Response of mouse spermatogonial stem cells to X-ray induction of heritable reciprocal translocations

Mutation Research, 126 (1984) 177-187 Elsevier

177

MTR 03833

Response of mouse spermatogonial stem cells to X-ray induction of heritable reciprocal translocations * W.M. Generoso, K.T. Cain, N.L.A. Cacheiro and C.V. Cornett Biology Division, Oak Ridge National Laboratory, P.O. Box Y, Oak Ridge, TN 37830 (U.S.A.)

with Appendix by D.G. Gosslee Computer Sciences, Union Carbide Corporation, Nuclear Division (Received 7 December 1982) (Revision received 17 October 1983) (Accepted 16 November 1983)

Summary Although heritable translocations are an important endpoint for the assessment of genetic risk from radiation, there has been a serious information gap with regard to their induction in spermatogonical stem cells, the most important cell stage in males for risk considerations. This led to uncertainty in estimating the magnitude of risk per unit exposure. Further, the relationship between the frequency of reciprocal exchanges scored by cytological analysis of the exposed male's meiocytes and the frequency of those transmitted to first-generation offspring needed to be re-examined. In order to fill in these gaps, two radiation studies, i.e., dose response and dose fractionation, were conducted on spermatogonial stem cells in which heritable and cytologically detected translocations were scored. The present data are by far the most extensive, to date, for heritable translocation induction in spermatogonial stem cells. The linearity of the rising portion of the dose-effect curve and the additivity of effects observed in the fractionation study allow a direct estimation of the number of transmissible translocations expected per unit exposure. Thus, the expected increase in heritable translocations per rad of acute X-rays is 3.89 x 10 -5 per gamete. The data also show a lack of consistency between cytologically and genetically scored translocations.

In mice, heritable reciprocal translocations are an important endpoint with respect to evaluation of hazards from radiation-induced chromosomal aberrations. It is, therefore, unfortunate that there had been a paucity of data on the actual rates at * Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. By acceptance of this article, the publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free licence in and to any copyright covering the article. 0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.

which heritable translocations are induced by ionizing radiations in spermatogonial stem ceils. Before the present study was completed, no data (for all practical purposes) were available for single doses up to 600 R (see Table 1). Where relatively more data are available, as for fractionated exposure, the number of progeny tested in each published study was quite low, averaging only 222 per treatment group (the largest group had 427 progeny tested). The low numbers of progeny tested must have been a major factor contributing to the wide range of values for the number of

178 TABLE 1 REPORTED STUDIES FROM LABORATORIES OTHER THAN ORNL ON I N D U C T I O N S OF HERITABLE TRANSLOCATIONS IN SPERMATOGONIAL STEM CELL OF MICE WITH ACUTE I O N I Z I N G RADIATIONS Radiation exposure

Stock of irradiated males

Number of progeny tested

Number of translocation carriers

Translocations per tad per gamete ( × 1 0 -~ )

Author

CBA Noninbred white mice Noninbred white mice ( C 3 H / H e H x 101/H)F 1 ( C 3 H / H e H × 101/H)F1 ( C 3 H / H e H × 101/H)F 1

1010 81 112 104 427 150

5 1 1 7 15 6

0.71 2.06 0.50 5.61 2.93 3.33

Reddi, 1965 Pomerantseva, 1969 Pomerantseva, 1969 Searle, 1964 Lyon et al., 1964 Ford et al., 1969

319

9

3.13

Pomerantseva et al., 1976

48 63 84

0 0 1

0 0 18.59

X - Radiation

700 600 600 600 600 600

R R R×3 R×2 Rx2 R×2

a b b b

T - Radiation

300 R × 3 a Intermediate

CBA neutrons

16 rad 32 rad 64 rad

Noninbred white mice Noninbred white mice Noninbred white mice

Pomerantseva, 1969 Pomerantseva, 1969 Pomerantseva, 1969

The interval between doses was 28 days. h The interval between doses was 8 weeks.

