Mutation Research, 243 (1990) 259-266 Elsevier
259
MUTLET 0324
The relation between induced reciprocal translocations and cell killing of mouse spermatogonial stem cells after combined treatments with hydroxyurea and X-rays P a u l P . W . v a n B u u l a n d J o h a n H. G o u d z w a a r d Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden (The Netherlandsj (Accepted 9 November 1989)
Keywords. Reciprocal translocation; Cell killing; Spermatogonial stem cells; (Mouse)
Summary The induction of reciprocal translocations in mouse spermatogonial stem cells, visualised in dividing primary spermatocytes, was studied after combined treatments with hydroxyurea (250 and 500 mg/kg) and X-rays (6, 8 and 9 Gy). The time intervals between the 2 treatments were 16 h (leading to extremely high cell killing) and 48 h (giving rise to less killing than irradiation alone). Comparison of the observed frequencies of translocations with reported data on stem cell killing (de Ruiter-Bootsma and Davids, 1981) show that the ratio between the probabilities that a radiation-induced basic lesion kills a cell or produces a translocation, theoretically calculated by Leenhouts and Chadwick (1981) to be about 10, can indeed be confirmed experimentally.
One of the central themes in the interpretation of experimental data on radiation-induced genetic damage in mammalian stem-cell spermatogonia is the assumption of coincidence of cell killing and induction of genetic damage in a heterogeneous cell population. With this hypothesis the humped dose-effect relationship as well as many observed dose-rate, dose-fractionation and radiation-quality effects can be explained. The framework for this understanding of the recovery pattern of genetic changes from male premeiotic germ cells has been Correspondence: Dr. P.P.W. van Buul, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands).
formulated by Russell (1956) and Oftedal (1968) for mutations and applied by Gerber and L6onard (1971) and Leenhouts and Chadwick (1981) to reciprocal translocations. Thus the spermatogonial stem cells are heterogeneous for both cell killing and mutation or translocation induction with the 2 sensitivities being correlated. With respect to dose-response kinetics this implies that at higher exposures aberration-carrying cells will be selectively eliminated, because of their sensitivity for cell killing, resulting in lower observed frequencies of translocations. The humped dose-effect relationship so obtained is secondary; it is a recovery curve and the peak yield of translocations is reached at the point where the induction is balanced by the elimination rate.
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
260
Spermatogonial stem-cell survival after radiation injury has been studied in rodents by (a) histological counts of surviving stem cells (Dym and Clermont, 1970; Oakberg, 1978), (b) counts of repopulated tubular cross sections or colonies (Withers et al., 1974; de Ruiter-Bootsma et al., 1976; Meistrich et al., 1978), (c) duration of the sterile period or recovered testis weight (Russell, 1954; Sheridan, 1971; Cattanach, 1974; Cattanach and Barlow, 1984; Cattanach and Kirk, 1987), (d) measuring levels of the X-isozyme of lactate dehydrogenase, L D H - X (Lu et al., 1980) and sperm counts (Lu et al., 1980; Zwanenburg et al., 1981; van Buul and de Boer, 1982). All these methods have certain advantages and disadvantages in that some do not register the functionality of the stem cells, some can only be used at higher doses and others are very indirect measures of testicular stem-cell survival. In most of the work dealing with correlations between stem-cell killing and the induction of reciprocal translocations or mutations dose-effect relationships were constructed. Indeed Cattanach (1974), Cattanach and Kirk (1987) and Lu et al. (1980) observed inflection points in the survival curves at exposure levels around the peak yields for the induction of genetic damage, but others did not (de Ruiter-Bootsma et al., 1976). There are some reports that describe other varying factors than single acute exposures. De Ruiter-Bootsma et al. (1979), using split doses, found a good correlation between stem-cell killing and the observed frequencies of translocations by Preston and Brewen (1976), but Oakberg (1978) concluded from splitdose experiments that mutation induction and cell killing were independent phenomena. Similarly, van Buul and de Boer (1982), using mice with different karyotypes, observed no correlation between stem-cell survival (measured as sperm counts) and the induction of translocations whereas Cattanach and Kirk (1987), using testis weight and sterile period, did find a positive correlation in different mouse strains and hybrids. In the present communication we describe the induction of reciprocal translocations in mouse spermatogonial stem cells by combined hydroxyurea
(HU) and X-ray treatments. The experimental set up is chosen in such a way that using the same concentrations of HU and the same X-ray doses but varying the time interval between the treatments, extreme differences in cell killing are obtained. The results indicate a 10:l ratio for radiation-induced cell killing and reciprocal translocations.
Materials and methods
Young mature 8-12-week-old Swiss randombred mice (Cpb(SE)S)) were injected i.p. with 250 or 500 m g / k g H U (BDH Chemicals Ltd.). H U was freshly dissolved in phosphate-buffered saline (PBS). 16 or 48 h later the mice received partialbody irradiation with X-ray doses of 6, 8 or 9 Gy. The time interval between H U treatment and Xirradiation was selected on the basis of experiments by de Ruiter-Bootsma and Davids (1981; personal communication), which indicated that H U given 16 h before irradiation reduced stem-cell survival to 3°70 of the level after irradiation alone, whereas an interval of 48 h between H U treatment and Xirradiation produced the same or even less cell killing than irradiation alone. Irradiation was given with an Orthovolt X-ray machine (Philips RT 250), operating at 200-250 kV and 15-20 mA, resulting in a half-value layer (HVL) of 1.0-2.5 mm Cu. The absorbed dose-rate was 0.6 G y / m i n . All irradiated animals received local testis irradiation. They were anaesthetised with 0.06 mg n e m b u t a l / g body weight and irradiation was limited to the posterior part of the body, not shielded with 6 m m lead. At various time intervals after irradiation (partly depending on the dose), meiotic preparations were made according to the air-drying method of Evans et al. (1964). No special effort was made to obtain spermatocyte populations immediately after recovery of the germinal epithelium, but in general for treatments expected to lead to more cell killing, longer sampling times were used. The mean sampling time for each treatment is given in Table 1. Coded slides, Cbanded with a modified technique of Sumner (1972), were analysed for the presence of
261
multivalent configurations in dividing primary spermatocytes. If possible 100 diakinesismetaphase I cells per testis were scored.
