EFFECTS OF DOSE ON THE INDUCTION OF DOMINANT-LETHAL MUTATIONS AND HERITABLE TRANSLOCATIONS WITH ETHYL METHANESULFONATE IN MALE MICE1

Transcription

1 EFFECTS OF DOSE ON THE INDUCTION OF DOMINANT-LETHAL MUTATIONS AND HERITABLE TRANSLOCATIONS WITH ETHYL METHANESULFONATE IN MALE MICE1 W. M. GENEROSO, W. L. RUSSELL, SANDRA W. HUFF, SANDRA K. STOUT AND D. G. GOSSLEE2 Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Manuscript received January 9, 1974 ABSTRACT Genetic damage by ethyl methanesulfonate (EMS) in male mice was measured at doses ranging from 50 to 300 mg/kg with dominant-lethal mutations and reciprocal translocations as endpoints. No appreciable increase in dominant-lethal mutations was detected following a dose of 100 mghg. Dominant lethals induced by EMS were convincingly detected only after a dose of 150 mg/kg, but in the translocation experiment an increase in the genetic effect was detectable at the 50 rng/kg dose. It is likely that dominant lethals had also been induced at the 50 and 100 mghg doses, but were no detected due to the relative insensitivity of the dominant-lethal procedure. Thus, for detection of low levels of EMS-induced chromosome breakage, translocations are a much more reliable endpoint than are dominant-lethal mutations. A procedure for large-scale screening of induced translocations is described.-the dominant-lethal dose-response curve, plotted on the basis of living embryos as a percentage of the control value, is clearly not linear as it is markedly concave downward. Similarly, the translocation dose-response curve showed a more rapid increase in the number of translocations with dose than would be expected on the basis d dosesquare kinetics. It is clear for both of these endpoints that the effectiveness of EMS in inducing chromosome breakage is proportionately much lower at low doses. INTRODUCTION EXPOSURE of human populations to chemicals occurs predominantly at chronic levels. For a proper evaluation of the genetic hazards to humans from chemicals, it is necessary to study in mammals the extent to which a variety of mutagenic chemicals differ in their efficacy at low, as well as high, doses. In other words, what are the shapes of the dose-effect curves for a variety of chemicals? In the case of mammalian germ cells, information on this question is meager. The present study is the first in a series to test certain known mutagenic compounds extensively over a range of doses. The study of chemical induction of chromosomal aberrations in the mouse is important in view of the many human genetic defects that result from various chromosome anomalies. So far, evaluation of chemica1;y induced chromosomai 1 Research jointly sponsored by the National Center for Toxicological Research and by the United States Atomic Energy Commission under contract with the Union Carbide Corporation. a Mathematics Research Staff, Computer Sciences Division, Union Carbide Corporation, Nuclear Division. Genetics 77: August, 1974.

