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Cytogenetic and genetic studies of radiation-induced chromosome damage in mouse oocytes. II. Induced chromosome loss and dominant visible mutations
Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER
Mutation Research 349 (1996)
155-162
Cytogenetic and genetic studies of radiation-induced chromosome damage in mouse oocytes. II. Induced chromosome loss and dominant visible mutations Charles Tease *, Graham Fisher MRC Radiobiology
Unit, Chilton,
Didcot.
OX1 I ORD, UK
Received 14 June 1995: revised 26 September 1995; accepted 28 September 1995
Abstract The rates of X-ray induced loss of chromosome 19 in mouse oocytes were investigated in 2 experiments using a genetic complementation test. After I Gy of acute X-rays to immediately preovulatory stage oocytes, chromosome 19 loss was estimated to have occurred in 1.68% of cells. In comparison, after 4 Gy of acute X-rays to dictyate stage oocytes, the rate was estimated at I. 18%. The slightly higher rate of chromosome loss in the former cell stage after a smaller dose of radiation reflects the known increased radiosensitivity of mouse oocytes in the period shortly before ovulation. Comparison of the observations here for chromosome I9 with published data for chromosome I suggests that chromosome length is one of the principal factors in determining the initial rate of induced loss in mouse oocytes. Ten dominant visible mutations were recovered among 1674 offspring following irradiation of preovulatory oocytes, and 8 in 2025 offspring after treatment of dictyate cells. Nine dominant mutations were karyotyped, 5 of these were found to be associated with a visible chromosome rearrangement. The data obtained in the present study show that radiation-induced chromosome anomalies in female germ cells are not all filtered out by prenatal embryonic death but that a proportion has the potential to contribute to the genetic burden of the next generation. Krvvordst
Mouse oocyte; X-rays; Chromosome loss; Dominant mutation
1. Introduction The various cytogenetic methods available for investigating chromosome segregation errors in mammalian germ cells have long been acknowledged to have an inherent weakness when used for
x Corresponding author.
0027-5107/96/$15.00 SSDI 0027-5
0
1996 Elsevier Science B.V.
107(95)00183-2
analysis of chromosome loss events. The principal problem relates to the difficulty of distinguishing chromosome loss resulting from a segregation error from that produced artefactually during cell preparation for microscopy. Cytogenetic analyses are therefore generally more appropriate for assessment of chromosome gain, and other approaches have had to be developed to study spontaneous or induced chromosome loss in germ cells. Initially, a procedure to
All rights reser,ved
assess sex chromosome aneuploidy in mice was devised by Russell (1976); subsequent developments have produced genetic assays for detecting autosomal chromosome loss. Detailed descriptions of the principles of these latter tests have been provided by various authors (e.g., Cattanach et al.. 1984; Lyon, 1984; Tease and Cattanach, 1986). In brief. the assays make use of nullisomy. spontaneous or induced. for a particular chromosome in the gametes of one parent being complemented at fertilization by the appropriate disomy in the gametes from the other parent. To elevate the rate of disomic gametes and thus increase the probability of complementation, mice heterozygous for one or more Robertsonian (Rb) translocations are employed. The use of marker genes allows easy identification of any offspring that result from such complementation. Cattanach et al. (1984) undertook a chromosome loss experiment in which female mice with a normal acrocentric karyotype were given an acute dose of X-rays in the range I to 4 Gy and mated to males heterozygous for Rb(l.3)lBnr. Chromosome I was marked in tester males with the recessive genes fuzzy (,fk> and leaden (In) to enable identification of progeny resulting from complementation of induced chromosome 1 loss in oocytes by Rb-mediated chromosome I nondisjunction in male meiosis. A linear, dose-related increase in the incidences of marked young was found. Cattanach et al. (1984) used an estimate of the proportion of sperm disomic for chromosome I to calculate the probability of complementation and thus the initial proportion of oocytes that suffered induced chromosome I loss. They concluded that 4 Gy of X-rays induced loss of chromosome 1 in approx. 3% of oocytes. This experiment therefore not only showed the efficacy of the method but also demonstrated a surprisingly high incidence of chromosome loss after X-irradiation. In addition to effects on chromosome segregation, Cattanach et al. (1984) also reported the occurrence of a few offspring carrying dominant visible mutations. Such mutations are regularly observed in radiation experiments and have been of value for making genetic risk estimations for the human population (Searle, 1974: Searle and Beechey, 1986: Selby. 1990). Recently, Cattanach et al. (1993) showed that dominant visible mutants found in mutagenesis experiments were frequently associated with chromo-
some aberrations particularly deletions. Subsequent analyses have also shown that mutants whose principal phenotypic effect was growth retardation can carry chromosome aberrations (Burtenshaw et al., 1994; Evans et al.. 1995). This observation is of interest in view of the suggestion of Searle and Beechey (1986) that growth retardation might be a phenotypic category meriting attention in mutagenesis experiments; the results from the recent cytogenetic analyses would appear to provide support for their suggestion. In a previous paper (Tease and Fisher, 1996). the effects of X-rays on mouse oocytes were assayed using various cytogenetic methods to analyse germ cells and embryos at different stages of development, The work reported here continues and extends this analysis. firstly by evaluating induced chromosome loss using a genetic method and secondly, by screening progeny for dominant visible mutations. Some of the animals falling into the latter category were investigated further by detailed karyotypic analysis of G-banded chromosomes. The data generated from these experiments complement those from the cytogenetic studies. and together they provide a comprehensive description of the types of chromosome damage initially present in mouse oocytes after irradiation and of the frequencies and categories of genetic anomalies transmitted to the F, progeny.
2. Materials
and methods
In the present study, induced loss of chromosome 19 was examined in female mice with a normal. acrocentric chromosome complement. The tester males had monobrachial homology (MBH) for 2 translocations. Rb(8.19)ICt and different Rb(9.19)163H. In part, use of chromosome 19 was a pragmatic choice dictated by the fact that few MBH combinations are male fertile and also the need to avoid use of chromosomes that show parental imprinting effects; in addition. however, this choice offered the possibility of comparing the relative responses of chromosomes of widely differing sizes as data for the largest chromosome of the mouse complement, chromosome 1, has already been published (Cattanach et al., 1984).
157
2.1. Experiment
I: Immediutely
preoLdutory
oocytes
and screening the phenotypes of the offspring. Suspected dominant mutations were tested for inheritance by mating to 3Hl animals and screening a minimum of 30 offspring for the novel phenotype. If the mating yielded fewer than 30 progeny, the mutation was regarded as incompletely tested. Some of the dominant mutations were further analysed by G-band karyotyping of lymphocyte or bone marrow chromosome preparations to screen for visible chromosome anomalies associated with the mutation. Spermatocyte preparations (Evans et al., 1964) were made from six randomly chosen males of the MBH stock. The cells were C-banded (Sumner, 1972) and chromosome numbers counted in metaphase II spermatocytes to determine the rate of chromosome 19 nondisjunction from which the proportion of spermatozoa with chromosome 19 disomy could be estimated.