Results The testis weights and results of the spermatocyte analysis are presented in Table I. They show that HU alone did not induce translocations or any loss in testis weights at the sampling times used. Combined exposures of HU and X-rays clearly give rise, for the induction of translocations, to interactive processes. With 6 Gy the 16-h interval was not different from irradiation alone, but the 48-h interval reduced the frequency of recoverable translocations. With the 8- and 9-Gy doses the picture is different. Although for both exposures the 48-h interval produced lower yields of translocations than irradiation alone, the differences were not significant (Student's t-test, p > 0.05). On the other hand, with the 16-h interval the frequencies of radiation-induced reciprocal translocations were drastically enhanced. For testis weight at the 6-Gy exposure level no clear effect of HU pretreatment seems to be pres-
ent. At the 8- and 9-Gy levels the 48-h time interval tends to increase the testis weight compared to Xirradiation alone, whereas the 16-h interval indicates a decreasing effect (Student's t-test, p < 0.05) for both exposures. The distributions of translocations among cells and the ratio of ring versus chain configurations for the various treatments are given in Table 2. Tests for goodness-of-fit with a Poisson distribution of the number of translocations per spermatocyte indicated that irradiation alone produced a significant deviation at the 8- and 9-Gy levels. It is also evident that HU given 48 h before such exposures gives rise to better Poisson fits. The ratio of ring versus chain translocation configurations seems not to be correlated with any type of treatment.
Discussion The results obtained after exposure to HU alone agree well with the observation of Lu and Meistrich (1979) that HU does not affect spermatogonial stem cells in a substantial way. Both parameters studied by us, translocation yield and testis
TABLE 1 I N D U C T I O N OF T R A N S L O C A T I O N S IN STEM-CELL S P E R M A T O G O N I A A N D TESTIS W E I G H T S A F T E R C O M B I N E D T R E A T M E N T S W I T H HU A N D X-RAYS Treatment
Time interval (h)
Number of mice
Mean sampling time (days)
Number of cells analysed
% Translocations +_ SEM
Testis weights (mg) ± SEM
0 250 HU
-
5 5
99
1000 1000
500 HU
-
4
105
800
6Gy 500 HU + 6 Gy 500 HU + 6 Gy
16 48
3 5 6
84 120 91
500 900 900
11.2 ± 2.1 13.8 ± 1.7 4.2 ± 0.8
64 + 2 71 ± 4 66 ± 3
8 Gy 500 HU + 8 Gy 500 HU + 8 Gy
16 48
9 7 3
109 157 195
1400 1300 450
10.9 ± 1.8 2 2 . 1 ± 3.9 8.9 ± 2.7
77 ± 5 63 _+ 6 95 ___ 8
9 Gy 250 HU + 9 Gy 250 HU + 9 Gy
16 48
7 6 3
118 168 188
1400 1100 450
8.5 + 4.4 20.5 ± 3.4 5.6 ± 2.0
81 + 3 47 ± 3 93 _ 3
0 0.1 ± 0.0
115 ± 3 114 ± 3
0
113 ± 3
262
TABLE 2 D I S T R I B U T I O N OF T R A N S L O C A T I O N S A M O N G CELLS AND N A T U R E OF T R A N S L O C A T I O N C O N F I G U R A T I O N S A F T E R C O M B I N E D T R E A T M E N T S W I T H HU AND X-RAYS Treatment
Interval (h)
°7o Translocations
Number of translocations per cell 0
6 Gy
1
2
Poisson fit
Ratio ring/chain
3
-
11.2
449
46
5
(1
+
0.5
500 HU + 6 Gy
16
13.8
784
108
8
0
+
0.7
500 HU + 6 Gy
48
4.2
863
36
1
(1
+
2.2
8 Gy 500 HU + 8 Gy
16
10.9 22.1
1268 1056
113 208
17 29
2 7
500 HU + 8 Gy
48
8.9
415
30
5
0
8.5
1297
88
14
1
20.5 5.6
901 427
174 21
23 2
2 0
9 Gy 250 HU + 9 Gy 250 HU + 9 Gy
16 48
weights, were not influenced by doses up to 500 m g / k g . The induction of translocations following single X-ray exposure confirms our earlier work in that for Swiss random-bred mice 6 Gy is just before the h u m p of the dose-effect curve, whereas 8 and 9 Gy are beyond it (van Buul and Goudzwaard, 1986). With respect to the combined treatments there is some difference between the 6-Gy data on one hand and the 8- and 9-Gy data on the other. At the 6-Gy level, pretreatment with H U does not have an effect on testis weight and only in the case of a pretreatment interval of 48 h does the translocation yield seem to be reduced. However, at the 8- and 9-Gy levels both testis weights and translocation yields are clearly affected by H U pretreatment. The testis weights perfectly parallel the expectation based on the cell-survival analysis by de RuiterBootsma and Davids (1981) and de RuiterB o o t s m a (personal communication) in that H U given 16 h before 1.5 Gy of neutron irradiation reduces stem-cell survival to 3% of the level observed after irradiation alone, whereas a 48-h interval produces less killing than irradiation alone. The translocation data using a 16-h interval show enhanced induction rates, which suggests a positive correlation between cell killing and the induction of translocations. Similarly at a 48-h interval,
1.0 0.8 _+
0.5 0.5
+
1.0 1.1
where less cell killing occurred, the yields of translocations seem to be somewhat lower than after irradiation alone. It should be mentioned here that some differences in sampling times exist between the different treatment groups (Table 1) and this might influence testis weight. On the other hand the recorded differences at the 16-h interval between H U and X-rays are so clear that it is unlikely that the relatively small variations in sampling times will be of main importance. F r o m a quantitative point of view the correlation of our translocation data with the stem-cell survival data of de Ruiter-Bootsma and Davids (1981) seems less perfect. They observed about 30 times more cell killing at a 16-h interval and we observed only 2-4 times more translocations, so a ratio of about 10:1 emerges. I n f o r m a t i o n about the quantitative relationship between spermatogonial stem-cell killing and translocation induction can be obtained from the theoretical studies of Leenhouts and Chadwick (1981). They developed a model for the spermatogonial stem-cell population in relation to its radiation response. By fitting data from the literature on radiation-induced translocations in mouse stem-cell spermatogonia to their model, they were able to show that the 2 most important parameters were (1) the proportions of radio-
