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MUTATION STUDIES UPON SPERMATOGONIAL STEM CELLS OF MAMMALS AND GENETIC TESTS FOR NON-DISJUNCTION IN THE MOUSE

Objective

This project is a continuation of Contract Number B16*0173 (Dr C Tease), Karyotypic analysis of spontaneous and radiation-induced chromosome anomalies in mouse foetuses, which has been combined with BI6*0143.

The aim of the present work is to examine further the various factors that influence the response of mouse oocytes to radiation-induced genetic damage. Such information should prove of value when using data from female mice for human genetic risk estimations.
Studies upon strain differences in genetic response to radiation may facilitate extrapolation of mouse data to man. The 101/H strain gives a different response from other mice tested. The basis of this difference is being studied.

The results of a 3 + 3 Gy study have shown that 101/H males had far longer sterile periods than the standard hydrid indicating higher levels of stem cell killing, but the specific locus mutation response was not significantly different.

Whether the stem cells remaining in the testis 24h after population depletion by X-rays or chemical agents are uniformly sensitive to genetic damage and cell killing and wherther the stem cells present in the testis 4 or more days after population depletion are in the process of repopulating the germinal epithelium are both questions that have been answered by giving hydroxyured (HU) pretreatments (500 + 500 mg/kg, 3h interval) 3h prior to 6 Gy X-irradiation. Population depletion was brought about by TEM treatment (2 mg/kg). Translocations were the genetic end pooint, and cell killing was assessed by reduction in recovered testis weight. Comparison of treatment groups show that the HU pretreatments enhanced cell killing 24h after population depletion suggesting that not all cells were in a sensitive non-dividing state but that some cells were in the S phase and therefore subject to killing by HU. However, no associated increase in the translocation yield was observed. The TEM + X-ray 24h treatment gave a much higher yield than obtained in prrevious studies. Therefore, an heterogeneity may, overall by indicated. Comparison of groups also show that the HU enhanced the level of cell killing 4 days after population depletion, again indicating that a substantial proportion of cells must be undergoing division. In this instance an enhanced translocation yield was obtained consistent with enrichment of G1 phase cells that are sensitive to translocation-induction by X-rays.

Research has been carried out into the development of 2 systems of genetic (complementation) tests using Robertsonian (Rb) translocations in tester animals to detect nondisjunction and chromosome loss events in normal mice. The 2 methods are being evaluated for detecting chromosome 11 loss, with 1 method being used to compare the frequency of chromosomes 11 and 13 loss following X-irradiation of males and females.

The Rb tester method, used to detect chromosome 11 and 13 loss following X-irradiation o chromosomally normal males or females, employed the Rb(11.13)4Bnr translocation carried heterozygeously in the tester animals. The chromosome 11 and 13 markers were estigial tail (vt) and satin (sa), respectively. Following irradiation with 4 Gy X-rays the normal wild type animals were mated for 1 week to the tester mice and the progeny screened for the vt and sa young, these indicated chromosome 11 and 13 loss events. The same mating regime and X-ray dose was used with monobrachial homology (MBH) tester method but here the tester animals carried both the Rb(11.13)4Bnr and RB(10.11)8Bnr translocations heterozygously. Chromosome 11 loss again was detected using vt as the marker.

Chromosome 11 loss was detected in all 4 types of cros but the frequencies were generally lower than found with several other chromosomes previously tested. In females at least, the MBH tester system appears to be more effective than the Rb tester system for detecting chromosome 11 loss. Difficulties were encountered in breeding from Rb tester females, necessitating reestablishment of the stock on a more vigorous background. This has now been achieved and the experimental work is in progress.

An understanding of factors that influence the response of mouse oocytes to radiation induced genetic damage may assist human risk assessment. It is not known whether a strain dependent variability occurs with female germ cells, as found in males. A study has therefore been initiated to compare the incidence of structural chromosome anomalies following X-irradiation of oocytes in 2 inbred strains. Female mice of the inbred strains C3H/HeH and 101/H were given either 2 or 4 Gy of acute X-rays. 14 days after treatment the females were induced to ovulate and metaphase I stage cells collected for assessment of induced chromosome damage. Age matched unirradiated females were used as controls. The slides prepared from control and irradiated females were coded and randomized prior to analysis. As analysis is not yet complete, the experiment has not been decoded.

Female mice of the F1 hybrid type C3H/HeH x 101/H were induced to ovulate and 3 h after human chorionic gonadtrophin (HCG), when the oocyts were at diakinesis of meiosis, they were given 1 Gy of acute X-rays. The females were mated overnight to males of the genotype Rb(8.19)1Ct ru/ Rb(9.19) ru and their offspring screened for the ru phenotype which occurs when maternal loss of chromosome 19 is complemented by paternal gain. The data clearly demonstrate the ability of X-rays to induce chromosome 19 loss in immediately preovulatory oocytes. The rate of chromosome loss is greater than that found in earlier experiments where dictyate stage oocytes were given 4 Gy of X-rays. This trend is consistent with the expectations of cytogenetic analyses which have shown that immediately preovulatoryoocytes are considerably more sensitive to radiation induced structural chromosome anomalies than cells at earlier stages of germ cell development.
Firstly, the influence of mouse strain on oocyte radiosensitivity is being investigated. Two inbred strains, C3H/HeH and 101/H, are being used initially; information from F1 hybrid females between these strains is already available for comparison. The animals are being given either 2 or 4 Gy of X-rays and the effect of the treatment is being assessed by analysis of metaphase I stage oocytes for chromosome aberrations.

Secondly, induced loss of chromosome 19 is being examined using a genetic complementation test. Female mice are being given 1 Gy of X-rays a few hours prior to ovulation; germ cells are known to be extremely radiosensitive at this point of development. The induced incidence of chromosome 19 loss can be estimated from the frequency of genetically marked offspring resulting from complementation. This information will be useful for :
assessing the importance of chromosome size as a factor influencing induced genetic damage;
comparing the radiosensitivities of oocyte at different stages of follicle development;
determining the relative sensitivities of different assays to detect radiation-induced genetic damage in oocytes.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Medical Research Council (MRC)
Address
20 Park Crescent
W1N 4AL London
United Kingdom