translocations per rad per gamete (0.5-5.6 x 10-5) in the X-ray fractionation studies. Certainly, in order to obtain an accurate estimate of the rate of

heritable translocation induction per unit acute exposure, it is essential to base this estimate on considerably more data (thus improving the stan-

TABLE 2 RECIPROCAL TRANSLOCATIONS SCORED IN DESCENDANT MEIOCYTES OF I R R A D I A T E D SPERMATOGON1AL STEM CELLS Study

Dose response ~

Dose fraction d

h c d e f

X-Ray exposure (R)

0 150 300 600 1 200 500X 4 ~ 600×2 r

Number of cells scored a

Number of cells with translocations

Number of translocations per cell b

0

1

2

>2

600 600 600 800 600

600 585 553 672 577

0 15 47 107 19

0 0 0 16 4

0 0 0 5 0

0 0.025 0.078 0,195 0.045

600 325

385 241

158 73

48 9

9 2

0,468 (0.029) 0,298 (0.030)

25 cells per testis were scored. Values in parentheses are standard errors. Cytological data were published previously by Crocker (1982). Data courtesy of R.J. Preston of the Biology Division, ORNL. Interval between doses was 4 weeks. Interval between doses was 8 weeks.

(0.006) (0.011) (0.017) (0.010)

179

dard error of the estimate of the rate) and to determine if the rate is affected by changing the conditions of radiation exposure. This is the primary reason why the present study was conducted. Secondarily, there is a definite need to check out the existing interpretation regarding the relationship between the incidence of reciprocal translocations scored in meiocytes that descended from spermatogoniai stem cells of exposed males and that which is actually transmitted to the next generation - - i.e., the frequency of heritable translocations expected on the basis of cytological data is about twice that which is actually transmitted to progeny (Ford et al., 1969). This interpretation, although based upon extensive cytological data, was also based upon rather low numbers of progeny tested. Furthermore, only one condition of radiation exposure was used - - i.e. 600 R + 600 R, given 8 weeks apart. Thus in order to have a better estimate of the rate of induction of heritable translocations in spermatogonical stem cells per rad of acute exposure and a better understanding of the relationship between cytological and transmitted frequencies, we studied the responses of spermatogonial stem cells under two conditions of radiation exposure - - i.e. varying doses and dose fractionation. Preliminary heritable translocation data from the dose-response study (Generoso et al., 1974) were used by Brewen and Preston (1975) in an

earlier report. The cytological dose-response data used for comparison in the present report were published earlier by Preston and Brewen (1973) and the cytological dose-fractionation data in Table 2 were kindly provided by R.J. Preston. Materials and methods

Dose-response study Male (101 x C3H)F~ mice, 12-15 weeks old, were given partial-body irradiation of 150, 300, 600, or 1200 R X-rays delivered at 96 R / m i n . The radiation source was a G.E. Maxitron 250 operated at 250 kVp, 30 MA. This study was conducted in two series (A and B). In series A, all dose groups were included. The control group for this series was also used in earlier heritable translocation studies. Series B, which was conducted later, was a repeat of control and 150 and 300 R doses. Control and irradiated males were caged individually with young virgin (SEC x C57BL)F 1 females at intervals indicated in Table 3. Each breeding pair was allowed to produce young continuously until the desired number of male progeny had been produced. Thus, the parental males and their mates were progeny-tested automatically for any pre-existing translocations.

Dose-fractionation study The stocks of mice, irradiation procedure, radi-

TABLE 3 L E N G T H OF INTERVALS BETWEEN THE TIME MALES A N D FEMALES WERE CAGED T O G E T H E R A N D APPEARANCE OF FIRST LITTERS Exposure (R)

Dose response Control series 150 series series 300 series series 600 series 1 200 series

B A B A B A A

Dose fractionation 500 x 4 600 x 2

Number of males exposed

Interval between end of treatment of males and introduction of females (days)

Interval between caging of males with females and appearance of first litters (ave. in days)

24 54 47 47 34 42 47

42 50 42 77 42 42

23.3 24.4 22.2 28.6 22.0 60.9 122.4

48 47

50 50

139.2 56.2

180

ation source and mating procedure used in this study are the same as with the dose-response study. Groups of mice were exposed to 500 R x 4 (4-week interval between doses) or 600 R × 2 (8week interval between doses). Irradiated males used in the cytological study of R.J. Preston were selected at random and killed for the preparation of meiotic spermatocytes at the earliest posttreatment intervals when sufficient diakinesismetaphase I spermatocytes were found in the slide preparations. Testicular materials were prepared for cytological examination as described in similar experiments of Preston and Brewen (1973).