263 resistant and radio-sensitive cells, and (2) the ratio o f the probabilities that a radiation-induced basic lesion in the DNA will cause cell death (p) or form a recoverable translocation (c). The best fit to the data was obtained when the p/c ratio was assumed to be 13. This means that the probability for spermatogonial cell death is 13 times higher than the probability that a translocation will be recovered and this is in close agreement with the ratio of 10 obtained in the present study. Such a quantitative comparison is only allowed if similar radiation responses exist in CBA mice (used by de Ruiter-Bootsma and Davids, 1981) and our Swiss random-bred mice. There have been several reports in the literature on clear strain or F1 hybrid differences in radiation response (Meistrich et al., 1984; Bianchi et al., 1986; Rutledge et al., 1986; Cattanach and Kirk, 1987; Favor et al., 1987) and the effect of these types of differences on our interstrain comparison is difficult to predict. However, the data on testis weights suggest that radiation-induced cell killing in Swiss random-bred mice is comparable to that reported for CBA mice. Moreover it should be mentioned that the ratio of 13 supposed by Chadwick and Leenhouts is also based on data from a number of different mouse strains or hybrids. In fact a 1: 1 ratio for cell killing and the production of a particular type of chromosomal aberration cannot be expected since the concurrently induced unstable type of chromosome aberrations, leading to cell killing, will influence this ratio. It has long been suggested that the principal mechanism of cell killing with ionising radiation is through the induction of lethal chromosome aberrations. In some studies the observed frequencies of unstable chromosome aberrations could indeed fully account for the decrease in cell survival (Carrano, 1973) whereas in others it could not (Bedford et al., 1978). The most detailed analysis in this field has been performed by Joshi et al. (1982). They studied in individual diploid Syrian hamster ceils the effects of chromosomal damage on reproductive capacity and concluded that all cell death could be attributed to loss of chromosome fragments. Scott and Zampetti-Bosseler (1987) confirmed
these results but showed concurrently that for diploid human fibroblasts a substantial amount of radiation-induced cell death is not mediated through visible chromosome aberrations. If this last observation holds for mouse spermatogonial stem cells, it will also influence the relation between cell killing and the induction of reciprocal translocations. The fact that the experimental results obtained at the 6-Gy level are somewhat different from those at the 8- and 9-Gy levels suggests differences between relatively radio-sensitive cells and more resistant survivors. Similar observations have been made by Cattanach (1974) and Cattanach and Crocker (1979) using fractionated X-ray exposures and sterile periods and translocations as biological endpoints. However, the 8- and 9-Gy exposures, which are clearly above the level producing the peak yield in the dose-effect relationship for single exposures, i.e., around 7 Gy, are better comparable to the 1.5-Gy neutron dose applied by de Ruiter-Bootsma and Davids (1981), which is also clearly above the peak yield obtained for neutrons at about 1 Gy (Searle et al., 1969). The biological processes underlying the variation in radiation response of spermatogonia with different time intervals between HU and X-ray treatments are thought to be (a) synchronisation of cells with respect to their position in the cell cycle, or (b) a reflection of a transitional phase of stem cells to alter cell kinetics for repopulation purposes. Since the experiments of Cattanach and Crocker (1980) and van Buul (1984) explanation (b) seems to be the most probable for time intervals around 24 h and the present results obtained with a 16-h interval indicate that even shorter after treatment than 24 h, the transitional phase can be traced back. With a 48-h interval the stem ceils have already established altered cell-cycle kinetics which make them relatively insensitive to the production of translocations (Cattanach and Mosely, 1974; Cattanach et al., 1976; Preston and Brewen, 1976) or as indicated by the present data to cell killing. The transitional phase of stem-cell spermatogonia 16-24 h after cell depletion is, among other things, characterised by homogeneity of the surviving stem
264
cells and evidence for this can be obtained by Poisson analysis of the distribution of observed translocations among scored cells. In general a homogeneous population (16-24 h after pretreatment) will lead to good fits to a Poisson distribution, whereas heterogeneity (48 h after pretreatment) will give rise to deviations f r o m such a distribution (Searle et al., 1968, 1974; Cattanach and Crocker, 1977). F r o m Table 2 it can be seen that analysis of our data points to the contrary in that the 16-h interval gives significant deviation f r o m a Poisson fit at the higher exposure levels whereas at a 48-h interval a much better fit is obtained suggesting homogenisation. An explanation for this unexpected result would be that overdispersion mainly reflects high degrees of cell killing, which increases the effect of clonal proliferation of the relatively few surviving stem cells and after combined H U - X - r a y treatments is no indication for homogeneity or heterogeneity of the spermatogonia. The results on combined treatments of X-rays and adriamycin or cyclophosphamide (van Buul, 1984) or fractionated X-ray exposures (van Buul and L6onard, 1984) indicate that indeed severe cell killing can cause deviations from a Poisson distribution of the number of induced translocations per cell. An alternative model for stem-cell c o m p a r t m e n talisation in relation to radiation-induced cell killing comes f r o m the work of Van Beek et al. (1986) and Bootsma and Davids (1988). They suppose 3 subpopulations of stem cells, a resting one (Go), a cycling one consisting of Gt, S and GE cells and one committed for cycling or stimulated. In a normal testis, the radiation response is dominated by the radiation-resistant 'stimulated for cycling' group (Do = 0.75 Gy of neutrons). After irradiation with relatively high doses no resting stem cells are formed any more, and thus no stimulation is occurring, but a permanent cycling population is created for several weeks where the intermediately sensitive S phase of the cycling cells (Do = 0.40 Gy of neutrons) determines the response of the stem-cell population. Shortly after H U treatment (2-24 h) Sphase cells are killed and for a short period the stimulation of Go cells is inhibited, possibly via in-
hibition of RNA polymerases. This creates and extremely sensitive stem-cell population (Do = 0.25 Gy of neutrons). At longer time intervals between H U treatment and irradiation (about 48 h), stemcell survival is even higher than in normal testis probably due to extra radio-resistant 'stimulated for cycling' cells as a proliferation response for cell loss. This model can explain the present results as well as most of the data on the effects of dose fractionation on translocation induction (for review, see van Buul, 1983). In addition, the data of Cattanach et al. (1989) for the induction of specificlocus mutations after combined H U and X-ray treatment of spermatogonia fits the model very well.
Conclusions Through the use of combined H U and X-ray treatments our data clearly demonstrate a correlation between spermatogonial stem-cell killing and recovered genetic damage in the mouse. They further show that such treatments with optimal timing can bring about high yields of chromosomal aberrations. In addition, our results provide some experimental evidence that in mouse spermatogonial stem cells, at the dose levels used, the probability that a radiation-induced basic lesion gives rise to cell killing is about 10 times the probability that a recoverable reciprocal translocation will be formed.