2 742 w. M. GENEROSO et al. aberrations as genetic hazards has been made primarily from dominant-lethal tests in mice or rats and from cytogenetic studies of somatic and germ cells of certain mammals. Although useful, these two test systems do not measure transmissable genetic effects, and it is not known what their results mean in terms of hazards. Obviously, the most important mutagenic effects are the permanent, transmissable ones. Thus, there is a need to obtain information on heritable chromosomal aberrations that directly represent hazards. An important aspect of the present study is that it permits comparison of the effects of different doses of EMS on the induction of dominant lethals on the one hand and heritable reciprocal translocations on the other, and thus a direct cotmparison olf their relative efficiency in measuring induced chromosomal aberrations. MATERIALS AND METHODS Twelve-week-old (101 x C3H)Fl male mice were injected intraperitoneally with either a 50, 100, 150, 200, 250, or 300 mg/kg dose of EMS in Hanks balanced salt solution (HBSS). Control mice were given a comparable volume of HBSS. The frequencies of dominant-lethal mutations were measured in germ cells treated as early spermatozoa and late spermatids, while heritable translocations were measured only in germ cells treated in the early spermatozoa stage. In the dominant-lethal experiment, males were mated with (101 x C3H)F, virgin females during days after injection with EMS. The females were killed during pregnancy for uterine analysis. In the translocation experiment, males were mated with normal (SEC x C57BL)F, females during days after injection and F, male offspring were tested for translocation heterozygosity. Random-bred T-stock females were also used in limited numbers to produce F, progeny. F, males were tested for complete or partial sterility by caging each one with a IO- to 12- week-old (SEC x C57BL)F1 female that was allowed to produce at least four litters. The litters were examined soon after birth and discarded immediately. Since the majority of females mate immediately after parturition, the litters were spaced about 20 days apart. The criteria used for full fertility of F, males were arbitrarily drawn on the basis of previous knowledge of the fertility of normal (SEC x C57BL)F1 females (GENFXOSO, STOUT and HUFF 1971). All F, males were declared fully fertile if at least three litters out of four were comprised of 10 or more young, or if the total number of young for the four litters was 40 or more. F, males that were either sterile or exhibited fertility lower than the above criteria were further tested by mating each one to three (C3H x C57BL)F, females. These females were killed 17 days after putting them with the male. If none of the three females was pregnant, sterility of the male was confiied. If less than all three were pregnant, or if one or more females were in the early stages in pregnancy, in which classification of embryos is difficult, additional females were added. A minimum of three pregnancies was analyzed before a decision on fertility was made. The criteria used for full fertilitjr of F, males in this test was also drawn arbitrarily. These were based on the repm ductive nature of (C3H x C57BL)Fl females mated with normal (101 x C3H)F, males (Table 1). F, males were given at least three more females to have a total of six analyzed pregnancies if, among the first three pregnancies, (a) all had one or more dead implants, (b) two of the pregnancies had a total of four or more dead implants, or (c) at least one pregnancy had three or more dead implants. Otherwise, the test males were classified fully fertile an the basis of the three analyzed females. Where six pregnancies were analyzed, an unambiguous determination of partial sterility has always been possible. Males confirmed as partially sterile were progenytested and finally killed, the testes were weighed, and cytogenetic analysis of diakinesis and metaphase I spermatocytes was made. Sterile males were killed soon after confirmation. The two epididymides were examined for presence and quality of sperm and the testes were weighed. The testes of the first four sterile F, males were prepared for cytogenetic analysis. From this, it became apparent that cytogenetic analysis of EMS-induced sterility is very difficult, because of either very few diakinesis and

3 EMS DOSE EFFECTS ON ABERRATIONS TABLE 1 Reproduction data on normal (C3H x C57BL)F, females mated to normal (IO1 X C3H)F, males' 743 A. Live Total Number of embryos Number of Frequency per litter females (Per cent) B. Dead Total w.0 * Females were killed for uterine analysis 12 to 15 days after observation of vaginal plug. metaphase I cells or stoppage of spermatogenic development at earlier meiotic stages. Cytogenetic analysis of sterile males was, therefore, dropped in most cases. In a few instances where testes weight was normal and there was an abundance of sperm in the epididymis, the testes of sterile F, males were prepared for cytogenetic analysis. Male and female parents of sterile and partially sterile F, males in the controls and in the group exposed to an EMS dose of 50 mghg were progeny-tested to ensure against the possibility of preexisting translocations. At higher EMS doses, progeny-testing of parents was not deemed necessary because the incidence of induction was high. RESULTS EMS dose effect on the induction of dominant lethals: The induction of dominant lethal mutations at various doses of EMS was determined at spermatogenic stages that were found in previous studies to be most sensitive to EMS (EHLING, CUMMING and MALLING 1968; CATTANACH, POLLARD and ISAACSON 1968; GENE- ROSO and RUSSELL 1969), i.e., days after treatment. The 6.5- to 7.5-day period corresponds to treated young spermatozoa while days correspond mainly to treated late spermatids (OAKBERG 1960; OAKBERG and DIMINNO 1960), although there might be an admixture with treated young spermatozoa. The dominant-lethal effects of each EMS dose were calculated using the average number of living embryos as a percentage of control. As shown in the last two columns of Table 2, the average numbers of living embryos were calculated either on the basis of fertile matings only or on the basis of all mated females (fertile and sterile). For the lower doses (up to 200 mg/kg) where the frequencies of fertile matings were normal, the average number of living embryos based on