Testing for translocation carriers Only male progeny born 69 days or more after irradiation were tested for translocation heterozygosity. They were tested for complete or partial sterility by use of the sequential procedure employed routinely in our laboratory (Generoso et al., 1981). All males from all groups (except those from 300 R, series B) that showed evidence of

partial sterility were progeny-tested for transmission of the partial sterility characteristic. A few male progeny from 300 R, series B that had inconclusive fertility data were also progeny-tested. A minimum of 12 sons were tested for each suspect partially sterile male, except in very few cases where the suspect male either became completely sterile or died before the desired number of male progeny could be produced. As soon as production of progeny was completed, each suspect was killed and the testes were prepared for cytological analysis using the air-dry technique of Evans et al. (1964). The same procedure was followed wherever the size of testes of sterile males was normal. Steriles whose testes were obviously smaller than normal were analyzed cytologically by use of either spermatogonia metaphases from squash preparation of testicular tubules or metaphases from cultured kidney cells (Cacheiro et al., 1974; Cacheiro and Russell, 1975). Sterile males from the control group of series A were not analyzed cytologically.

TABLE 4 DOSE EFFECTS OF ACUTE X-RAYS ON I N D U C T I O N OF HERITABLE RECIPROCAL TRANSLOCATIONS IN MOUSE SPERMATOGONIA X-Ray exposure (R)

Series

Number of male progeny tested

0

A

4 392

1

3~

0.023

B

1041

0

5

0

Total

5 433

1

8

0.018 (0, 0.04)

A B Total

993 2085 3078

6 13 19

4 7 11

0.60 0.62 0.62 (0.35, 0.89)

4.01 (0.94)

A B Total

1018 2 324 3 342

8 35 43

5d 9~ 14

0.88 1.59 1.38 (0.99, 1.77)

4.34 (0.67)

600

A

1 075

18

7r

1.86 (1.06, 2.66)

3.07 (0.69)

1 200

A

1038

5

4 '~

0.58 (0.11, 1.05)

0.47 (0.20)

150

300

b c a f

Number of partially sterile males

Number of sterile males

Frequency of translocations (%) a

Translocations per rad per gamete (xlO -5)b

The frequencies for the irradiated groups were based on all partially sterile males plus the steriles that were cytologically confirmed. Values in parentheses are 95% confidence limits. Values in parentheses are standard errors. No cytology was done on these 3 steriles. 1 sterile male was cytologically confirmed as a translocation heterozygote. Two were cytologically confirmed as translocation carriers. Two others died before cytological examination was performed. 2 sterile males were cytologically confirmed as translocation heterozygotes.

181

Results

2,8

¸

Dose-response study The frequencies of heritable translocations recovered after various doses of X-rays are shown in Table 4. In the irradiated groups and in the control group of series B the frequencies were based on all partially sterile males that were confirmed either cytologically or genetically, partially steriles that bad a clear-cut reduction in fertility and sterile males that were cytologically confirmed as translocation heterozygotes. The 3 steriles from series A control group were not cytologically analyzed but they are not likely to be translocation carriers as none of the 5 steriles in series B control group, which were cytologically analyzed, were found to carry translocations. All partially sterile males that were progeny-tested, with the exception of two, had produced partially sterile sons (Table 5). One of the two partially sterile males became completely sterile before a progeny test could be done, but the cytology unequivocally proved translocation heterozygosity. The other died before the progeny test and cytology were done, but the fertility data on 3 females mated to him are con-

o

20

o m

z u LU

0

150

300

600 X-RAY DOSE (R)

1200

Fig. 1. Dose-response curve for heritable translocations induced in spermatogonial stem cells. Vertical bars represent 95% confidence limits. The linear model Y = a + b D was used to fit the responses at doses 0-600 R. Weighted least squares estimates of the slope and intercept are 0.00385 (0.00047) and 0.0195 (0.0196), respectively, with the standard errors indicated in parentheses. The weight at each dose is equal to the reciprocal of the variance.