Acknowledgements We thank our colleagues Prof. Dr. A.T. Natarajan, Dr. A.D. Tates and Dr. A.L. Bootsma (Utrecht) for their constructive comments on the manuscript. This work has been supported by the Association of Euratom and the University of Leiden, Contract BIO-E-406-81 NL.
References Bedford, J.S., J.B. Mitchell, H.G. Griggs and M . A Bender
265
(1978) Radiation-induced cellular reproductive death and chromosome aberrations, Radiat. Res., 76, 573-586. Beek, M.E.A.B. van, J.A.G. Davids and D.G. de Rooy (1986) Variation in the sensitivity of the mouse spermatogonial stem cell population to fission neutron irradiation during the cycle of the seminiferous epithelium, Radiat. Res., 108,282-295. Bianchi, M., J.I. Delic, G. Hurtado de Catalfo and J.H. Hendry 0985) Strain differences in the radiosensitivity of mouse spermatogonia, Int. J. Radiat. Biol., 48, 579-588. Bootsma, A.L., and J.A.G. Davids (1988) The cell cycle of spermatogonial colony forming stem cells after neutron irradiation, Cell Tissue Kinet., 21, 105-113. Buul, P.P.W. van (1983) Induction of chromosome aberrations in stem cell spermatogonia of mammals, in: T. Ishihara and M.S. Sasaki (Eds.), Progress and Topics in Cytogenetics, Vol. 4, Radiation-induced Chromosome Damage in Man, Alan R. Liss, New York, pp. 369-400. Buul, P.P.W. van (1984) Enhanced radiosensitivity for the induction of translocation in mouse stem cell spermatogonia following treatment with cyclophosphamide or adriamycin, Mutation Res., 128,207-211. Buul, P.P.W. van, and P. de Boer (1982) The induction by Xrays of chromosomal aberrations in somatic and germ cells of mice with different karyotypes, Mutation Res., 92, 229-241. Buul, P.P.W. van, and J.H. Goudzwaard (1986) Dose-effect relationship for X-ray-induced translocations in spermatogonia of normal and T70H translocation heterozygous mice, Mutation Res., 173, 41-48. Buul, P.P.W. van, and A. L6onard (1984) Effects of unequally fractionated X-ray exposures on the induction of chromosomal rearrangements in mouse spermatogonia, Mutation Res., 127, 65-72. Carrano, A.V. (1973) Chromosome aberrations and radiationinduced cell death. II. Predicted and observed cell survival, Mutation Res., 17, 355-366. Cattanach, B.M. (1974) Spermatogonial stem cell killing in the mouse following single and fractionated X-ray doses, as assessed by length of sterile period, Mutation Res., 25, 53-62. Cattanach, B.M., and J.H. Barlow (1984) Evidence for the reestablishment of a heterogeneity in radiosensitivity among spermatogonial stem cells repopulating the mouse testis following depletion by X-rays, Mutation Res., 127, 81-91. Cattanach, B.M., and A.J.M. Crocker (1979) Translocation yield from mouse spermatogonial stem cells following unequal-sized X-ray fractionations: evidence of radiationinduced loss of heterogeneity, Mutation Res., 60, 73-82. Cattanach, B.M., and A.J.M. Crocker (1980) Modified genetic response to X-irradiation of mouse spermatogonial stem cells surving treatment with TEM, Mutation Res., 70, 211-220. Cattanach, B.M., and M.J. Kirk (1987) Enhanced spermatogonial stem cell killing and reduced translocation yield from X-irradiated 101/H mice, Mutation Res., 176, 69-79. Cattanach, B.M., and H. Moseley (1974) Sterile period, translocation and specific locus mutation in the mouse following fractionated X-ray treatments with different frac-
tionation intervals, Mutation Res., 25, 63-72. Cattanach, B.M., C.M. Heath and J.M. Tracey (1976) Translocation yield from the mouse spermatogonial stem cell following fractionated X-ray treatments: influence of unequal fraction size and of increasing fractionation interval, Mutation Res., 35,257-268. Cattanach, B.M., J. Peters and C. Rasberry (1989) Induction of specific locus mutations in mouse spermatogonial stem cells by combined chemical X-ray treatments, Mutation Res., 212, 91-101. Dym, M., and Y. Clermont (1970) Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of t h e r a t testis, Am. J. Anat., 128, 265-282. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3, 289-294. Favor, J., A. Neuh~iuser-Klaus and U.H. Ehling (1987) Radiation-induced forward and reverse specific locus mutations and dominant cataract mutations in treated strain BALB/c and DBA/2 male mice, Mutation Res., 177, 161-169. Gerber, G.B., and A. L6onard (1971) Influence of selection, non-uniform cell population and repair on dose-effect curves of genetic effects, Mutation Res., 12, 175-182. Joshi, G.P., W.J. Nelson, S.H. Revell and C.A. Shaw (1982) Xray-induced chromosome damage in live mammalian cells, and improved measurements of its effects on their colonyforming ability, Int. J. Radiat. Biol., 41, 161-181. Leenhouts, H.P., and K.H. Chadwick (1981) An analytical approach to the induction of translocations in spermatogonia of the mouse, Mutation Res., 82, 305-321. Lu, C.C., and M.L. Meistrich (1979) Cytotoxic effects of chemotherapeutic drugs on mouse testis cells, Cancer Res., 39, 3575-3582. Lu, C.C., M.L. Meistrich and H.D. Thames (1980) Survival of mouse testicular stem cells after y or neutron irradiation, Radiat. Res., 81,402-415. Meistrich, M.L., N.R. Hunter, N. Suzuki, P.K. Trostle and H.R. Withers (1978) Gradual regeneration of mouse testicular stem cells after exposure to ionizing radiation, Radiat. Res., 74, 349-362. Meistrich, M.L., M. Finch, C.C. Lu, A.L. de Ruiter-Bootsma and D.G. de Rooy (1984) Strain differences in the response of mouse testicular stem cells to fractionated radiation, Radiat. Res., 97, 478-487. Oakberg, E.F. (1978) Differential spermatogonial stem cell survival and mutation frequency, Mutation Res., 50, 327-340. Oftedal, P. (1968) A theoretical study of mutant yield and cell killing after treatment of heterogeneous cell populations, Hereditas, 60, 177-210. Preston, R.J., and J.G. Brewen (1976) X-ray-induced translocations in spermatogonia. II. Fractionation responses in mice, Mutation Res., 36, 333-344. Ruiter-Bootsma, A.L. de, and J.A.G. Davids (1981) Survival of spermatogonial stem cells in the CBA mouse after combined
266
exposure to I-MeV fission neutrons and hydroxyurea, Radiat. Res., 85, 38-46. Ruiter-Bootsma, A.L. de, M.F. Kramer, D.G. de Rooy and J . A . G . Davids (.1976) Response of stem cells in the mouse testis to fission neutron of I MeV mean energy and 300 kV Xrays. Methodology, dose-response studies, relative biological effectiveness, Radiat. Res., 67, 56-68. Ruiter-Bootsma, A.L. de, M.F. Kramer, D.G. de Rooy and J .A.G. Davids (1977) Survival of spermatogonial stem cells in the m o u s e after split-dose irradiation with fission neutrons of 1 MeV mean energy or 300 kV X-rays, Radiat. Res., 71, 579-592. Russell, W.L. (1954) Genetic effects of radiation in m a m m a l s , in: A. Hollaender (Ed.), Radiation Biology, Vol. 1, McGrawHill, New York, pp. 825-859. Russell, W.L. (1956) Lack of linearity between mutation rate a n d dose for X-ray induced mutations in mice, Genetics, 41, 658-659. Rutledge, J.C., K.T. Cain, L.A. Hughes, P . W . Braden and W . M . Generoso (1986) Difference between two hybrid stocks of mice in the incidence of congenital abnormalities following X-ray exposure of stem-cell spermatogonia, Mutation Res., 163,299-302. Scott, D., and F. Zampetti-Bosseler (1987) Radiation-induced c h r o m o s o m e damage and cell death in h u m a n and hamster cells, in: Radiation Research, Proc. 8th ICRR Edinburgh, Vol. 1, Taylor and Francis, London, p. 229.