Female mice of the F, hybrid type C3H/HeH X IO 1/H (abbreviated to 3H1) were induced to ovulate using 2 IU of pregnant mares serum gonadotrophin (PMS) followed 48 h later by 2 IU of human chorionic gonadotrophy (HCG).They were given 1 Gy of X-rays 3 h after HCG injection and then mated overnight to MBH males carrying Rb(8.19)lCt and Rb(9.19) 163H. and also homozygous for the chromosome 19 marker gene ruby eye (nl). The following morning the females were inspected for the presence of a vaginal plug as indication of successful mating, and then separated from the males. Pregnancy was allowed to go to term and liveborn offspring screened for the presence of homozygosity for t-14 as occurs when induced loss of the maternal chromosome 19 is complemented by the presence of two paternally-derived chromosomes 19. The offspring were also screened for the presence of dominant mutations affecting external appearance. 2.2. Experiment
3. Results
2: Dictyate oocytes
3. I. Chromosome
The results from Experiment 1 are presented in Table 1. Overall, 1603 offspring from unirradiated females and 1674 offspring from females given 1 Gy of X-rays were screened. No ru exceptions were found in the unirradiated, control series whereas 6 animals with this phenotype were found in the irradiated group. All 6 animals appeared to possess the karyotype expected of complementation, that is, 36 acrocentric and 2 Robertsonian metacentric chromosomes. However, one of the t-u exceptions produced a wild-type mouse when tested in the backcross. As
3Hl females were given 4 Gy of X-rays and mated to males of the same genotype as above. The matings were continued for 4 weeks, at which time the males were removed. As before, offspring were screened for nl exceptions and possible dominant mutations. was made Confirmation of the ru classification initially by chromosome analysis of cultured lymphocytes to show the presence of the 2 patemally-derived Robertsonian chromosomes in diploid cells, and then by backcrossing to ru homozygous mice Table
loss
I
X-ray induced loss of chromosome Dose(Gy)
Experiment
Interval
19 in mouse oocytes No. of
I.II/YLI exceptions
No. of
Mean litter
offspring
(c/r)
litters
size
1 286
5.60
415
3.52
Control
_
1603
0
I Experiment
0.5 days
1673
6
Control
_
1510
1 (0.07)
232
6.51
4
<
I463
3 (0.21)
253
5.78
4 Total (irrad)
(0.36)
2
I week I-4 weeks
562
2 (0.36)
172
3.21
2025
5 (0.25)
425
4.76
Table 2 The proportions of metaphase II spermatocytes with different chromosome constitutions in 6 male mice with monobrachial homology for Rb(8.19)l Ct and Rb(9.19Jl63H % cells with chromosome
complement:
I7+Rb+Y
17+Rb+Xl8+Y
lX+Xl6+2Rb+Y
16+2Rb+X
34.2 ,’
34.0 h
10.3
7.0 d
Sample included: cells. I6+Rb+X: l.++Rb+X
7.2
7.3 i
‘I 6 cells, I6 + Rb + Y: 2 cells. I8 + Rb+ Y. h 4 I cell, 17+Rb. ’ I cell. IS+?Rb+Y. ” I cell.
the breeding test showed this latter animal was not a product of complementation, the simplest explanation is that it was a trisomic conceptus that survived because of mosaicism following loss of the acrocentric chromosome 19 in some cell populations during embryonic development. This ru exception was not included in the subsequent attempt to calculate the initial incidence of chromosome 19 loss in X-irradiated oocytes. The rate of chromosome nondisjunction in the tester males was determined in the present study through analysis of metaphase II spermatocytes in 6 animals (Table 2). There was no indication in the data of significant variation between males in the frequency of aneuploid cells. Thus there is no reason to suspect that in the experimental matings the probability of complementation might vary between the different males used in the actual test crosses. From this analysis, an estimated 14.3% of spermatozoa produced by MBH males are anticipated to be disomit for chromosome 19. Cattanach et al. (1984) developed a statistical method through which to determine the proportion of germ cells with induced chromosome loss. This method exploits knowledge of the rate of chromosome nondisjunction in germ cells of the tester parent. Using this statistic it is possible to estimate the proportion of cells with induced chromosome loss that would be necessary to account for the observed rate of complementation. From the estimate of 14.3% of disomic sperm in the present study, it was calculated that approx. 1.68% (9.5% confidence limits, 0.64 and 3.39) of oocytes must have undergone induced loss of chromosome 19 to account for the observed rate of 0.309 YCIexceptions in Experiment 1. The progeny of irradiated females in Experiment
2 are separated in Table 1 into those born within 28 days of irradiation, and therefore presumed to be conceptions in the first week after treatment, and those born in later weeks. This was done to determine whether the variability in dictyate oocyte sensitivity to radiation-induced chromosome damage at different stages of maturation (Brewen et al., 1976) could also be discerned using this genetic method. Overall, 1 ru exception was found in the progeny of control females and 6 among those of irradiated females. As above, ru exceptions were tested both cytogenetically and genetically and one animal from the irradiated series was found to be mosaic; again this animal was not included in the analysis. The frequency of ru exceptions was 0.2 I c/oin the first week and 0.36% for the remainder of the sampling interval (Table 1). The difference is clearly nonsignificant given the relatively small number of ru exceptions that were found. nevertheless, the occurrence of a higher rate at the longer sampling intervals is in line with the previous description of a greater radiosensitivity of these cells for induced chromosome damage compared with oocytes within a few days of ovulation (Brewen et al., 1976). Using the approach of Cattanach et al. (1984), an overall estimate was obtained of 1.I 8% (95% confidence limits 0.40 and 2.59) of oocytes with induced loss of chromosome 19 as a consequence of X-irradiation. 3.2. Dominant
mutatiorls
A number of phenotypically deviant mice were found among the litters of control and irradiated females from both experiments and where possible these animals were tested for inheritance of the novel phenotypes (Table 3). The most common category recorded was that of small body size. This classification is obviously somewhat subjective but in an attempt to introduce an element of objectivity it was decided to categorise as small any animal that weighed less than 75% of the average litter weight on the basis of previous reports (Kirk and Lyon, 1982; Searle and Beechey, 1986). Clearly, this approach was only of value where litter sizes were large enough to provide a meaningful average; where litter sizes were small, the element of subjectivity remained in the classification.