elusive indication of translocation heterozygosity (58% of the 10.3 implants per female were dead, as

TABLE 5 ANALYSIS OF P A R T I A L L Y STERILE M A L E P R O G E N Y Exposure (R)

N u m b e r confirmed by: progeny testing only a

cytology only

Unconfirmed b

Total

progeny testing and cytology

D o s e - effect stud>'

0, series A 150, series A series B 300, series A series B 600, series A 1 200, series A

1

1

6 12 8

1 1 2 1

34

5 6

2

15 4

1c

6 13 8 35 18 5

Dose-fractionation study

500x4 600 × 2 " b c d

75 55

2d

N o multivalent chromosome association was found in a m i n i m u m of 25 cells scored per animal. These mice died before progeny test and cytological verification were conducted. Average percent dead implantations from 3 pregnancies was 58%. Average percent dead implantations were 44% (from 3 pregnancies) and 60% (from 5 pregnancies).

82 63

182 "FABLE6 DOSE-FRACTIONATION EFFECTS OF ACUTE X-RAYS ON INDUCTION OF HERITABLE RECIPROCAL TRANSLOCATIONS IN MOUSE SPERMATOGONIA X-Ray exposure (R)

Interval between doses (weeks)

Number of male progeny tested

Number of partially sterile males

Number of sterile males

Frequency of translocations (%) ~

Translocations per tad per gamete b (× 10 5)

500 z 4 600 × 2

4 8

1135 1198

82 63

14 ~ 8d

7.40 (5.87, 8.93) 5.34 (4.69, 5.99)

3.69 (0.39) 4.44 (0.54)

a Frequencies were based on all partially sterile males plus the steriles that were cytologically confirmed. Values in parentheses are 95% confidence limits. b Values in parentheses are standard errors. 2 steriles were confirmed as translocation carriers cytologically. 1 sterile male was confirmed as translocation carrier cytologically.

compared to about 5-8% dead implants for normal animals). Because the corresponding frequencies in series A and B were not significantly different from one another, all comparisons were made based on pooled data. The frequencies of heritable translocations at all doses of X-rays studied were significantly higher than the spontaneous level. The frequency observed at 300 R was significantly higher than that observed at 150 R but the frequency observed at 600 R is not significantly higher than that observed at 300 R. The dose-effect curve (Fig. 1) is clearly h u m p e d as the frequency observed at 600 R was significantly higher than that observed at 1200 R. Even though the response at 600 R is on the descending portion of the curve, the responses up to this dose do not deviate significantly from the linear function.

Dose-fractionation study Results of the heritable translocation study are shown in Table 6. The criteria used in classifying translocation carriers are the same in the d o s e - r e sponse study. A m o n g partially sterile males, a total of 11 did not show multivalent chromosome association in at least 25 diakinesis-metaphase I cells scored although partial sterility proved to be transmissible, two were confirmed cytologically but not genetically because of the early onset of sterility, and two (with clear-cut partial sterility data from uterine analysis) died before progeny testing and cytological analysis were conducted. The frequencies of translocation carriers observed at the

two fractionation exposures are clearly higher than both the spontaneous frequency and that observed or expected for each individual fraction.

