons, conceived within a week of the treatment, were tested for fertility and their testes examined cytologically for chromosome aberrations in spermatocytes. 5557 of those diagnosed as semi-sterile and 3540 of those diagnosed as sterile were judged to be heterozygous for one or more reciprocal translocations. Numbers of 0, 1, 2… translocations per mouse showed a good fit to a Poisson distribution, in contrast to previous findings with spermatogonial irradiation. Although the dose response fitted a linear relationship, the power law equation of best fit had a dose-exponent of 1.41. Further analysis along similar lines to those used previously in Drosophila by Catcheside, Lea and Haldane, which assumed random rejoining of breaks and direct proportionality between dosage and number of breaks, gave a close fit between the actural results and those expected if αq = 2.8·103−/rad, where α is the mean number of breaks per nucleus and q is the proportion which rejoin or restitute. By combining these data with those for litter-size reduction in F1 (taken as a measure of induced dominant lethality) α was estimated to be 3.4 × 10−3 per rad. When compared with the value of 0.8 × 10−3 per rad obtained in Drosophila by Haldane and Lea, this suggested that mouse haploid nuclei are more radiosensitive to chromosome breakage than Drosophila haploid nuclei by a factor of about 4. The mean number of implants per pregnant female mated to cytologically abnormal males was about 15% lower than with normal males. This pre-implantation loss was thought to be mainly the result of a reduction in the rate of fertilization in this group rather than to early death of unbalanced zygotes. There was no evidence for the induction of any undetected types of chromosomal aberration or gene mutation which could cause intrauterine death in the progeny of F1 males.

This chapter surveys the methods for analyzing mouse chromosomes, both mitotic and meiotic. In addition, references for methodology and examples of the kinds of information that can be obtained using the various methods are also provided in the chapter. Both genome sequencing and the availability of sequence have contributed to a virtual explosion in a number of cytogenetic applications. Cytogenetic mapping can be used to relate genetic linkage maps to cytologically identified chromosomal regions, reciprocal translocations, and genes being scored in the same linkage cross. However, new resources are continually becoming available and the explosion of activity in mouse and mammalian molecular cytogenetics points towards recent knowledge in this field. Advances in genomics and imaging and nanotechnology continue to bring new and useful methods to the field. Nonetheless, the basic technologies reviewed in the chapter remain useful for probing the structure of mouse chromosomes and for evaluation of de novo and heritable defects of chromosome structure and number.

Translocation frequencies were studied in spermatocytes derived from the spermatogonia of mice given 300 rad X-rays in two equal fractions separated by intervals of 0–8 h. In the first experiment (0–2 h intervals) there was a significant fall from 14.5% to 8.1% in the frequency of affected cells when there was an 0.5-h interval between doses instead of a single dose, followed by a just significant rise to 10.8% at a 2-h interval. However, the translocation frequency after a single 300-rad dose was higher than expected from previous experiments at this level (see paper III of this series). In the second experiment (0–8 h intervals) there was again an initial fall (from 8.8% at 0 h to 6.6% at 1 h) and a rise later (to 8.1% at 6 h and at 8 h intervals), but the changes in frequency did not reach a significant level. Results are compared with those obtained in similar recent experiments by Léonard and Deknudt (500 R total exposure) in which an initial decrease in yield was also found, but with more pronounced fluctuations in frequency later. The relative importance of radiation-induced changes in the spermatogonial population and of decreased oppurtunities for interaction of lesions on fractionation is discussed with reference to the observed effects.

The rate of induction of reciprocal translocations by 56–816 R exposures of mouse spermatogonia to acute γ-irradiation (95 R/min) was determined by cytological examination of descendant spermatocytes. The dose-response relationship did not differ significantly from linearity and had a regression coefficient of 1.8·10−4 per R with respect to translocations per spermatocyte. Further analysis at exposures below 816 R (considered less likely to produce distortion) showed that the quadratic regression of best fit had too small a square-law component to account for the very low frequency of translocations obtained after chronic γ-exposures in a previous experiment. The possibility is discussed that there is some extra factor, besides the diminution of the square-law component, which operates to reduce the yield after protracted exposures.

