A.G. Searle

STUDIES ON THE INDUCTION OF TRANSLOCATIONS IN MOUSE SPERMATOGONIA. I. THE EFFECT OF DOSE-RATE.

Abstract 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. Frequencies of 0, 1, 2 translocations per spermatocyte did not fit a Poisson distribution since there were less then expected in the 1-class, but more in higher classes. This was probably a consequence of differential radiosensitivity of the irradiated spermatogonia, although preferential clonal proliferation may also be involved.

A comparison of the mutagenic effectiveness of chronic neutron- and γ-irradiation of mouse spermatogonia

Abstract Hybrid male mice were given a dose of 214 rad fast fission neutrons (plus 93 rad γ-contamination) over twelve weeks, while brothers were given 606 rad γ-radiation (plus 2.5 rad neutron contamination) over the same period. Six weeks later they were mated to females homozygous for recessive alleles at seven specific loci. Neutron-irradiated mice remained sterile for a further four weeks but γ-irradiated mice showed no sterile period. Testis weight fell to less than 20% during neutron irradiation, but to about half normal during the γ-irradiation. The frequency of specific locus mutations was 22.9 · 10 −5 /locus in the neutron series and 3.9 · 10 −5 /locus in the γ-series, from which induced mutation rates of 100.6 · 10 −8 /rad for neutrons and 4.4 · 10 −8 /rad for γ-rays were derived, after allowing for spontaneous mutations. Thus the RBE for neutrons is about 23 in these circumstances. A figure of about 20 was arrived at by analysing data on dominant visible mutations. There were indications that chronic neutron exposures were also much more effective than chronic γ for the induction of reciprocal translocations and other chromosomal changes. However, the sex ratio was very similar in the two series. Most of the dominant and recessive mutations tested proved to be homozygous lethals; there were also signs that carriers of newly arisen specific locus mutations were more likely to die before weaning age than non-carriers. So the true specific locus mutation frequencies are probably higher than those given here.

The effect of hypothermia on the induction of chromosomal mutations by acute x-irradiation of mice.

Abstract Small mammals can survive cooling to o°, at which temperature their tissues are virtually anoxic. Thus hypothermia provides a method for studying the role of oxygen tension differences in determining genetic radiosensitivity of different mammalian germ-cell stages. Male mice were given 600 rad acute X-irradiation when hypothermic or at normal body temperature. In mice irradiated when hypothermic the yield of dominant lethal mutations from treated spermatozoa was slightly but not significantly reduced, while the incidence of cytologically identifiable reciprocal translocation configurations in primary spermatocytes derived from irradiated A type spermatogonia was halved (8.3% instead of 17.1%). The usual post-radiation sterile period after 600 rad was abolished in the males made hypothermic and the testis weights showed less reduction than in those given X-rays alone. This evidence for the protection of spermatogonia from somatic and genetic radiation injury by hypothermia shows that oxygen tension differences are important in determining the extent of both types of damage in these immature germ cells. With mature germ cells, however, these and previous findings agree in showing that hypothermia or other methods of oxygen deprivation provide little if any protection from genetic radiation damage to spermatozoa, presumably because they are normally hypoxic.

The ineffectiveness of chronic irradiation with neutrons and gamma rays in inducing mutations in female mice

Abstract Hybrid female mice were exposed to 412 rads 60Co γ rays or 80 rads fast neutrons (+ γ contamination) in 12 weeks and the amount of genetic damage in offspring was compared with controls. Frequencies of specific locus recessive and of dominant visible mutations were very low in the irradiated series, differing little from the control frequencies of nil. There were no significant differences between the sex-ratios of offspring in the three series, thus no evidence for the induction of sex-linked lethal mutations. No estimates can be given of the relative genetic effectiveness of chronic neutron and γ exposures in female mice, but the specific locus mutation frequency after the neutron dose was less than 5 per cent of that obtained in male mice after spermatogonial irradiation with a similar dose over the same 12-week period. It is now clear that both acute and chronic exposures of the immature mouse oocyte to both high and low LET radiations result in minimal genetic damage. It is still not clear w...

A test for mutagenicity of caffeine in mice

Summary1.The results are given of an experiment comparing the specific locus mutation-rates in male and in female mice having 0.1% caffeine dissolved in their drinking-water up to the age of ten weeks. Their parents had the same treatment from the time of mating, so that the tested germ-cells might be exposed to caffeine during embryonic development.2.The mutation-rates in males and females did not differ significantly from each other, nor from the known spontaneous rate; thus there was no evidence for induction of mutations by the caffeine treatment. Neither was there evidence for the induction of dominant lethals following caffeine treatment of males.3.The treatment did not noticeably affect reproduction, but some mice developed aggressive tendencies towards their cage-mates. Some mice were kept on 0.1% caffeine throughout life: they continued to breed satisfactorily on the whole.