Discussion

Heritable translocations per rad per gamete The primary objective of the present study is to estimate the magnitude of risk per unit exposure -i.e., the n u m b e r of heritable translocations expected per rad per gamete. In order to do so, it is essential to determine how the rate of induction is affected by the important conditions of radiation exposure such as dose, dose rate and dose fractionation. Furthermore, it is important that there is a fairly g o o d estimate of the spontaneous frequency, With the exception of d o s e - r a t e effects, the three other factors needed for an accurate determination of an estimate have been satisfied in the present study. First, we now have a good estimate of the shape of the heritable translocation dose-effect curve. The ascending portion of the ' h u m p e d ' dose-effect curve fits the linear model very closely. Second, the frequencies of heritable translocations observed in the 600 R x 2 and 500 R × 4 fractionation experiments appear to be within reasonable bounds of what might be expected from additivity. In the case of 600 R x 2 exposure, the frequency observed (Table 6) does not differ significantly from twice the frequency observed from a single 600 R exposure (Table 4), Similar direct comparison cannot be made be-

183 tween 500 R x 4 and the single 500 R exposure because no data are available for the latter. However, it should be reasonable to assume that 500 R is in the ascending portion of the heritable translocation dose-effect curve and that the expected frequency can be calculated on the basis of linearity. When this is done, the frequency observed for the fractionated exposure is, again, not significantly different from that expected from 4 times that expected for single dose. And thirdly, the spontaneous frequency of heritable translocation of 1.8 x 10 -4 should be the most reliable figure available as it is based on highest number of progeny tested. The linearity of the ascending portion of the dose-effect curve and the additivity of effects observed in the dose-fractionation experiments permit a direct calculation of the rates of induction of heritable translocations per gamete per rat of acute exposure. These values, given in the last column of Tables 4 and 6, were calculated by subtracting the spontaneous from the observed frequency and dividing the result by the total dose received. The translocations per rad per gamete value for 1200 R single exposure is clearly lower than the rest of the values. This is expected since the observed frequency falls at the tail end of the descending portion of the humped dose-effect curve. All other values, on the other hand, are very close to one another, ranging from 3.07 x 10 -5 to 4.44 x 10 -s. Because there are no significant differences between the values within this range, the average translocations per rad per gamete value weighted by the reciprocal of the variances is estimated to be 3.89 ( + 0 . 2 5 ) x 10 -5.

Relationship between frequencies of cytologically scored and transmitted reciprocal translocations The first attempt to determine if the number of translocations transmitted to progeny following irradiation of spermatogonial stem cells can be predicted from data obtained through cytological analysis of direct descendant meiocytes was made by Ford et al. (1969) from an X-ray fractionation study (600 R x 2, 8 weeks between exposures). It was concluded that the frequency of translocation heterozygotes in the progeny of irradiated male mice sired subsequent to the recovery of fertility is only about half of that expected from the frequen-

cies of multivalent configurations observed in their spermatocytes. The favored interpretation for the discrepancy was that there might exist a selective process that is operating on diploid rather than haploid genomes but perhaps taking effect on the haploid spermatids or sperm. Brewen and Preston (1975), using their data on the ratio of X-ray-induced reciprocal translocations in primary spermatocytes to dicentrics in peripheral leukocytes of the mouse, went further and concluded that the yield of dicentrics in human leukocytes after radiation exposure can be corrected by approximately 1/32 to arrive at an estimate of recovered translocations in the offspring of irradiated men. From a theoretical standpoint it does seem reasonable to assume that a close relationship between cytological and transmitted data might exist in the mouse. Thus, the cytological work of Preston and Brewen (1973) and the preliminary heritable translocation experiment of Generoso et al. (1974), reported together by Brewen and Preston (1975), were aimed at reanalyzing this relationship in a dose-response study. It was found that the ratio between the number of heritable translocations per gamete scored and the numbers of cytologically scored translocations per cell were 0.11-0.13 for 300, 600 and 1200 R and 0.25 for 150 R. Using the same assumptions as those of Ford et al. (1969) the ratio of 0.25 is the value obtained when exactly the expected number of heritable translocations are recovered. Brewen and Preston (1975) tentatively concluded that this ratio might be dose-dependent. The preliminary data of Generoso et al. (1974) are the same as those of series A in Table 4. The series A data are not satisfactory for such comparison because the frequencies of heritable translocations observed for 150 R and for 300 R are not significantly different. This prompted additional data to be obtained (series B). The pooled series A and B data, in which differences in the observed frequencies of heritable translocations between these two doses are significant, were now used to obtain the ratio between the cytological and the transmitted frequencies. These ratios and the similarly obtained ones from the fractionation experiments are shown in the second column of Table 7. The ratios ranged from 0.095 to 0.248 and it appears that there is no particular pattern in the