4 744 w. M. GENEROSO et al. pregnant females only gives a closer estimate of the true dominant-lethal effects. On the other hand, for the two high doses (250 and 300 mg/kg) where high dominant-lethal effects led to preimplantation embryonic death and consequent classification of the majority of mated females as sterile, the average numbers of living embryos based on all mated females obviously give a closer estimate. The induction of dominant-lethal mutations was generally similar at the two mating intervals throughout the EMS dose range used (Table 2). In both stages, the lowest dose that induced a detectable increase in dominant-lethal mutations was 150 mg/kg and the frequencies of dominant-lethal mutations were comparable at each dose, as indicated by significant reductions in the number of living embryos accompanied by increases in the frequency of dead implantations. The dose of 250 mg/kg appears to be the saturation point above which the dominantlethal-eff ect determination is no longer accurate. For the determination of the dose-effect curve, results for the two mating intervals were combined and the value calculated for the dose of 300 mg/kg was not included. The value used for the 250 mg/kg dose was obtained by taking all mated females (fertile and sterile) into consideration, while for all lower doses the values obtained on the basis of fertile matings only were used. As in the separate mating intervals, no appreciable increase in dominant-lethal mutations TABLE 2 Effect of EMS dose on the induction of dominant lethal mutations Living embryos as per cent of control Treatment-to- Number Number fntiliratiim EM s of of Total Living Dead Among Among interval dose mated fer!ile implants embry;s implants fertile all mated (days) (mg/kg) females matings (avg)* (avg) (Per cent) matlngs females e w $ 3.5 4' t C' t $ t 3.5t Combined ( ) " f t f * Per fertile matings. tp < $ P < 0.05.

5 EMS DOSE EFFECTS ON ABERRATIONS EO J 0 K + z 0 U ll tn 0 > K * 40 5 W z_ 2 J 20 0 I I I I I EMS DOSE (mg/kg) FIGURE 1.-Dose-response curve for EMS-induced dominant-lethal mutations. 95 % confidence intervals are indicated by vertical bars. can be detected at the dose of 100 mg/kg. Increase in induced dominant-lethal mutations was first detected at 150 mg/kg and thereafter the effect increased very sharply with dose. The dominant-lethal dose-response curve is clearly not linear-it is markedly concave downward (Figure 1 ). EMS dose efject on the induction of translocations: In contrast to dominantlethal mutations, a significant increase in the frequency of translocations was already detectable at the 50 mg/kg dose (Table 3), but like dominant lethals the TABLE 3 Effect of EMS dose on the induction of reciprocal translccations Number of translocations EMS dose Number of Partially Frequency (mg/kg) Fl males tested sterile Sterile Total (Per cent) ' w ' * The testes weight of this animal is 0.M grams. + P =

6 746 w. M. GENEROSO et al. frequency increased sharply and nonlinearly with dose. The translocation doseresponse curve (Figure 2) showed that there is a more rapid increase in the number of translocations with dose than would be expected on the basis of dosesquarekinetics (P forlackof fit, < 0.01, (~~(3d.f.) = 16.2). A total of 148 EMS-induced translocations were recovered. Of these, about onethird (50) were completely sterile and two-thirds (98) were partially sterile. The distribution of sterile and partially sterile translocations at each dose is given in Table 3. There are slightly more sterile than partially sterile F, males in the 50 and 100 mg/kg doses although the numbers are low and the differences are not significant. On the other hand, there were significantly more partially sterile than sterile males at the 150 and 200 mg/kg doses (P for difference < 0.01 in both doses). The great majority of male sterile translocations had distinctly small testes. Forty-eight out of 50 sterile males were so classified, and the 44 whose testes weights were obtained had weights of the two testes ranging from 0.03 to 0.12 gram (Table 4). Pourteerr of the steriles had very few sperm in the epididymis while in 34 no sperm were observed. Among those with few sperm, most of the sperm were nonmotile and in many cases a high frequency of sperm with bent tail or other abnormality was observed. The other two sterile F, males had normal testes weights (0.22 and 0.24 gram) and many normal-looking sperm were found in the epididymis. Cytogenetic analysis confirmed that these two males were translocation heterozygotes. / FIGURE 2.-Dose-response curve for EMS-induced reciprocal translocations. 95% confidence intervals are indicated by vertical bars. Broken line shows the curve, Pi = 0.03 f 3.22 X Ik4Di2, estimated by least squares using binomial weights.