APPARENT polyploid primary Spermatocytes at diakinesis-metaphase are seen frequently in air-dried preparations from testes of mice and other mammalian species1. Most seem to be tetraploids but some higher “polyploids” occur, including “hexaploids” and even “decaploids”. Deviation from the geometric series suggests that these cells, or at least some of them, may arise as a result of cell fusion rather than such processes as endoreduplication or the formation of restitution nuclei, which would double the chromosomes. Although apparent polyploid spermatogonia are also seen, such cells will not necessarily give rise to Spermatocytes which can reach diakinesis-metaphase. Indeed, the possibility that polyploid germ cells, spermatogonia and Spermatocytes are all an artefact of preparation arising from the spreading together of the contents of adjoining cells is not excluded on present evidence. Artefactual origin is nevertheless unlikely because of the uniformity in degree of contraction and appearance of the chromosomes or bivalents and the regularity of many of the spreads.

A sterile mouse was found to be a chromosomal mosaic with two cell lines, one containing 39 chromosomes, the other 41. Both types were present in approximately equal numbers in the bone marrow, but the 41-chromosome line alone was identified in spermatogonia and spermatocytes. Evidence from chromosome morphology at mitotic metaphase and from spermatocytes at diakinesis and metaphase I identified the two cell-lines as XO and XYY respectively. The alternative possibilities of exclusion of the XO line from the seminiferous elements by chance and by selection are discussed and the latter interpretation favoured. The possibility is raised that the Y chromosome of mammals carries genetic information essential for normal spermatogenesis, as distinct from its established function in embryonic gonadal determination.

Journal Article RECIPROCAL TRANSLOCATIONS Get access C. E. FORD, D.Sc. F.R.S., C. E. FORD, D.Sc. F.R.S. Medical Research Council Radiobiological Research unit HarwellDidcot, Berkshire Search for other works by this author on: Oxford Academic PubMed Google Scholar HILARY M. CLEGG, B.Sc. HILARY M. CLEGG, B.Sc. Medical Research Council Radiobiological Research unit HarwellDidcot, Berkshire Search for other works by this author on: Oxford Academic PubMed Google Scholar British Medical Bulletin, Volume 25, Issue 1, January 1969, Pages 110–114, https://doi.org/10.1093/oxfordjournals.bmb.a070659 Published: 01 January 1969

The effect of dose-rate on the induction of reciprocal translocations in mouse A type spematogonia by 600 RX- and γ-irradiation was studied by scoring multivalent configurations in descendant spermatocytes. With X-irradiation over a range of dose-rates from 0.8 to 913 R/min there was no significant change in the frequency of affected spermatocytes, which averaged 12.8%. With γ-irradiation, however, there was a steady increase in frequency from 1.4% at 0.02 R/min to 12.1% at 86 R/min, the points fitting a straight line on a semi-log plot. At 0.08 R/min the X-ray yield was twice that for γ-rays. Possible reasons for these differences are discussed.

Lethally irradiated CBA mice were injected with a mixture of two cell suspensions, syngeneic with each other and with the host, but distinguished by the presence of either one or two T6 marker chromosomes. One of the cell suspensions was derived from adult bone marrow (10 5 cells), the other from adult thymus or pooled lymph nodes or thoracic duct lymph (10 7 cells). The recipients were killed between one day and one year after irradiation, having been injected 1 ½ h previously with Colcemid. Their bone marrow, spleen, thymus and lymph nodes were studied cytologically, and counts were made of the numbers of mitotic cells derived from the two donor cell suspensions and from the irradiated host. Bone marrow, spleen and thymus were all recolonized predominantly or exclusively by descendants of injected bone marrow cells. Descendants of injected lymphoid cells were seen in substantial numbers only in the lymph nodes, where they formed the majority of the total dividing cells between 1 and 3 weeks after irradiation. After that the proportion of such cells in the lymph nodes decreased gradually, in favour of bone marrow-derived cells, but they did not disappear completely. Lymphoid cells were equally unsuccessful at recolonizing the myeloid and thymic tissues of mice given a high sublethal dose of irradiation (800 rad) without bone marrow therapy. The normality of the repopulated lymph nodes and thymus was verified histologically and by two functional tests involving the capacity of cells rapidly to recolonize the lymph nodes or form macroscopic haematopoietic nodules in the spleen of further lethally irradiated mice. A small and decreasing amount of haematopoiesis was found in the lymph nodes during the first 2 months after irradiation, but not in the thymus.

Preparations were obtained from the testes of six sterile males of the T(X; ?) 16H stock. 81.7% of 694 primary spermatocytes in stages from diakinesis to first metaphase contained chain quadrivalents composed of a terminal Y chromosome and three other elements that were distinguishable in length. A normal XY pair was not seen in any cell. These observations and others indicate that the stock carries an unequal reciprocal translocation between the X chromosome and one of the shortest autosomes, the break points being approximately median in both chromosomes. This suggests that the locus of tabby (Ta), which is known to be closely linked with the breakage point, lies near the middle of the X chromosome.