184

ratio of transmitted and cytologically scored chromosomal effects. Another way of analyzing the data is by comparing the observed heritable translocation frequencies with those expected from the cytological frequencies, the expected values derived following the same assumptions regarding meiotic segregation and gametic selection outlined by Ford et al. (1969). This comparison is of considerable interest in view of the generally adopted observation of Ford et al. (1969) that only half of the expected frequency was actually transmitted. Tests of the hypotheses that (a) the observed frequencies (Tables 4 and 6) are the same as those expected (Table 7) and (b) that the observed frequencies are only one-half of those expected are shown in Fig. 2. Again, there appears to be no specific pattern that would suggest a close association between cytological and transmitted frequencies. In the dose-response study the observed frequencies for 300 and 1200 R are not significantly different from either the expected or half of the expected value, for 150 R the observed frequency is significantly more than half of that expected, and for 600 R the observed frequency is significantly lower than that expected but not significantly different from half of expected. In the dose-fractionation

TABLE 7 R E L A T I O N S H I P BETWEEN C Y T O L O G I C A L L Y G E N E T I C A L L Y SCORED T R A N S L O C A T 1 O N S Exposure (R)

Heritable translocations per g a m e t e / translocations per spermatocyte a

Dose response 0 0 150 0.248 300 0.177 600 0.095 1 200 0.129

(0.085) (0.036) (0.023) (0.059)

Dose fractionation 500 X 4 0.158 (0.019) 600 X 2 0.179 (0.028)

AND

Expected frequency of heritable translocations b (%)

0 0.63 2.04 4.18 0.94

(0.17) (0.29) (0.32) (0.18)

10.42 (0.52) 7.23 (0.65)

a Values in parentheses are standard errors. ~' Calculated from the cytological data in Table 2 using the method employed by Ford et al. (1969). Values in parentheses are standard errors.

14.

0,2

do

~o 68o 12oo 5oo,4 6oo,2 X-RAY EXPOSURE (R)

Fig. 2. Ratios between observed and expected frequencies of heritable translocations. The expected frequencies were calculated from the cytological data in Table 2 using the method employed by Ford et al. (1969). Vertical bars represent 95% confidence limits.

study both observed frequencies are significantly lower than the corresponding expected frequencies but significantly higher than half of expected frequencies. The ratios between heritable translocations per gamete and cytologically scored translocations per spermatocyte and the ratios between the observed and the expected frequencies of heritable translocations show no significant heterogeneity, which appears to contradict the Brewen and Preston (1975) suggestion that the nature of the relationship between cytologically scored and transmitted translocations at low doses may be different from that at high doses. It should be noted, however, that the cytological and heritable translocation studies were not completely parallel. For example, there is a difference in the sampling time between the corresponding cytological and genetic studies - - i.e., in the cytological study mice were killed for testicular preparation as soon as a sufficient number of spermatocytes could be seen in the slides (consequently from spermatogonial stem cell repopulation) while in the genetic study male progeny were collected over a period of several months from the time irradiated males recovered their fertility. Whether or not this sampling time difference has any effect at all has not really been established satisfactorily. It is cogent to bring up that there is a difference in the shapes of the