C. Tease, G. Fishrr/Mutation
Table
IS9
3
The numbers
and categories
of mice identified
Category
Inherited
Treatment group Experiment Control
Experiment Control
as phenotypically
abnormal
Not inherited
and the results of breeding Trisomy mosaic
tests
Sterile
Died, untested
Total
20 0 1 35 1 8
29 4 5 60
2
5
1 small tail kinks other small tail kinks other
l CY
ICY
Research 349 (19961 155-162
I7
2 small tail kinks small tail kinks other
A total of 29 animals from unirradiated females in Experiment 1 and 5 from Experiment 2 were classified as small. Only 4 animals from the former experiment were tested for fertility, the remainder died either pre- or post-weaning, or failed to breed. None of the small animals that were tested from control females transmitted the phenotype. There is an interesting discrepancy in the proportions of animals from control mothers that were recorded as small in the 2 experiments. It may be that the use of hormones to induce ovulation in Experiment 1 increased the chance of small embryos surviving to term or that the risk of small size was increased because of uterine conditions being less optimal following induction of ovulation without regard to the normal fertility cycle. For irradiated females, an increased proportion of animals were classified as small and some of these were shown to be heritable (Table 3). Inheritance of the phenotype was proved in 4/ 12 animals in Experiment 1 and 2/9 animals in Experiment 2. In addition to animals that were classified as small, a variety of other phenotypically deviant mice were found (Table 3). These ranged from minor effects such as slight tail kinking through to more extreme effects such as syndactylism. In all cases, the mutant animals were somewhat smaller than phenotypically normal sibs. In controls of Experiment 1, 8/ 9 potential mutants proved not to be heritable, the other died before mating; in the irradi-
0
0
0
0
3 26 7 10
15 0
0
0
4
ated females, 5/ 14 tested proved heritable, 10 others gave no result (Table 3). A similar situation prevailed in Experiment 2 with no proved dominant mutations in the control group, and 6/l 1 tested in the irradiated group being inherited, with 6 others
Table 4 Phenotypes of the mutations selected for karyotypic analysis and the outcome of the cytogenetic investigation (NF, no rearrangement found) Phenotype
Experiment 1 Small with eye nomalies Small with eye anomalities Brachyury Domed head Syndactyly Experiment Steeloid
Chromosome rearrangement
Reference
Deletion, Chromosome Deletion, Chromosome Deletion, Chromosome NF NF
Tease and Fisher, 1993b Tease and Fisher, 1993b 13 Fisher and Tease, I992 17
2
Small with eye anomalies Small with tail kinks and white spotting Dark footpads and genetalia
Deletion, Chromosome Duplication, Chromosome NF
NF
Tease and Fisher, 1993b 10 Tease and Fisher, I994 16 Tease and Fisher. l993a
giving no result (Table 3). Detailed karyotypic analyses were performed on mutants whose phenotypic effects were greater than simply size reduction or slight tail kinking. From the 2 experiments. 9 mutants were screened and in 5 a visible chromosome rearrangement was found to be associated with the phenotypic effect (Table 4). A chromosome I deletion was also found in a sterile male from Experiment I (Table 3) that had a white head spot and a white patch on its thorax (Tease. 1993). Another interesting chromosome anomaly was recovered in one of the r~l exceptions of Experiment I. When this animal was screened for the presence of the 2 Robertsonian translocations, a very small supernumerary chromosome was also seen to be present in the cells. This microchromosome has proved to be heritable and has no overt effect on external phenotype (Tease and Fisher. in preparation).
4. Discussion X-ray induced chromosome damage has been studied previously in preovulatory and dicytate stage oocytes of 3H I female mice. Tease and Fisher ( 1986) reported that structural chromosome aberrations were present in metaphase I oocytes at a rate of I .28/ cell after I Gy to preovulatory stage oocytes treated similarly to the present study. A subsequent investigation yielded aberration induction rates of 0.26,’ cell and O.lO/cell after a 4 Gy treatment of dictyate stages 16.5 days and 3.5 days, respectively. before ovulation (Tease and Fisher, 1991). If these induction rates are transformed to a per Gy basis to facilitate comparison of the relative sensitivities of the different oocyte developmental stages. then preovulatory oocytes are seen to yield approx. 5 to I3 times more aberrations than the 2 dictyate stages. The present study has produced 2 measures of X-ray induced genetic damage in mouse oocytes, firstly, the rate of chromosome 19 loss. and secondly. the incidence of dominant visible mutations. For the former, the respective rates in preovulatory and dictyate oocytes were 1.68% and 1.18%. Again, if the rates are transformed to a per Gy basis. then the frequency of chromosome I9 loss was approx. 6 times greater in preovulatory than dictyate oocytes. With regard to dominant visible mutations, IO were
found in the 1674 mice screened in Experiment I and 8 in the 2025 mice of Experiment 2. These figures translate into rates of 5.97 X IO-’ and 9.88 X IO-’ per Gy, respectively, which again indicate that preovulatory oocytes were approx. 6 times more sensitive to induction of dominant mutations than the combined dictyate stages. Both measures of genetic damage in the present study therefore fell within the range obtained by comparing the outcomes of a cytogenetic analysis. Obviously this comparison is limited by the need to combine results of dictyate stages which are known to vary in their radiosensitivity (Brewen et al.. 1976; Tease and Fisher. 1991). Nevertheless, it is of interest that the various assays, with their widely differing endpoints, all tend to give a reasonably similar estimate of the comparative sensitivity of preovulatory and dictyate oocytes. The only previous study in mouse oocytes using this genetic complementation assay was carried out by Cattanach et al. ( 1984) who screened for induced loss of chromosome I. They estimated that 4 Gy of X-rays to dictyate stage cells of 3Hl females caused chromosome I loss in approx. 3.5% of oocytes. By comparison, in the present study the same dose of X-rays to oocytes at a similar stage of maturation yielded a rate of chromosome I9 loss of I. 18%. If the rate of induced chromosome loss is correlated with the size of the target chromosome then obviously a higher rate would be expected for chromosome I compared with chromosome 19. Given that chromosome I9 is approx. 40% of the length of chromosome I, then from Cattanach et al.‘s (1984) data an expectation of 1.4% of cells with chromosome 19 loss is generated. The observed rate does not differ dramatically from that expected. Overall. the present study confirms the initial finding of Cattanach et al. (1984) that X-irradiation can induce chromosome loss in a surprisingly high proportion of cells. Comparison of the data in the 2 studies indicates that chromosome length is an important factor in determining the initial rate of induced chromosome loss. Searle and Beechey (1986) argued for the inclusion of growth retardation as an informative category of dominant visible mutation following parental exposure to radiation. Following X-irradiation of spermatogonial stem cells, they showed that approx. 40% of the offspring that had been classified as small
C. Tetrse. G. Fisher/Mututim
transmitted the growth retardation phenotype. Our data are similar with 7 out of 1.5 fully tested smalls in the 2 experiments proving heritable. However, as Searle and Beechey ( 1986) also acknowledged, testing for heritability of growth retardation poses problems as many of the animals either fail to survive to weaning or result in incomplete testing, as was also the case here (Table 3). An additional complicating factor in the present work was the presence of mice which proved upon cytogenetic investigation, or through breeding, to be mosaic for trisomy 19 (Table 3). It is not certain what proportion of the small mice that died before testing were also mosaic. Nevertheless. the data in the present study supports the suggestion of Searle and Beechey (1986) that, following parental X-irradiation, a substantial proportion of the F, animals classified as small have a genetic basis for their growth retardation Recently, Cattanach et al. (1993) found that a large proportion of dominant visible mutations were associated with a visible chromosome aberration, most often a deletion. Further studies have confirmed and extended this initial report to show that deletions or duplications of practically all members of the chromosome complement could be found after exposure of male germ cells to X-rays (see Beechey, 1995). Cytogenetic analysis of some of the dominant mutants recovered in the present experiments has also revealed the presence of chromosome aberrations (Table 4). Karyotypic analysis of 14 day postimplantation embryos from female mice treated as in Experiment 1 showed that approx. 13.6% of the embryos had a visible chromosome anomaly (Tease and Fisher, 1996). What proportion of these complete gestation and give rise to dominant visibles is unknown but the present work provides clear evidence that a significant fraction are capable of achieving full term. Moreover, it is a reasonable supposition that many of the animals with putative dominant visible mutations but which died in the pre-weaning or early post-weaning period (Table 3), carried some form of chromosome damage. It is clear from these experiments that not only does a significant proportion of germ cells with X-ray induced chromosome damage contribute to the genetic burden of the next generation, but there is also a strong likelihood that they are a causative factor in premature death in the immediately postnatal period.