Searle, A.G., E.P. Evans, C.E. l:ord and B.J. West, with ap pendix by D.G. Papworth (1968) Studies on the induction of translocations in mouse spermatogonia. 1. The effect of doserate, Mutation Res., 6, 427-436. Searle, A.G., E.P. Evans and B.J. West (1969) Studies on te induction of translocations in mouse spermatogonia. 11. Effects of fast neutron irradiation, Mutation Res., 7,235-240. Searle, A.G., C.E. Ford, E.P. Evans, C.V. Beechey, M.D. Burtenshaw and H.M. Glegg (1974) The induction of translocations in mouse spermatozoa. 1. Kinetics of doseresponse with acute X-irradiation, Mutation Res., 22, 157-174. Sheridan, W. (1971) The effects of the time interval in fractionated X-ray treatment of mouse spermatogonia, Mutation Res., 13, 163-169. Sumner, A.T. (1972) A simple technique for demonstrating centromeric heterochromatin, Exp. Cell. Res., 75, 304-306. Withers, H.R., N. Hunter, H.T. Barkley Jr. and B.O. Reid (1974) Radiation survival and regeneration characteristics of spermatogenic stem cells of mouse testis, Radiat. Res., 57, 88-103. Zwanenburg, T.S.B., P. de Boer and P. Stam (1981) Clonal analysis of radiation-induced translocations in stem-cell sperm a t o g o n i a of normal and T70H translocation heterozygous mice, Mutation Res., 83, 207-219. C o m m u n i c a t e d by F.H. Sobels
259
MUTLET 0324
The relation between induced reciprocal translocations and cell killing of mouse spermatogonial stem cells after combined treatments with hydroxyurea and X-rays P a u l P . W . v a n B u u l a n d J o h a n H. G o u d z w a a r d Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden (The Netherlandsj (Accepted 9 November 1989)
Keywords. Reciprocal translocation; Cell killing; Spermatogonial stem cells; (Mouse)
Summary The induction of reciprocal translocations in mouse spermatogonial stem cells, visualised in dividing primary spermatocytes, was studied after combined treatments with hydroxyurea (250 and 500 mg/kg) and X-rays (6, 8 and 9 Gy). The time intervals between the 2 treatments were 16 h (leading to extremely high cell killing) and 48 h (giving rise to less killing than irradiation alone). Comparison of the observed frequencies of translocations with reported data on stem cell killing (de Ruiter-Bootsma and Davids, 1981) show that the ratio between the probabilities that a radiation-induced basic lesion kills a cell or produces a translocation, theoretically calculated by Leenhouts and Chadwick (1981) to be about 10, can indeed be confirmed experimentally.
One of the central themes in the interpretation of experimental data on radiation-induced genetic damage in mammalian stem-cell spermatogonia is the assumption of coincidence of cell killing and induction of genetic damage in a heterogeneous cell population. With this hypothesis the humped dose-effect relationship as well as many observed dose-rate, dose-fractionation and radiation-quality effects can be explained. The framework for this understanding of the recovery pattern of genetic changes from male premeiotic germ cells has been Correspondence: Dr. P.P.W. van Buul, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands).
formulated by Russell (1956) and Oftedal (1968) for mutations and applied by Gerber and L6onard (1971) and Leenhouts and Chadwick (1981) to reciprocal translocations. Thus the spermatogonial stem cells are heterogeneous for both cell killing and mutation or translocation induction with the 2 sensitivities being correlated. With respect to dose-response kinetics this implies that at higher exposures aberration-carrying cells will be selectively eliminated, because of their sensitivity for cell killing, resulting in lower observed frequencies of translocations. The humped dose-effect relationship so obtained is secondary; it is a recovery curve and the peak yield of translocations is reached at the point where the induction is balanced by the elimination rate.