7 EMS DOSE EFFECTS ON ABERRATIONS 74 7 TABLE 4 Testes weight of EMS-induced sterile and partially sterile translocations Class Number of Average testes Standard Fl males weight* (grams) deviation Sterile 44.f Partially sterile 943.w.M Normal @AI * Weights are for the two testes per male. 9 There were a total of 50 sterile F, males. The two sterile males with normal testes weights were not included. Four of the animals clearly had small testes but weights were not taken. $ There were a total of 98 partially sterile. Testes weights for four of the animals were not taken. Testes weights of the partially sterile animals were, on the average, similar to those of normal males. However, it appears that there is more variation in testes weights among those partially sterile than in normals, as indicated by a significantly higher standard deviation (P <.Ol). Although there was no overlap in testes weights between the sterile males with small testes and the partially sterile ones, the weight of the testes of one of the latter was only 0.14 gram which is considerably smaller than normal. For 96 out of 98 partially sterile males, translocation heterozygosity was confirmed by cytogenetic examination of diakinesis and metaphase I spermatocytes. Furthermore, partial sterility of all 96 F, males was found to be transmissable to the following generation. The other two confirmed partially sterile mice, one each from 200 and 150 mg/kg doses, died before they could be progeny-tested. TABLE 5 Auerage fertilify of partially sterile mules bused on fgur litters Number of partially Litter size Percent Litter no. sterile males (a%)* of normal c s C Average 43.2 * For comparison with litter size of normal males refer to Table 7. TABLE 6 Auerage fertility of partially sterile males bmed on six females killed Number Implants Living embryos Dead implants Class of males (avd (awl (Percent) Partially sterile * Normal * P < 0.01 us. controls.

8 748 w. M. GENEROSO et al. Two sets of fertility data (Tables 5 and 6) are available for the partially sterile F, progeny. The first set was obtained by caging each male with a young female of the (SEC X C57BL)F, strain to produce at least four litters. The second set was obtained by mating each one to at least six virgin (C3H X C57BL)Fl females that were killed during pregnancy for uterine analysis. On the basis of live births, the average fertility of partially sterile translocation heterozygotes was 43.2% that of normal males. This is comparable to the results obtained on sacrificed females. The average nuzber of living embryos for the partially sterile group is 44.1 % that of normal controls. Data on sacrificed females (Table 6) show that fertilization involving sperm carrying unbalanced chromosome constitutions leads to death of embryos shortly be ore or shortly after implantation in most cases. This is indicated by the observation that reduction in the number of living embryos is attributable mainly to an accompanying increase in dead implantation. However, a small proportion of embryonic loss occurred in early cleavage stages, as indicated by a significantly lower number of implantations in the partially sterile group. DISCUSSION The dominant-lethal and translocation dose-effect curves clearly indicate that the process involved from the time the chemical is administered until the genetic damage is transmitted through the germ cells is a complex one. The dominantlethal dose-effect curve departs markedly from linearity while the translocation curve significantly differs from the one expected on the basis of a two-hit kinetic. It is obvious for both endpoints that the effectiveness of EMS is proportionately much lower at low doses and that there is a point in EMS dosage beyond which additional chemical results in a very sharp increase in mutation rates. That dose appears to be between 100 and 150 mg/kg. This observation suggests that there might be a mechanism operating at low doses of EMS that reduces either the production of chromosonial damage or transmission of the damage. Such a mechanism may manifest itself before the chemical reaches the target cell, or within the target cell before damage to the chromosomes, or after damage to the chromosome has been done. At the moment there is no substantial information available to help explain the shapes of the dose-effect curves. It is clear that genetic damage can be induced at doses as low as 50 mg/kg, which was the lowest dose used in this experiment. The threshold for genetic effects of EMS, if there is any, must be at a lower dose. One important aspect of this dose-response study from the practical standpoint is the comparison in efficiency between dominant-lethal mutations and heritable translocations as meascres of induced chromosome aberrations in male mice. The dominant-lethal test carried out in male mice is generally regarded as a very impel-tant mammalian test in the evaluation of mutagenic hazards of chemicals to man. Whereas induced dominant-lethal mutations were convincingly detected at 150 mg/kg and above, a significant increase in induced translocations was already detectable at the 50 mg/kg dose. Similar dominant-lethal results in mice were obtained by EHLING, CUMMING and MALLING (1968) who were unable to detect any increase in dominant lethal mutations after an EMS dose of 100 mg/kg