Resrnrch
349 (I9961
155-162
161
A contrary conclusion was advanced by Reichert et al. (I 984) from their study of radiation-induced chromosome anomalies in post-implantation mouse embryos. They were unable to detect such rearrangements and therefore suggested that conceptuses with induced chromosome aberrations must invariably fail to survive to parturition. The cytogenetic data described in our earlier paper (Tease and Fisher, 1996) in combination with the observations here clearly show that the conclusion of Reichert et al. (1984) is incorrect. Rather, by extrapolation from our data, we suggest that any dose of radiation that is capable of causing structural chromosome rearrangements in female germ cells must inevitably also present a significant risk of heritable chromosome aberrations to the next generation.
Acknowledgements We thank Peter Adams for carrying out the irradiations, David Papworth for the statistical calculations, and Dr Bruce Cattanach for his comments on the manuscript. This work was supported in part by Euratom contracts Bi6- I43 and F 13P-CT920005.
References Beechey, C.V. (1995) Maps of chromosome anomalies in the mouse. Mouse Genome, 93, 67-88. Brewen. J.G., H.S. Payne and R.J. Preston (1976) X-ray induced chromosome aberrations in mouse dictyate oocytes. I. Time and dohe relationships. Mutation Res.. 35. I 1 I- 120. Cattanach. B.M., D. Papworth and M. Kirk (1984) Genetic tests for autosomal non-disjunction and chromoome loss in mice, Mutation Res., 126. 189-204. Cattanach, B.M.. M.D. Burtenahaw, C. Rasberry and E.P. Evans ( 1993) Large deletions and other gross form5 of chromosome imbalance are compatible with viability and fertility in the mouse, Nature Genet., 3. 56-61. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations for mammalian testes, Cytogenetics. 33. 289-294. Fisher G. and C. Tease (1992) T’5H: a radiation-induced deletion affecting the T. qk and Tme loci. Mouse Genome 90, 209-2 10. Kirk. M. and M.F. Lyon (I 982) Induction of congenital anomalies in offspring of female mice exposed to varying doses of X-rays, Mutation Res.. 106, 73-83. Lyon. M.F. (1984) The use of Robertsonian translocations for
studies of nondisjunction. in: T. laihara (Ed.). Radiation-Induced Chromosome Damage in Man. Alan R. Liss, New York, pp. 321-346. Reichert, W., W. Buselmaier and F. Vogel (1984) Elimination of X-ray-induced chromosomal aberrations in the progeny of female mice, Mutation Res., 139, 87-93. Russell. L.B. (1976) Numerical sex chromosome anomalies in mammals: their spontaneous occurrence and use in mutagenesis studies. in: A Hollander (Ed.), Chemical Mutagenesis. Vol. 4. Plenum Press. New York. pp. 55-91. Searle. A.G. (1973) Mutation induction in mice. Ad\. Radiat. Biol.. 4, 131-207. Searle. A.G. and C. Beechey (1986) The role of dominant visihles in mutagenicity testing. in: C. Ramel. B. Lambert and J. Magnusson (Eds.), Genetic Toxicology and Environmental Mutagens. Alan R. Liss, New York, pp. 51 I-518. Selby. P.B. (1990) Experimental induction of dommant mutations in mammals by ionizing radiations and chemicals, Issues Rev. Teratol.. 5. I8 I-253 Sumner. A.J. (1972) A simple technique for demonstrating centromeric heterochromatin, Exp. Cell Res.. 75, 304-306. Tease. C. (1993) A cytogenetically visible, radiation-induced deletion on chromosome I. Mouse Genome. 9 I, I IS.
Tease, C. and B.M. Cattanach (1986) Mammalian genetic and cytogenetic tests for non-disjunction, in: F.J. de Serres (Ed.), Chemical Mutagens, Vol. IO. Plenum Prcsh. New York. pp. 21.5-283. Tease. C. and G. Fisher (1986) X-ray-induced chromosome aherrations in immediately preovulatory oocyte\. Mutation Res., 173. 21 l-215. Tease. C. and G. Fisher (I991 1The influence 01‘maternal age on radiation-induced chromosome aberrations in mouse oocytea. Mutation Rea.. 262. 57-62. Tease. C. and G. Fisher (1993a) Tail kinks. small size, and white spotting (Tks): a radiation-induced mutation located on chromosome 9. Mouse Genomc, 9 I. 137-13X. Tease. C. and G. Fisher (1993b) Cytogenetic detection of four new. viable deletions in the progeny of X-irradiated females. Mouse Genome, 9 I. 855. Tease, C. and G. Fisher (1994) Presumptive duplication\ after X-irradiation of females, Mouse Gcnome, 92. 3.50 Teaae C. and G. Fisher Cytogenetic and genetic studies of radiation-induced chromosome damage in mouse oocytcs. 1. Numerical and btructul-al chromosome anomalies in metaphase II oocyteh. pre- and post-implantation cmbryoh. Mutation Res.. thih i\\ue.