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
260
Spermatogonial stem-cell survival after radiation injury has been studied in rodents by (a) histological counts of surviving stem cells (Dym and Clermont, 1970; Oakberg, 1978), (b) counts of repopulated tubular cross sections or colonies (Withers et al., 1974; de Ruiter-Bootsma et al., 1976; Meistrich et al., 1978), (c) duration of the sterile period or recovered testis weight (Russell, 1954; Sheridan, 1971; Cattanach, 1974; Cattanach and Barlow, 1984; Cattanach and Kirk, 1987), (d) measuring levels of the X-isozyme of lactate dehydrogenase, L D H - X (Lu et al., 1980) and sperm counts (Lu et al., 1980; Zwanenburg et al., 1981; van Buul and de Boer, 1982). All these methods have certain advantages and disadvantages in that some do not register the functionality of the stem cells, some can only be used at higher doses and others are very indirect measures of testicular stem-cell survival. In most of the work dealing with correlations between stem-cell killing and the induction of reciprocal translocations or mutations dose-effect relationships were constructed. Indeed Cattanach (1974), Cattanach and Kirk (1987) and Lu et al. (1980) observed inflection points in the survival curves at exposure levels around the peak yields for the induction of genetic damage, but others did not (de Ruiter-Bootsma et al., 1976). There are some reports that describe other varying factors than single acute exposures. De Ruiter-Bootsma et al. (1979), using split doses, found a good correlation between stem-cell killing and the observed frequencies of translocations by Preston and Brewen (1976), but Oakberg (1978) concluded from splitdose experiments that mutation induction and cell killing were independent phenomena. Similarly, van Buul and de Boer (1982), using mice with different karyotypes, observed no correlation between stem-cell survival (measured as sperm counts) and the induction of translocations whereas Cattanach and Kirk (1987), using testis weight and sterile period, did find a positive correlation in different mouse strains and hybrids. In the present communication we describe the induction of reciprocal translocations in mouse spermatogonial stem cells by combined hydroxyurea
(HU) and X-ray treatments. The experimental set up is chosen in such a way that using the same concentrations of HU and the same X-ray doses but varying the time interval between the treatments, extreme differences in cell killing are obtained. The results indicate a 10:l ratio for radiation-induced cell killing and reciprocal translocations.
Materials and methods
Young mature 8-12-week-old Swiss randombred mice (Cpb(SE)S)) were injected i.p. with 250 or 500 m g / k g H U (BDH Chemicals Ltd.). H U was freshly dissolved in phosphate-buffered saline (PBS). 16 or 48 h later the mice received partialbody irradiation with X-ray doses of 6, 8 or 9 Gy. The time interval between H U treatment and Xirradiation was selected on the basis of experiments by de Ruiter-Bootsma and Davids (1981; personal communication), which indicated that H U given 16 h before irradiation reduced stem-cell survival to 3°70 of the level after irradiation alone, whereas an interval of 48 h between H U treatment and Xirradiation produced the same or even less cell killing than irradiation alone. Irradiation was given with an Orthovolt X-ray machine (Philips RT 250), operating at 200-250 kV and 15-20 mA, resulting in a half-value layer (HVL) of 1.0-2.5 mm Cu. The absorbed dose-rate was 0.6 G y / m i n . All irradiated animals received local testis irradiation. They were anaesthetised with 0.06 mg n e m b u t a l / g body weight and irradiation was limited to the posterior part of the body, not shielded with 6 m m lead. At various time intervals after irradiation (partly depending on the dose), meiotic preparations were made according to the air-drying method of Evans et al. (1964). No special effort was made to obtain spermatocyte populations immediately after recovery of the germinal epithelium, but in general for treatments expected to lead to more cell killing, longer sampling times were used. The mean sampling time for each treatment is given in Table 1. Coded slides, Cbanded with a modified technique of Sumner (1972), were analysed for the presence of
261
multivalent configurations in dividing primary spermatocytes. If possible 100 diakinesismetaphase I cells per testis were scored.
Results The testis weights and results of the spermatocyte analysis are presented in Table I. They show that HU alone did not induce translocations or any loss in testis weights at the sampling times used. Combined exposures of HU and X-rays clearly give rise, for the induction of translocations, to interactive processes. With 6 Gy the 16-h interval was not different from irradiation alone, but the 48-h interval reduced the frequency of recoverable translocations. With the 8- and 9-Gy doses the picture is different. Although for both exposures the 48-h interval produced lower yields of translocations than irradiation alone, the differences were not significant (Student's t-test, p > 0.05). On the other hand, with the 16-h interval the frequencies of radiation-induced reciprocal translocations were drastically enhanced. For testis weight at the 6-Gy exposure level no clear effect of HU pretreatment seems to be pres-
ent. At the 8- and 9-Gy levels the 48-h time interval tends to increase the testis weight compared to Xirradiation alone, whereas the 16-h interval indicates a decreasing effect (Student's t-test, p < 0.05) for both exposures. The distributions of translocations among cells and the ratio of ring versus chain configurations for the various treatments are given in Table 2. Tests for goodness-of-fit with a Poisson distribution of the number of translocations per spermatocyte indicated that irradiation alone produced a significant deviation at the 8- and 9-Gy levels. It is also evident that HU given 48 h before such exposures gives rise to better Poisson fits. The ratio of ring versus chain translocation configurations seems not to be correlated with any type of treatment.
Discussion The results obtained after exposure to HU alone agree well with the observation of Lu and Meistrich (1979) that HU does not affect spermatogonial stem cells in a substantial way. Both parameters studied by us, translocation yield and testis
TABLE 1 I N D U C T I O N OF T R A N S L O C A T I O N S IN STEM-CELL S P E R M A T O G O N I A A N D TESTIS W E I G H T S A F T E R C O M B I N E D T R E A T M E N T S W I T H HU A N D X-RAYS Treatment
Time interval (h)
Number of mice
Mean sampling time (days)
Number of cells analysed
% Translocations +_ SEM
Testis weights (mg) ± SEM
0 250 HU
-
5 5
99
1000 1000
500 HU
-
4
105
800
6Gy 500 HU + 6 Gy 500 HU + 6 Gy
16 48
3 5 6
84 120 91
500 900 900
11.2 ± 2.1 13.8 ± 1.7 4.2 ± 0.8
64 + 2 71 ± 4 66 ± 3
8 Gy 500 HU + 8 Gy 500 HU + 8 Gy
16 48
9 7 3
109 157 195
1400 1300 450
10.9 ± 1.8 2 2 . 1 ± 3.9 8.9 ± 2.7
77 ± 5 63 _+ 6 95 ___ 8
9 Gy 250 HU + 9 Gy 250 HU + 9 Gy
16 48
7 6 3
118 168 188
1400 1100 450
8.5 + 4.4 20.5 ± 3.4 5.6 ± 2.0
81 + 3 47 ± 3 93 _ 3