Genetic effects of spermatogonial x-irradiation on productivity of f1 female mice.

Abstract 1. (1) Hybrid male mice were given two doses of 600 R high intensity X-rays eight weeks apart and outcrossed 12 weeks after the second dose. Daughters were also outcrossed and effects on their productivity studied by obtaining data from their first 4 litters at birth and from their fifth at about the 14-day foetal stage. Controls had exactly the same treatment apart from the irradiation. 2. (2) The average number of liveborn per tested female fell by 4.8% from 26.0 in the controls to 24.7 in the irradiated series. The mean litter-size fell significantly by 5.5%. 3. (3) The main factor reducing productivity was inherited semi-sterility in 6.7% of daughters of irradiated males, with none in controls. When combined with data on sons of the same irradiated males this gives an estimated rate of induction of translocations in spermatogonia of 6.4.10 −5 /genome/R after two doses of 600 R. This is about one-sixth that reported for spermatozoal irradiation at similar single doses. It is suggested that this differences is mainly due to differential radio-sensitivity of spermatogonial mitotic stages, with only the most condensed stages having a similar sensitivity to that of spermatozoa. 4. (4) The sex-ratio at birth fell significantly from 52.0% in progeny of control daughters to 49.4% in the irradiated series. Other data, however, gave little evidence for the action of recessive sex-linked lethals, or teh presence of XO mice. It was concluded that the sex-ratio decrease was probably due mainly to other unknown causes and that a large-scale experiment with sex-linked markers is necessary if the rate of induction of sex-linked lethals is to be accurately determined. 5. (5) Dominant sub-lethality, sterlity or reduced fertility other than semi-sterility did not seem to play part in the reduction of productivity in daughters of irradiated males.

The mutagenic effectiveness of fast neutrons in male and female mice

Abstract Previous work has shown that protracted doses of fast neutrons induce high frequencies of specific locus mutations and of reciprocal translocations in mouse spermatogonia. However, they are mutagenically very ineffective when the dictyate oocyte is the stage at risk. The mutagenic response of similar germ-cell stages in the 2 sexes, namely the mitotically dividing primordial spermatogonia and oogonia with their precursors, has now been compared following irradiation for 1 week before the 12th day of embryonic life with a mean dose of 108.5 rad fast neutrons +20.5 R γ-contamination. The large clusters of specific locus mutations found in both series show conclusively t that mutations can be readily induced in female early embryonic germ cells as well as in male. Cluster size was used to estimate the mean number of germ cell at risk, from which mutation frequencies were calculated. These were 5.3·10 −5 per locus in males and 6.4·10 −5 per locus in females, from which induced mutation rates of 42·10 −8 and 58.10 −8 per rad respectively can be calculated. If dose attenuation is allowed for, higher figures are obtained. Those given are somewhat lower than the rate after protracted fast neutron irradiation of spermatogonia in the adult (109·10 −8 per rad) but higher than the mutation rates after low linear-energy transfer irradiation of spermatogonia and much higher than frequencies after protracted irradiation of dictyate oocytes. Air-dried preparations from testicular cell suspensions of 20 males irradiated as embryos were scored for presence of multivalent configurations indicating translocations. 2 had high and 2 had low frequencies of translocations; in addition, 1 mouse proved to be XYY and another had a clone of XO cells. The overall translocation frequency was 1.2%, which is lower than would be expected with irradiation of adult spermatogonia. It is concluded that the intrinsic radiosensitivity of male and female genetic material is similar in mice and that in the male these early embryonic stages are not much less sensitive than spermatogonia in the adult. It seems likely that the immature dictyate oocyte is the only germ-cell stage in the female from which mutations cannot be recovered.

Genetic Effects of High-LET Radiations in Mice

Despite pioneering work by Snell and Aebersold (1, 2) nearly thirty years ago, our ignorance of the genetic effects of high-LET radiations in mammals was until recently almost complete. Large-scale studies now in progress on the genetic effects of fast neutrons in mice are rapidly changing the situation, however, so that a fairly coherent picture of one aspect of this subject is already emerging. This picture is one of high genetic effectiveness under most, but not all, conditions of irradiation. In the present paper we shall give the latest results of our own work in this field, then shall discuss these results in conjunction with those of other workers. In this manner, we hope to arrive at an overall view of the present state of knowledge, especially with regard to the immature germ-cell stages which are most important from the standpoint of long-term risk to populations. Preliminary results of a comparison of the genetic effectiveness of chronic neutron and y-ray exposures have already been published (3), and a full account of this experiment will appear shortly (4).