ELSEVIER
Mutation Research 349 (1996)
155-162
Cytogenetic and genetic studies of radiation-induced chromosome damage in mouse oocytes. II. Induced chromosome loss and dominant visible mutations Charles Tease *, Graham Fisher MRC Radiobiology
Unit, Chilton,
Didcot.
OX1 I ORD, UK
Received 14 June 1995: revised 26 September 1995; accepted 28 September 1995
Abstract The rates of X-ray induced loss of chromosome 19 in mouse oocytes were investigated in 2 experiments using a genetic complementation test. After I Gy of acute X-rays to immediately preovulatory stage oocytes, chromosome 19 loss was estimated to have occurred in 1.68% of cells. In comparison, after 4 Gy of acute X-rays to dictyate stage oocytes, the rate was estimated at I. 18%. The slightly higher rate of chromosome loss in the former cell stage after a smaller dose of radiation reflects the known increased radiosensitivity of mouse oocytes in the period shortly before ovulation. Comparison of the observations here for chromosome I9 with published data for chromosome I suggests that chromosome length is one of the principal factors in determining the initial rate of induced loss in mouse oocytes. Ten dominant visible mutations were recovered among 1674 offspring following irradiation of preovulatory oocytes, and 8 in 2025 offspring after treatment of dictyate cells. Nine dominant mutations were karyotyped, 5 of these were found to be associated with a visible chromosome rearrangement. The data obtained in the present study show that radiation-induced chromosome anomalies in female germ cells are not all filtered out by prenatal embryonic death but that a proportion has the potential to contribute to the genetic burden of the next generation. Krvvordst
Mouse oocyte; X-rays; Chromosome loss; Dominant mutation
1. Introduction The various cytogenetic methods available for investigating chromosome segregation errors in mammalian germ cells have long been acknowledged to have an inherent weakness when used for
x Corresponding author.
0027-5107/96/$15.00 SSDI 0027-5
0
1996 Elsevier Science B.V.
107(95)00183-2
analysis of chromosome loss events. The principal problem relates to the difficulty of distinguishing chromosome loss resulting from a segregation error from that produced artefactually during cell preparation for microscopy. Cytogenetic analyses are therefore generally more appropriate for assessment of chromosome gain, and other approaches have had to be developed to study spontaneous or induced chromosome loss in germ cells. Initially, a procedure to
All rights reser,ved
assess sex chromosome aneuploidy in mice was devised by Russell (1976); subsequent developments have produced genetic assays for detecting autosomal chromosome loss. Detailed descriptions of the principles of these latter tests have been provided by various authors (e.g., Cattanach et al.. 1984; Lyon, 1984; Tease and Cattanach, 1986). In brief. the assays make use of nullisomy. spontaneous or induced. for a particular chromosome in the gametes of one parent being complemented at fertilization by the appropriate disomy in the gametes from the other parent. To elevate the rate of disomic gametes and thus increase the probability of complementation, mice heterozygous for one or more Robertsonian (Rb) translocations are employed. The use of marker genes allows easy identification of any offspring that result from such complementation. Cattanach et al. (1984) undertook a chromosome loss experiment in which female mice with a normal acrocentric karyotype were given an acute dose of X-rays in the range I to 4 Gy and mated to males heterozygous for Rb(l.3)lBnr. Chromosome I was marked in tester males with the recessive genes fuzzy (,fk> and leaden (In) to enable identification of progeny resulting from complementation of induced chromosome 1 loss in oocytes by Rb-mediated chromosome I nondisjunction in male meiosis. A linear, dose-related increase in the incidences of marked young was found. Cattanach et al. (1984) used an estimate of the proportion of sperm disomic for chromosome I to calculate the probability of complementation and thus the initial proportion of oocytes that suffered induced chromosome I loss. They concluded that 4 Gy of X-rays induced loss of chromosome 1 in approx. 3% of oocytes. This experiment therefore not only showed the efficacy of the method but also demonstrated a surprisingly high incidence of chromosome loss after X-irradiation. In addition to effects on chromosome segregation, Cattanach et al. (1984) also reported the occurrence of a few offspring carrying dominant visible mutations. Such mutations are regularly observed in radiation experiments and have been of value for making genetic risk estimations for the human population (Searle, 1974: Searle and Beechey, 1986: Selby. 1990). Recently, Cattanach et al. (1993) showed that dominant visible mutants found in mutagenesis experiments were frequently associated with chromo-
some aberrations particularly deletions. Subsequent analyses have also shown that mutants whose principal phenotypic effect was growth retardation can carry chromosome aberrations (Burtenshaw et al., 1994; Evans et al.. 1995). This observation is of interest in view of the suggestion of Searle and Beechey (1986) that growth retardation might be a phenotypic category meriting attention in mutagenesis experiments; the results from the recent cytogenetic analyses would appear to provide support for their suggestion. In a previous paper (Tease and Fisher, 1996). the effects of X-rays on mouse oocytes were assayed using various cytogenetic methods to analyse germ cells and embryos at different stages of development, The work reported here continues and extends this analysis. firstly by evaluating induced chromosome loss using a genetic method and secondly, by screening progeny for dominant visible mutations. Some of the animals falling into the latter category were investigated further by detailed karyotypic analysis of G-banded chromosomes. The data generated from these experiments complement those from the cytogenetic studies. and together they provide a comprehensive description of the types of chromosome damage initially present in mouse oocytes after irradiation and of the frequencies and categories of genetic anomalies transmitted to the F, progeny.
2. Materials
and methods
In the present study, induced loss of chromosome 19 was examined in female mice with a normal. acrocentric chromosome complement. The tester males had monobrachial homology (MBH) for 2 translocations. Rb(8.19)ICt and different Rb(9.19)163H. In part, use of chromosome 19 was a pragmatic choice dictated by the fact that few MBH combinations are male fertile and also the need to avoid use of chromosomes that show parental imprinting effects; in addition. however, this choice offered the possibility of comparing the relative responses of chromosomes of widely differing sizes as data for the largest chromosome of the mouse complement, chromosome 1, has already been published (Cattanach et al., 1984).
157
2.1. Experiment
I: Immediutely
preoLdutory
oocytes
and screening the phenotypes of the offspring. Suspected dominant mutations were tested for inheritance by mating to 3Hl animals and screening a minimum of 30 offspring for the novel phenotype. If the mating yielded fewer than 30 progeny, the mutation was regarded as incompletely tested. Some of the dominant mutations were further analysed by G-band karyotyping of lymphocyte or bone marrow chromosome preparations to screen for visible chromosome anomalies associated with the mutation. Spermatocyte preparations (Evans et al., 1964) were made from six randomly chosen males of the MBH stock. The cells were C-banded (Sumner, 1972) and chromosome numbers counted in metaphase II spermatocytes to determine the rate of chromosome 19 nondisjunction from which the proportion of spermatozoa with chromosome 19 disomy could be estimated.