0 0.1 ± 0.0
115 ± 3 114 ± 3
0
113 ± 3
262
TABLE 2 D I S T R I B U T I O N OF T R A N S L O C A T I O N S A M O N G CELLS AND N A T U R E OF T R A N S L O C A T I O N C O N F I G U R A T I O N S A F T E R C O M B I N E D T R E A T M E N T S W I T H HU AND X-RAYS Treatment
Interval (h)
°7o Translocations
Number of translocations per cell 0
6 Gy
1
2
Poisson fit
Ratio ring/chain
3
-
11.2
449
46
5
(1
+
0.5
500 HU + 6 Gy
16
13.8
784
108
8
0
+
0.7
500 HU + 6 Gy
48
4.2
863
36
1
(1
+
2.2
8 Gy 500 HU + 8 Gy
16
10.9 22.1
1268 1056
113 208
17 29
2 7
500 HU + 8 Gy
48
8.9
415
30
5
0
8.5
1297
88
14
1
20.5 5.6
901 427
174 21
23 2
2 0
9 Gy 250 HU + 9 Gy 250 HU + 9 Gy
16 48
weights, were not influenced by doses up to 500 m g / k g . The induction of translocations following single X-ray exposure confirms our earlier work in that for Swiss random-bred mice 6 Gy is just before the h u m p of the dose-effect curve, whereas 8 and 9 Gy are beyond it (van Buul and Goudzwaard, 1986). With respect to the combined treatments there is some difference between the 6-Gy data on one hand and the 8- and 9-Gy data on the other. At the 6-Gy level, pretreatment with H U does not have an effect on testis weight and only in the case of a pretreatment interval of 48 h does the translocation yield seem to be reduced. However, at the 8- and 9-Gy levels both testis weights and translocation yields are clearly affected by H U pretreatment. The testis weights perfectly parallel the expectation based on the cell-survival analysis by de RuiterBootsma and Davids (1981) and de RuiterB o o t s m a (personal communication) in that H U given 16 h before 1.5 Gy of neutron irradiation reduces stem-cell survival to 3% of the level observed after irradiation alone, whereas a 48-h interval produces less killing than irradiation alone. The translocation data using a 16-h interval show enhanced induction rates, which suggests a positive correlation between cell killing and the induction of translocations. Similarly at a 48-h interval,
1.0 0.8 _+
0.5 0.5
+
1.0 1.1
where less cell killing occurred, the yields of translocations seem to be somewhat lower than after irradiation alone. It should be mentioned here that some differences in sampling times exist between the different treatment groups (Table 1) and this might influence testis weight. On the other hand the recorded differences at the 16-h interval between H U and X-rays are so clear that it is unlikely that the relatively small variations in sampling times will be of main importance. F r o m a quantitative point of view the correlation of our translocation data with the stem-cell survival data of de Ruiter-Bootsma and Davids (1981) seems less perfect. They observed about 30 times more cell killing at a 16-h interval and we observed only 2-4 times more translocations, so a ratio of about 10:1 emerges. I n f o r m a t i o n about the quantitative relationship between spermatogonial stem-cell killing and translocation induction can be obtained from the theoretical studies of Leenhouts and Chadwick (1981). They developed a model for the spermatogonial stem-cell population in relation to its radiation response. By fitting data from the literature on radiation-induced translocations in mouse stem-cell spermatogonia to their model, they were able to show that the 2 most important parameters were (1) the proportions of radio-
263 resistant and radio-sensitive cells, and (2) the ratio o f the probabilities that a radiation-induced basic lesion in the DNA will cause cell death (p) or form a recoverable translocation (c). The best fit to the data was obtained when the p/c ratio was assumed to be 13. This means that the probability for spermatogonial cell death is 13 times higher than the probability that a translocation will be recovered and this is in close agreement with the ratio of 10 obtained in the present study. Such a quantitative comparison is only allowed if similar radiation responses exist in CBA mice (used by de Ruiter-Bootsma and Davids, 1981) and our Swiss random-bred mice. There have been several reports in the literature on clear strain or F1 hybrid differences in radiation response (Meistrich et al., 1984; Bianchi et al., 1986; Rutledge et al., 1986; Cattanach and Kirk, 1987; Favor et al., 1987) and the effect of these types of differences on our interstrain comparison is difficult to predict. However, the data on testis weights suggest that radiation-induced cell killing in Swiss random-bred mice is comparable to that reported for CBA mice. Moreover it should be mentioned that the ratio of 13 supposed by Chadwick and Leenhouts is also based on data from a number of different mouse strains or hybrids. In fact a 1: 1 ratio for cell killing and the production of a particular type of chromosomal aberration cannot be expected since the concurrently induced unstable type of chromosome aberrations, leading to cell killing, will influence this ratio. It has long been suggested that the principal mechanism of cell killing with ionising radiation is through the induction of lethal chromosome aberrations. In some studies the observed frequencies of unstable chromosome aberrations could indeed fully account for the decrease in cell survival (Carrano, 1973) whereas in others it could not (Bedford et al., 1978). The most detailed analysis in this field has been performed by Joshi et al. (1982). They studied in individual diploid Syrian hamster ceils the effects of chromosomal damage on reproductive capacity and concluded that all cell death could be attributed to loss of chromosome fragments. Scott and Zampetti-Bosseler (1987) confirmed
these results but showed concurrently that for diploid human fibroblasts a substantial amount of radiation-induced cell death is not mediated through visible chromosome aberrations. If this last observation holds for mouse spermatogonial stem cells, it will also influence the relation between cell killing and the induction of reciprocal translocations. The fact that the experimental results obtained at the 6-Gy level are somewhat different from those at the 8- and 9-Gy levels suggests differences between relatively radio-sensitive cells and more resistant survivors. Similar observations have been made by Cattanach (1974) and Cattanach and Crocker (1979) using fractionated X-ray exposures and sterile periods and translocations as biological endpoints. However, the 8- and 9-Gy exposures, which are clearly above the level producing the peak yield in the dose-effect relationship for single exposures, i.e., around 7 Gy, are better comparable to the 1.5-Gy neutron dose applied by de Ruiter-Bootsma and Davids (1981), which is also clearly above the peak yield obtained for neutrons at about 1 Gy (Searle et al., 1969). The biological processes underlying the variation in radiation response of spermatogonia with different time intervals between HU and X-ray treatments are thought to be (a) synchronisation of cells with respect to their position in the cell cycle, or (b) a reflection of a transitional phase of stem cells to alter cell kinetics for repopulation purposes. Since the experiments of Cattanach and Crocker (1980) and van Buul (1984) explanation (b) seems to be the most probable for time intervals around 24 h and the present results obtained with a 16-h interval indicate that even shorter after treatment than 24 h, the transitional phase can be traced back. With a 48-h interval the stem ceils have already established altered cell-cycle kinetics which make them relatively insensitive to the production of translocations (Cattanach and Mosely, 1974; Cattanach et al., 1976; Preston and Brewen, 1976) or as indicated by the present data to cell killing. The transitional phase of stem-cell spermatogonia 16-24 h after cell depletion is, among other things, characterised by homogeneity of the surviving stem