9 EMS DOSE EFFECTS ON ABERRATIONS 749 and RAY et al. (1972) who were unable to detect dominant-lethal effects until the 150 mg/kg dose was used. It is likely that dominant-lethal mutations had also been induced at the 100 nig/kg or lower doses but were not detected owing to the relative insensitivity of the dominant-lethal procedure. It should be pointed out that the germ-cell stages sampled in this study are most sensitive to dominantlethal induction but may not necessarily be so to the translocation induction. In addition to the higher sensitivity of the translocation procedure, translocations are a much more reliable endpoint in terms of human hazards than dominantlethal mutations because, unlike the latter, they represent transmissible genetic damage. Thus, for the detection of low levels of chromosome breakage, translocations are a more reliable endpoint than dominant-lethal mutations. There is no doubt that EMS-induced partial sterility among F, male progeny, as determined by the procedure described here, is associated with induced reciprocal translocations. Cytogenetic analysis of partially sterile animals, the transmission o partial sterility, and the study of CATTANACH, POLLARD and ISAACSON (1968) on EMS-induced partial sterility support this. Similarly, partial sterility in F, male progeny produced by radiation and other chemicals to male parents has almost always been associated with induced reciprocal translocations (see review by RUSSELL 1962). These observations clearly establish that partial sterility alone can be taken as an unequivocal indicator of a reciprocal translocation. But what about induced complete sterility in F, male progeny? It may be recalled that of the combined completely and partially sterile F, male progeny in the EMS experiment about one-third were of the completely sterile kind. In a much lower number of F, males tested, CATTANACH, POLLARD and ISAACSON (1968) obtained about equal numbers of sterile and partially sterile animals. Thus there can be little doubt that complete sterility among F, male progeny comprises a significant proportion of EMS-induced effects and it is important to know the basis for the induced sterility. There are now strong reasons to believe that at least the great majority of induced sterility among F, males, like partial sterility, is attributable to induced chromosomal rearrangement. Some of the evidence had already been discussed by CATTANACH, POLLARD and ISAACSON in In addition, through cytogenetic analysis of gonia1 and meiotic cells, they established the presence of translocations in seven out of 10 sterile F, males. The three that did not have observable abnormal karyotypes did not have meiotic cells. Since mitotic cells were analyzed, it is possible that rearrangements may have been present in these three cases but may not have resulted in obvious changes in chromosome size. Additional data on the cytological causes of F, male sterility after mutagenic treatment have recently been obtained by CAcHhmo, RUSSELL and SWARTOUT (1974) who found that the great majority of sterile sons produced by fathers post-meiotically treated with EMS or X-rays were carriers of a translocation and that a majority of the translocations were of the autosome-autosome type. Undoubtedly, the most sensitive method for studying the ability of low doses of EMS to induce chromosomal breakage in male postmeiotic stages is screening male progeny of treated males for translocation heterozygosity. Our earlier results from a similar study with triethylene melamine (TEM) disclose a similar

10 750 w. M. GENEROSO et al. situation; (these results will be published elsewhere). Both lines of evidence indicate that translocations are a much more sensitive endpoint for measurement of low levels of chemically induced chromosome breakage than are dominantlethal mutations. We have developed a new procedure, a modification of the one used in this study, for large-scale screening of translocations. The most common procedure for detecting translocation heterozygotes is to mate F, male progeny to three or more different females. Each female is opened at midpregnancy and living and dead implantations are counted. From the fertility data on three or more females, detection of complete sterility is straightforward. A partially sterile male is expected to sire, on the average, less than half as many living embryos as a normal male, with accompanying increases in the number of dead implantations. When only low numbers of F, males are to be tested, this procedure is the most suitable one. But when large numbers of F, males are involved, this is obviously an expensive method and requires a good deal of animal handling and record keeping. The procedure described below reduces these problems, hence it is more suitable for large-scale screening and experimentation. The key to this screening procedure is the use of a strain 01 females with exceptional fertility such as the (SEC X C57BL)F,. The normal fertility of the (SEC x C57BL) F, females has previously been described ( GENEROSO, STOUT and HUFF 1971). Additional information is given in Table 7. Each F, male to be tested is caged with a female of the (SEC x C57BL)F1 strain. The females must be 10 to 12 weeks old and can continue to be used through the time the 12th litter is produced. If the size of the first litter is large enough, the male is declared fertile and discarded immediately after the litter is scored. If the first litter is not large enough, a second litier is produced. If the second litter size is large enough the male is declared fertile and immediately discarded. (When neither the first nor the second litter is large enough, the male is a suspect and is tested further by mating him to 3 virgin females, of any suitable strain, which are killed during pregnancy.) In any case, another male is placed with the female one week after TABLE 7 Reproductive performance of (SEC X C57BL)F, females mated with normal mules Litter no. hlean Total Number less Number more Proportion with in succession size litters than 10 than 9 more than a a