Female mice of the F, hybrid type C3H/HeH X IO 1/H (abbreviated to 3H1) were induced to ovulate using 2 IU of pregnant mares serum gonadotrophin (PMS) followed 48 h later by 2 IU of human chorionic gonadotrophy (HCG).They were given 1 Gy of X-rays 3 h after HCG injection and then mated overnight to MBH males carrying Rb(8.19)lCt and Rb(9.19) 163H. and also homozygous for the chromosome 19 marker gene ruby eye (nl). The following morning the females were inspected for the presence of a vaginal plug as indication of successful mating, and then separated from the males. Pregnancy was allowed to go to term and liveborn offspring screened for the presence of homozygosity for t-14 as occurs when induced loss of the maternal chromosome 19 is complemented by the presence of two paternally-derived chromosomes 19. The offspring were also screened for the presence of dominant mutations affecting external appearance. 2.2. Experiment
3. Results
2: Dictyate oocytes
3. I. Chromosome
The results from Experiment 1 are presented in Table 1. Overall, 1603 offspring from unirradiated females and 1674 offspring from females given 1 Gy of X-rays were screened. No ru exceptions were found in the unirradiated, control series whereas 6 animals with this phenotype were found in the irradiated group. All 6 animals appeared to possess the karyotype expected of complementation, that is, 36 acrocentric and 2 Robertsonian metacentric chromosomes. However, one of the t-u exceptions produced a wild-type mouse when tested in the backcross. As
3Hl females were given 4 Gy of X-rays and mated to males of the same genotype as above. The matings were continued for 4 weeks, at which time the males were removed. As before, offspring were screened for nl exceptions and possible dominant mutations. was made Confirmation of the ru classification initially by chromosome analysis of cultured lymphocytes to show the presence of the 2 patemally-derived Robertsonian chromosomes in diploid cells, and then by backcrossing to ru homozygous mice Table
loss
I
X-ray induced loss of chromosome Dose(Gy)
Experiment
Interval
19 in mouse oocytes No. of
I.II/YLI exceptions
No. of
Mean litter
offspring
(c/r)
litters
size
1 286
5.60
415
3.52
Control
_
1603
0
I Experiment
0.5 days
1673
6
Control
_
1510
1 (0.07)
232
6.51
4
<
I463
3 (0.21)
253
5.78
4 Total (irrad)
(0.36)
2
I week I-4 weeks
562
2 (0.36)
172
3.21
2025
5 (0.25)
425
4.76
Table 2 The proportions of metaphase II spermatocytes with different chromosome constitutions in 6 male mice with monobrachial homology for Rb(8.19)l Ct and Rb(9.19Jl63H % cells with chromosome
complement:
I7+Rb+Y
17+Rb+Xl8+Y
lX+Xl6+2Rb+Y
16+2Rb+X
34.2 ,’
34.0 h
10.3
7.0 d
Sample included: cells. I6+Rb+X: l.++Rb+X
7.2
7.3 i
‘I 6 cells, I6 + Rb + Y: 2 cells. I8 + Rb+ Y. h 4 I cell, 17+Rb. ’ I cell. IS+?Rb+Y. ” I cell.
the breeding test showed this latter animal was not a product of complementation, the simplest explanation is that it was a trisomic conceptus that survived because of mosaicism following loss of the acrocentric chromosome 19 in some cell populations during embryonic development. This ru exception was not included in the subsequent attempt to calculate the initial incidence of chromosome 19 loss in X-irradiated oocytes. The rate of chromosome nondisjunction in the tester males was determined in the present study through analysis of metaphase II spermatocytes in 6 animals (Table 2). There was no indication in the data of significant variation between males in the frequency of aneuploid cells. Thus there is no reason to suspect that in the experimental matings the probability of complementation might vary between the different males used in the actual test crosses. From this analysis, an estimated 14.3% of spermatozoa produced by MBH males are anticipated to be disomit for chromosome 19. Cattanach et al. (1984) developed a statistical method through which to determine the proportion of germ cells with induced chromosome loss. This method exploits knowledge of the rate of chromosome nondisjunction in germ cells of the tester parent. Using this statistic it is possible to estimate the proportion of cells with induced chromosome loss that would be necessary to account for the observed rate of complementation. From the estimate of 14.3% of disomic sperm in the present study, it was calculated that approx. 1.68% (9.5% confidence limits, 0.64 and 3.39) of oocytes must have undergone induced loss of chromosome 19 to account for the observed rate of 0.309 YCIexceptions in Experiment 1. The progeny of irradiated females in Experiment
2 are separated in Table 1 into those born within 28 days of irradiation, and therefore presumed to be conceptions in the first week after treatment, and those born in later weeks. This was done to determine whether the variability in dictyate oocyte sensitivity to radiation-induced chromosome damage at different stages of maturation (Brewen et al., 1976) could also be discerned using this genetic method. Overall, 1 ru exception was found in the progeny of control females and 6 among those of irradiated females. As above, ru exceptions were tested both cytogenetically and genetically and one animal from the irradiated series was found to be mosaic; again this animal was not included in the analysis. The frequency of ru exceptions was 0.2 I c/oin the first week and 0.36% for the remainder of the sampling interval (Table 1). The difference is clearly nonsignificant given the relatively small number of ru exceptions that were found. nevertheless, the occurrence of a higher rate at the longer sampling intervals is in line with the previous description of a greater radiosensitivity of these cells for induced chromosome damage compared with oocytes within a few days of ovulation (Brewen et al., 1976). Using the approach of Cattanach et al. (1984), an overall estimate was obtained of 1.I 8% (95% confidence limits 0.40 and 2.59) of oocytes with induced loss of chromosome 19 as a consequence of X-irradiation. 3.2. Dominant
mutatiorls
A number of phenotypically deviant mice were found among the litters of control and irradiated females from both experiments and where possible these animals were tested for inheritance of the novel phenotypes (Table 3). The most common category recorded was that of small body size. This classification is obviously somewhat subjective but in an attempt to introduce an element of objectivity it was decided to categorise as small any animal that weighed less than 75% of the average litter weight on the basis of previous reports (Kirk and Lyon, 1982; Searle and Beechey, 1986). Clearly, this approach was only of value where litter sizes were large enough to provide a meaningful average; where litter sizes were small, the element of subjectivity remained in the classification.