185 dose-effect curves obtained by Preston and Brewen (1973) on one hand, which deviated significantly from linearity, and that obtained by other workers, on the other, which generally used relatively longer sampling times and which do not deviate significantly from linearity (see reviews by Brewen and Preston (1973) and UNSCEAR Report (1977)). Thus, we may conclude that the present cytological and heritable translocation data do not permit a reliable extrapolation from cytological data to what might be expected among progeny. Taking an overall look at the data of Ford et al. (1969) together with the present results and considering the fact that the cytological scoring must have missed a good number of cells that had translocations, it does appear that, in general, the observed frequencies are lower than the expected ones. This discrepancy may mean that either certain of the assumptions are not operational or other forces not related to segregation are responsible. With respect to the latter, Ford et al. (1969) suggested that it could be attributable to selection during the postmeiotic stages against translocation-carrying gametes while Crocker (1982) suggested that the frequency of translocations scored cytologically may be spuriously elevated as a result of metaphase I delay (i.e., spermatocytes carrying translocations take longer to pass through metaphase I). Neither of the suggestions were based on direct evidence. It may be noted that, in the case of the Crocker (1982) study, the delay was demonstrated for translocations involving Robertsonian chromosomes in comparison with those involving acrocentric chromosomes only but not for translocations involving acrocentric chromosomes only in comparison with nontranslocation chromosomes. We are following up on this problem. One study that needs to be repeated is the estimation of the actual proportion of unbalanced segregants by conducting a sizable dominant-lethal experiment simultaneously with a cytological study of the same exposed males. In order to determine accurately the rate of induction of dominant-lethal mutations by X-rays and to be able to explain satisfactorily whatever observation is made in connection with cytologically scored and transmitted translocations, it is essential to use optimum exposure conditions and size of the experiment in addition to using the most suitable

female mice. The heritable translocation and cytological results of the 500 R x 4 experiments are encouraging in this regard. Using the same assumptions of Ford et al. (1969) we would expect a dominant-lethal-type effect of about 20% from unbalanced segregants of reciprocal translocation. A study to check this expectation is now underway. A related study was prompted by the evidence that the size of the translocated segment, or the size of the chromosomes involved in the exchange, has some influence on the segregation of these chromosomes (Generoso et al., 1981). It appears that short translocation segments favor the type of segregation (i.e., alternate) that results in a relatively higher proportion of balanced segregants. In other words, recovery of reciprocal translocations among progeny after treatment of spermatogonial stem cells appears to be influenced, at least in part, by the position of the exchange and size of the translocated segment. Analysis of the translocation heterozygotes produced in the present study (from spermatogonia irradiation) and of translocation heterozygotes produced from irradiation of postmeiotic male germ cells will be reported elsewhere.

Acknowledgements We are grateful to Dr. R.J. Preston for the use of the previously unpublished X-ray fractionation cytological data and to Drs. M.D. Morris, E.F. Oakberg and G.A. Sega for discussion and review of the manuscript.

Appendix. Statistical methods D. G. Gosslee

(1) The statistical analyses are based on the assumption of a binomial or multinomial distribution of the observations, as appropriate. The standard errors of ratios are estimated by an approximation obtained using the first-order terms of a series expansion (Ashton, 1972). The notation used for the standard error and the variance of a statistic are SE and VAR, respectively; for example, [SE( p)]2 = VAR(p). (2) The number of translocations per cell, PL, in

186

Table 2 is calculated by the formula p, =p~ + 2p2 + 3p> where Pl, P2 and P3 are the proportions of cells with 1, 2, and 3 or more translocations, respectively, of N cells scored. The p~'s are multinomially distributed with means equal to ~r~, variances equal to % (1 - % ) / N , and covariances equal to -~r~rJN. Estimates of the variances and covariances are obtained by substituting Pi for % so that the variance of Pt is estimated by

1

VAR(pt) = ~ [ Pl( 1 - P l ) +4p2(1 - P z ) + 9p3(1 - P , ) - 2(2piP2 + 3pip3 + 6p2 p3)] (3) The 95% confidence limits on the frequency of translocations, f, in percent as shown in Tables 4 and 6 and in Fig. 1, were estimated by using the normal approximation to the binomial distribution and are f - 1.96SE(f) and f + 1.96SE(f), where V A R ( f ) = f ( I O O - f ) / N . The standard error of the proportion of translocations per rad per gamete in Tables 4 and 6 is S E ( f ) / 1 0 0 , divided by the X-ray exposure in rad. (4) The standard error of the ratio of heritable translocations per gamete to translocations per spermatocyte, R = H / T (Table 7), is estimated from the approximation, VAR(R)=R 2 VAR(H) + VAR(T) H2 T2 2COV( H, T) ]