11 EMS DOSE EFFECTS ON ABERRATIONS 75 1 the litter is born and the same procedure is followed until the last male is added no later than after the 10th litter. The lapse of one week is required so that parentage of litters will not be confused. Since the female is normally mated shortly after parturition, a maximum of three litters are scored per male-two litters if a male is declared fertile after scoring his first litter and three litters if the decision is made after scoring the second litter. In all cases checking for newly born mice is made only when they are expected, i.e., pens are examined daily during weekdays beginning 18 days after pairing or appearance of a litter. Young are discarded immediately after they are scored. From the EMS translocation experiment, it was found that out of 462 litters sired by 98 mice confirmed as partially sterile, only nine litters sired by independent males consisted of 10 or more young, and only four of the nine litters had 11 or more. The probability that a partially sterile male will sire a litter of 10 or more is then estimated as and of 11 or more is It is possible that these probabilities may not hold true for translocations induced by other agents but, if a litter of 10 or more is used as an indicator of full fertility, chances are that those that will be missed will be difficult to detect by any other means. Analysis of the fertility data of the (SEC X C57BL)F1 females mated with fully fertile males revealed that, if a litter size of 10 or more sired by a male in his first or second litter is used as the indicator of full fertility and each female is allowed to produce 12 consecutive litters, an average of 5.27 males from a sample clear of translocations may be tested per female. This average was calculated by considering all possible configurations of two and three litters required far males for each female. Since each configuration is not equally likely, the average is a weighted average. For each configuration the number of males that can be tested is weighted by the chance that it will occur. There are 21 combinations of two and three litter periods that can test 4,5 or 6 males. For example, 6 males can be tested in 6 periods of two litters, 5 males can be tested in one period of three litters followed by 4 periods of two litters, and 4 males can be tested in 4 periods of three litters. The chances of the three configurations are 0.293, and 0.002, respectively. The chances are computed by using the last column in Table 7, which gives the probability that a fertile male will be declared fertile and thus require only two litters. The three configurations above can be combined with the other 18 and grouped according to the number of males that can be tested. The estimates of the proportions for 4, 5 and 6 males that can be tested are , , and , respectively. The weighted average of 4, 5 and 6. by use of the above proportions, is Out of these, the number of test males per female that can be declared fully fertile is estimated as The remainder are classified as suspects and have to be tested further by mating them to at least three virgin females which will be killed during pregnancy. Obviously, the higher the frequency of translocations the lower the number of males that can be tested per female. Thus a large percentage of the F, males can be declared fertile by the simple procedure of counting the number of live births in at most two litters, with only small risk of declaring a partially sterile male as fertile. Since (SEC x C57BL) F, females normally produce 12 good litters, it is now possible to test several F,

12 752 w. M. GENEROSO et al. males per female instead of sacrificing three or more females per F, male as in the old procedure. The procedure described above is now routinely used in our laboratory for screening male translocation heterozygotes produced in chemical and radiation experiments. A modification of this procedure can be made depending upon the reproductive nature of the strain of females used in the screening and on the efficiency desired. The authors are grateful to DRS. RHODA F. GRELL and R. J. PRESTON for reviewing the manuscript. LITERATURE CITED CACHEIRO, N. L. A., L. B. RUSSELL and M. S. SWARTOUT, 1974 Translocations, the predominant cause of total sterility in sons of mice treated with mutagens. Genetics 76: CATTANACH, B. M., C. E. POLLARD and J. H. ISAACSON, 1968 Ethyl methanesulfonate-induced chromosome breakage in the mouse. Mutation Res. 6: EHLING, U. H., R. B. CUMMING and H. V. MALLING, 1968 Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res. 5: GENEROSO, W. M., SANDRA K. STOUT and SANDRA W. HUFF, 1971 Effects of alkylating chemicals on reproductive capacity of adult female mice. Mutation Res. 13: GENEROSO, W. M. and W. L. RUSSELL, 1969 Strain and sex variations in the sensitivity of mice to dominant-lethal induction with ethyl methanesulfonate. Mutation Res. 8: OAKBERG, E. F., 1960 Irradiation damage to animals and its effect on their reproductive capacity. J. Dairy Sci. (Suppl.) 43: OAKBERG, E. F. and R. L. DIMINNO, 1960 X-ray sensitivity of primary spermatocytes of the mouse. Intern. J. Radiation Biol. 2: RAY, V. A., H. E. HOLDEN, D. S. SALSBURG, J. H. ELLIS, L. J. JUST and M. H. VOYER, 1972 Comparative studies of induced mutations with host-mediated, dominant-lethal and cytogenetic assays. Program and Abstracts, 3rd Annual Meeting, Environmental Mutagen Society, March 26-29, 1972, p. 14. RUSSELL, L. B., 1962 Chromosome aberrations in experimental mammals. pp In: Progress in Medical Genetics. Vol. 2. Edited by A. G. STEINBERC and A. G. BEARN. Grune and Stratton, New York. Corresponding editor: E. H. Y. CHU