C. Tease, G. Fishrr/Mutation
Table
IS9
3
The numbers
and categories
of mice identified
Category
Inherited
Treatment group Experiment Control
Experiment Control
as phenotypically
abnormal
Not inherited
and the results of breeding Trisomy mosaic
tests
Sterile
Died, untested
Total
20 0 1 35 1 8
29 4 5 60
2
5
1 small tail kinks other small tail kinks other
l CY
ICY
Research 349 (19961 155-162
I7
2 small tail kinks small tail kinks other
A total of 29 animals from unirradiated females in Experiment 1 and 5 from Experiment 2 were classified as small. Only 4 animals from the former experiment were tested for fertility, the remainder died either pre- or post-weaning, or failed to breed. None of the small animals that were tested from control females transmitted the phenotype. There is an interesting discrepancy in the proportions of animals from control mothers that were recorded as small in the 2 experiments. It may be that the use of hormones to induce ovulation in Experiment 1 increased the chance of small embryos surviving to term or that the risk of small size was increased because of uterine conditions being less optimal following induction of ovulation without regard to the normal fertility cycle. For irradiated females, an increased proportion of animals were classified as small and some of these were shown to be heritable (Table 3). Inheritance of the phenotype was proved in 4/ 12 animals in Experiment 1 and 2/9 animals in Experiment 2. In addition to animals that were classified as small, a variety of other phenotypically deviant mice were found (Table 3). These ranged from minor effects such as slight tail kinking through to more extreme effects such as syndactylism. In all cases, the mutant animals were somewhat smaller than phenotypically normal sibs. In controls of Experiment 1, 8/ 9 potential mutants proved not to be heritable, the other died before mating; in the irradi-
0
0
0
0
3 26 7 10
15 0
0
0
4
ated females, 5/ 14 tested proved heritable, 10 others gave no result (Table 3). A similar situation prevailed in Experiment 2 with no proved dominant mutations in the control group, and 6/l 1 tested in the irradiated group being inherited, with 6 others
Table 4 Phenotypes of the mutations selected for karyotypic analysis and the outcome of the cytogenetic investigation (NF, no rearrangement found) Phenotype
Experiment 1 Small with eye nomalies Small with eye anomalities Brachyury Domed head Syndactyly Experiment Steeloid
Chromosome rearrangement
Reference
Deletion, Chromosome Deletion, Chromosome Deletion, Chromosome NF NF
Tease and Fisher, 1993b Tease and Fisher, 1993b 13 Fisher and Tease, I992 17
2
Small with eye anomalies Small with tail kinks and white spotting Dark footpads and genetalia
Deletion, Chromosome Duplication, Chromosome NF
NF
Tease and Fisher, 1993b 10 Tease and Fisher, I994 16 Tease and Fisher. l993a
giving no result (Table 3). Detailed karyotypic analyses were performed on mutants whose phenotypic effects were greater than simply size reduction or slight tail kinking. From the 2 experiments. 9 mutants were screened and in 5 a visible chromosome rearrangement was found to be associated with the phenotypic effect (Table 4). A chromosome I deletion was also found in a sterile male from Experiment I (Table 3) that had a white head spot and a white patch on its thorax (Tease. 1993). Another interesting chromosome anomaly was recovered in one of the r~l exceptions of Experiment I. When this animal was screened for the presence of the 2 Robertsonian translocations, a very small supernumerary chromosome was also seen to be present in the cells. This microchromosome has proved to be heritable and has no overt effect on external phenotype (Tease and Fisher. in preparation).
4. Discussion X-ray induced chromosome damage has been studied previously in preovulatory and dicytate stage oocytes of 3H I female mice. Tease and Fisher ( 1986) reported that structural chromosome aberrations were present in metaphase I oocytes at a rate of I .28/ cell after I Gy to preovulatory stage oocytes treated similarly to the present study. A subsequent investigation yielded aberration induction rates of 0.26,’ cell and O.lO/cell after a 4 Gy treatment of dictyate stages 16.5 days and 3.5 days, respectively. before ovulation (Tease and Fisher, 1991). If these induction rates are transformed to a per Gy basis to facilitate comparison of the relative sensitivities of the different oocyte developmental stages. then preovulatory oocytes are seen to yield approx. 5 to I3 times more aberrations than the 2 dictyate stages. The present study has produced 2 measures of X-ray induced genetic damage in mouse oocytes, firstly, the rate of chromosome 19 loss. and secondly. the incidence of dominant visible mutations. For the former, the respective rates in preovulatory and dictyate oocytes were 1.68% and 1.18%. Again, if the rates are transformed to a per Gy basis. then the frequency of chromosome I9 loss was approx. 6 times greater in preovulatory than dictyate oocytes. With regard to dominant visible mutations, IO were
found in the 1674 mice screened in Experiment I and 8 in the 2025 mice of Experiment 2. These figures translate into rates of 5.97 X IO-’ and 9.88 X IO-’ per Gy, respectively, which again indicate that preovulatory oocytes were approx. 6 times more sensitive to induction of dominant mutations than the combined dictyate stages. Both measures of genetic damage in the present study therefore fell within the range obtained by comparing the outcomes of a cytogenetic analysis. Obviously this comparison is limited by the need to combine results of dictyate stages which are known to vary in their radiosensitivity (Brewen et al.. 1976; Tease and Fisher. 1991). Nevertheless, it is of interest that the various assays, with their widely differing endpoints, all tend to give a reasonably similar estimate of the comparative sensitivity of preovulatory and dictyate oocytes. The only previous study in mouse oocytes using this genetic complementation assay was carried out by Cattanach et al. ( 1984) who screened for induced loss of chromosome I. They estimated that 4 Gy of X-rays to dictyate stage cells of 3Hl females caused chromosome I loss in approx. 3.5% of oocytes. By comparison, in the present study the same dose of X-rays to oocytes at a similar stage of maturation yielded a rate of chromosome I9 loss of I. 18%. If the rate of induced chromosome loss is correlated with the size of the target chromosome then obviously a higher rate would be expected for chromosome I compared with chromosome 19. Given that chromosome I9 is approx. 40% of the length of chromosome I, then from Cattanach et al.‘s (1984) data an expectation of 1.4% of cells with chromosome 19 loss is generated. The observed rate does not differ dramatically from that expected. Overall. the present study confirms the initial finding of Cattanach et al. (1984) that X-irradiation can induce chromosome loss in a surprisingly high proportion of cells. Comparison of the data in the 2 studies indicates that chromosome length is an important factor in determining the initial rate of induced chromosome loss. Searle and Beechey (1986) argued for the inclusion of growth retardation as an informative category of dominant visible mutation following parental exposure to radiation. Following X-irradiation of spermatogonial stem cells, they showed that approx. 40% of the offspring that had been classified as small
C. Tetrse. G. Fisher/Mututim
transmitted the growth retardation phenotype. Our data are similar with 7 out of 1.5 fully tested smalls in the 2 experiments proving heritable. However, as Searle and Beechey ( 1986) also acknowledged, testing for heritability of growth retardation poses problems as many of the animals either fail to survive to weaning or result in incomplete testing, as was also the case here (Table 3). An additional complicating factor in the present work was the presence of mice which proved upon cytogenetic investigation, or through breeding, to be mosaic for trisomy 19 (Table 3). It is not certain what proportion of the small mice that died before testing were also mosaic. Nevertheless. the data in the present study supports the suggestion of Searle and Beechey (1986) that, following parental X-irradiation, a substantial proportion of the F, animals classified as small have a genetic basis for their growth retardation Recently, Cattanach et al. (1993) found that a large proportion of dominant visible mutations were associated with a visible chromosome aberration, most often a deletion. Further studies have confirmed and extended this initial report to show that deletions or duplications of practically all members of the chromosome complement could be found after exposure of male germ cells to X-rays (see Beechey, 1995). Cytogenetic analysis of some of the dominant mutants recovered in the present experiments has also revealed the presence of chromosome aberrations (Table 4). Karyotypic analysis of 14 day postimplantation embryos from female mice treated as in Experiment 1 showed that approx. 13.6% of the embryos had a visible chromosome anomaly (Tease and Fisher, 1996). What proportion of these complete gestation and give rise to dominant visibles is unknown but the present work provides clear evidence that a significant fraction are capable of achieving full term. Moreover, it is a reasonable supposition that many of the animals with putative dominant visible mutations but which died in the pre-weaning or early post-weaning period (Table 3), carried some form of chromosome damage. It is clear from these experiments that not only does a significant proportion of germ cells with X-ray induced chromosome damage contribute to the genetic burden of the next generation, but there is also a strong likelihood that they are a causative factor in premature death in the immediately postnatal period.