HT

1

The covariance of H and T, COV(H, T), is zero in this case since H and T are estimated from independent data. (5) The dose-response line in Fig. 1 was estimated by weighted least squares where each frequency was weighted by the reciprocal of its variance. (6) The 95% confidence limits for the ratios displayed in Fig. 2 are the ratio +_ 1.96SE(ratio), where SE(ratio) is estimated by the procedure described above for estimating SE(R). (7) Translocations per gamete per rad for single

exposures of 150 through 600 R and for fractionated doses were averaged since the hypothesis of homogeneity could not be rejected at a level of significance of 1%. The weighted average of 3.89 × 10 5 was calculated by weighting each ratio by the reciprocal of its variance.

References Ashton, W.D. (1972) The Logit Transformation, Hafner Publishing Co., New York, Appendix 1. Brewen, J.G., and R.J. Preston (1975) The use of chromosome aberrations for predicting genetic hazards to man, in: (O.F. Nygaard, H.I. Adler and W.K. Sinclair (Eds.), Radiation Research: Biomedical, Chemical and Physical Perspectives, Academic Press, New York, pp. 926-936. Cacheiro, N.L.A, and L.B. Russell (1975) Evidence that linkage group IV as well as linkage group X of the mouse are in chromosome 10, Genet. Res., 25, 193-195. Cacheiro, N.L.A., L.B. Russell and M.S. Swartout (1974) Translocations the predominant cause of total sterility in some of mice treated with mutagens, Genetics, 76, 73 91. Crocker, M. (1982) Metaphase I delay as a factor influencing translocation yield from spermatogonial irradiation in mice carrying Robertsonian translocations, Mutation Res., 103, 339-343. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3, 289-294. Ford, C.E., A.G. Searle, E.P. Evans and B.J. West (1969) Differential transmission of translocations induced in spermatogonia of mice by irradiation, Cytogenetics, 8, 447-470. Generoso, W.M., K.T. Cain and S.W. Huff (1974) Dose effects of acute X rays on induction of heritable reciprocal translocations in mouse spermatogonia, Biology Division Annual Progress Report, Oak Ridge National Laboratory, pp. 136-138. Generoso, W.M., M. Krishna, K.T. Cain and C.W. Sheu (1981) Comparison of two methods for detecting translocation heterozygotes in mice, Mutation Res., 81,177 186. Lyon, M.F., R.J.S. Phillips and A.G. Searle (1964) The overall rates of dominant and recessive lethal and visible mutation induced by spermatogonial X-irradiation of mice, Genet. Res., 5, 448-467. Pomerantseva, M.D. (1969) The mutagenic effect of various types of radiations on the germ cells of male mice, Ill. Study of genetic damages in mice of the first generation obtained from males whose spermatogonia were subjected to irradiation, Genetika, 5, 30-37. Pomerantseva, M.D., L.K. Ramaiya and G.A. Vilkina (1976) Mutagenic effect of various types of radiation in the germ cells of male mice, X. Frequency of recessive lethal mutations and reciprocal translocations in the spermatogonia of mice subjected to fractioned gamma irradiation, Genetika, 12, 56-63. Preston, R.J., and J.G. Brewen (1973) X-ray-induced transloca-

187 tions in spermatogonia, I. Dose and fractionation responses in mice, Mutation Res., 9, 215-223. Reddi, O.S. (1965) Radiation-induced translocations in mouse spermatogonia, Mutation Res., 2, 95. Searle, A.G. (1964) Genetic effects of spermatogonial X-irradiation on productivity of F1 female mice, Mutation Res., 1, 99-108. United Nations Scientific Committee on the Effects of Atomic

Radiation (1972) Levels and Effects, 1972 report to the General Assembly, United Nations, New York. United Nations Scientific Committee on the Effects of Atomic Radiation (1977) Sources and Effects of Ionizing Radiation, 1977 report to the General Assembly, United Nations, New York.