Resrnrch
349 (I9961
155-162
161
A contrary conclusion was advanced by Reichert et al. (I 984) from their study of radiation-induced chromosome anomalies in post-implantation mouse embryos. They were unable to detect such rearrangements and therefore suggested that conceptuses with induced chromosome aberrations must invariably fail to survive to parturition. The cytogenetic data described in our earlier paper (Tease and Fisher, 1996) in combination with the observations here clearly show that the conclusion of Reichert et al. (1984) is incorrect. Rather, by extrapolation from our data, we suggest that any dose of radiation that is capable of causing structural chromosome rearrangements in female germ cells must inevitably also present a significant risk of heritable chromosome aberrations to the next generation.
Acknowledgements We thank Peter Adams for carrying out the irradiations, David Papworth for the statistical calculations, and Dr Bruce Cattanach for his comments on the manuscript. This work was supported in part by Euratom contracts Bi6- I43 and F 13P-CT920005.
References Beechey, C.V. (1995) Maps of chromosome anomalies in the mouse. Mouse Genome, 93, 67-88. Brewen. J.G., H.S. Payne and R.J. Preston (1976) X-ray induced chromosome aberrations in mouse dictyate oocytes. I. Time and dohe relationships. Mutation Res.. 35. I 1 I- 120. Cattanach. B.M., D. Papworth and M. Kirk (1984) Genetic tests for autosomal non-disjunction and chromoome loss in mice, Mutation Res., 126. 189-204. Cattanach, B.M.. M.D. Burtenahaw, C. Rasberry and E.P. Evans ( 1993) Large deletions and other gross form5 of chromosome imbalance are compatible with viability and fertility in the mouse, Nature Genet., 3. 56-61. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations for mammalian testes, Cytogenetics. 33. 289-294. Fisher G. and C. Tease (1992) T’5H: a radiation-induced deletion affecting the T. qk and Tme loci. Mouse Genome 90, 209-2 10. Kirk. M. and M.F. Lyon (I 982) Induction of congenital anomalies in offspring of female mice exposed to varying doses of X-rays, Mutation Res.. 106, 73-83. Lyon. M.F. (1984) The use of Robertsonian translocations for
studies of nondisjunction. in: T. laihara (Ed.). Radiation-Induced Chromosome Damage in Man. Alan R. Liss, New York, pp. 321-346. Reichert, W., W. Buselmaier and F. Vogel (1984) Elimination of X-ray-induced chromosomal aberrations in the progeny of female mice, Mutation Res., 139, 87-93. Russell. L.B. (1976) Numerical sex chromosome anomalies in mammals: their spontaneous occurrence and use in mutagenesis studies. in: A Hollander (Ed.), Chemical Mutagenesis. Vol. 4. Plenum Press. New York. pp. 55-91. Searle. A.G. (1973) Mutation induction in mice. Ad\. Radiat. Biol.. 4, 131-207. Searle. A.G. and C. Beechey (1986) The role of dominant visihles in mutagenicity testing. in: C. Ramel. B. Lambert and J. Magnusson (Eds.), Genetic Toxicology and Environmental Mutagens. Alan R. Liss, New York, pp. 51 I-518. Selby. P.B. (1990) Experimental induction of dommant mutations in mammals by ionizing radiations and chemicals, Issues Rev. Teratol.. 5. I8 I-253 Sumner. A.J. (1972) A simple technique for demonstrating centromeric heterochromatin, Exp. Cell Res.. 75, 304-306. Tease. C. (1993) A cytogenetically visible, radiation-induced deletion on chromosome I. Mouse Genome. 9 I, I IS.
Tease, C. and B.M. Cattanach (1986) Mammalian genetic and cytogenetic tests for non-disjunction, in: F.J. de Serres (Ed.), Chemical Mutagens, Vol. IO. Plenum Prcsh. New York. pp. 21.5-283. Tease. C. and G. Fisher (1986) X-ray-induced chromosome aherrations in immediately preovulatory oocyte\. Mutation Res., 173. 21 l-215. Tease. C. and G. Fisher (I991 1The influence 01‘maternal age on radiation-induced chromosome aberrations in mouse oocytea. Mutation Rea.. 262. 57-62. Tease. C. and G. Fisher (1993a) Tail kinks. small size, and white spotting (Tks): a radiation-induced mutation located on chromosome 9. Mouse Genomc, 9 I. 137-13X. Tease. C. and G. Fisher (1993b) Cytogenetic detection of four new. viable deletions in the progeny of X-irradiated females. Mouse Genome, 9 I. 855. Tease, C. and G. Fisher (1994) Presumptive duplication\ after X-irradiation of females, Mouse Gcnome, 92. 3.50 Teaae C. and G. Fisher Cytogenetic and genetic studies of radiation-induced chromosome damage in mouse oocytcs. 1. Numerical and btructul-al chromosome anomalies in metaphase II oocyteh. pre- and post-implantation cmbryoh. Mutation Res.. thih i\\ue.