264
cells and evidence for this can be obtained by Poisson analysis of the distribution of observed translocations among scored cells. In general a homogeneous population (16-24 h after pretreatment) will lead to good fits to a Poisson distribution, whereas heterogeneity (48 h after pretreatment) will give rise to deviations f r o m such a distribution (Searle et al., 1968, 1974; Cattanach and Crocker, 1977). F r o m Table 2 it can be seen that analysis of our data points to the contrary in that the 16-h interval gives significant deviation f r o m a Poisson fit at the higher exposure levels whereas at a 48-h interval a much better fit is obtained suggesting homogenisation. An explanation for this unexpected result would be that overdispersion mainly reflects high degrees of cell killing, which increases the effect of clonal proliferation of the relatively few surviving stem cells and after combined H U - X - r a y treatments is no indication for homogeneity or heterogeneity of the spermatogonia. The results on combined treatments of X-rays and adriamycin or cyclophosphamide (van Buul, 1984) or fractionated X-ray exposures (van Buul and L6onard, 1984) indicate that indeed severe cell killing can cause deviations from a Poisson distribution of the number of induced translocations per cell. An alternative model for stem-cell c o m p a r t m e n talisation in relation to radiation-induced cell killing comes f r o m the work of Van Beek et al. (1986) and Bootsma and Davids (1988). They suppose 3 subpopulations of stem cells, a resting one (Go), a cycling one consisting of Gt, S and GE cells and one committed for cycling or stimulated. In a normal testis, the radiation response is dominated by the radiation-resistant 'stimulated for cycling' group (Do = 0.75 Gy of neutrons). After irradiation with relatively high doses no resting stem cells are formed any more, and thus no stimulation is occurring, but a permanent cycling population is created for several weeks where the intermediately sensitive S phase of the cycling cells (Do = 0.40 Gy of neutrons) determines the response of the stem-cell population. Shortly after H U treatment (2-24 h) Sphase cells are killed and for a short period the stimulation of Go cells is inhibited, possibly via in-
hibition of RNA polymerases. This creates and extremely sensitive stem-cell population (Do = 0.25 Gy of neutrons). At longer time intervals between H U treatment and irradiation (about 48 h), stemcell survival is even higher than in normal testis probably due to extra radio-resistant 'stimulated for cycling' cells as a proliferation response for cell loss. This model can explain the present results as well as most of the data on the effects of dose fractionation on translocation induction (for review, see van Buul, 1983). In addition, the data of Cattanach et al. (1989) for the induction of specificlocus mutations after combined H U and X-ray treatment of spermatogonia fits the model very well.
Conclusions Through the use of combined H U and X-ray treatments our data clearly demonstrate a correlation between spermatogonial stem-cell killing and recovered genetic damage in the mouse. They further show that such treatments with optimal timing can bring about high yields of chromosomal aberrations. In addition, our results provide some experimental evidence that in mouse spermatogonial stem cells, at the dose levels used, the probability that a radiation-induced basic lesion gives rise to cell killing is about 10 times the probability that a recoverable reciprocal translocation will be formed.
Acknowledgements We thank our colleagues Prof. Dr. A.T. Natarajan, Dr. A.D. Tates and Dr. A.L. Bootsma (Utrecht) for their constructive comments on the manuscript. This work has been supported by the Association of Euratom and the University of Leiden, Contract BIO-E-406-81 NL.
References Bedford, J.S., J.B. Mitchell, H.G. Griggs and M . A Bender
265
(1978) Radiation-induced cellular reproductive death and chromosome aberrations, Radiat. Res., 76, 573-586. Beek, M.E.A.B. van, J.A.G. Davids and D.G. de Rooy (1986) Variation in the sensitivity of the mouse spermatogonial stem cell population to fission neutron irradiation during the cycle of the seminiferous epithelium, Radiat. Res., 108,282-295. Bianchi, M., J.I. Delic, G. Hurtado de Catalfo and J.H. Hendry 0985) Strain differences in the radiosensitivity of mouse spermatogonia, Int. J. Radiat. Biol., 48, 579-588. Bootsma, A.L., and J.A.G. Davids (1988) The cell cycle of spermatogonial colony forming stem cells after neutron irradiation, Cell Tissue Kinet., 21, 105-113. Buul, P.P.W. van (1983) Induction of chromosome aberrations in stem cell spermatogonia of mammals, in: T. Ishihara and M.S. Sasaki (Eds.), Progress and Topics in Cytogenetics, Vol. 4, Radiation-induced Chromosome Damage in Man, Alan R. Liss, New York, pp. 369-400. Buul, P.P.W. van (1984) Enhanced radiosensitivity for the induction of translocation in mouse stem cell spermatogonia following treatment with cyclophosphamide or adriamycin, Mutation Res., 128,207-211. Buul, P.P.W. van, and P. de Boer (1982) The induction by Xrays of chromosomal aberrations in somatic and germ cells of mice with different karyotypes, Mutation Res., 92, 229-241. Buul, P.P.W. van, and J.H. Goudzwaard (1986) Dose-effect relationship for X-ray-induced translocations in spermatogonia of normal and T70H translocation heterozygous mice, Mutation Res., 173, 41-48. Buul, P.P.W. van, and A. L6onard (1984) Effects of unequally fractionated X-ray exposures on the induction of chromosomal rearrangements in mouse spermatogonia, Mutation Res., 127, 65-72. Carrano, A.V. (1973) Chromosome aberrations and radiationinduced cell death. II. Predicted and observed cell survival, Mutation Res., 17, 355-366. Cattanach, B.M. (1974) Spermatogonial stem cell killing in the mouse following single and fractionated X-ray doses, as assessed by length of sterile period, Mutation Res., 25, 53-62. Cattanach, B.M., and J.H. Barlow (1984) Evidence for the reestablishment of a heterogeneity in radiosensitivity among spermatogonial stem cells repopulating the mouse testis following depletion by X-rays, Mutation Res., 127, 81-91. Cattanach, B.M., and A.J.M. Crocker (1979) Translocation yield from mouse spermatogonial stem cells following unequal-sized X-ray fractionations: evidence of radiationinduced loss of heterogeneity, Mutation Res., 60, 73-82. Cattanach, B.M., and A.J.M. Crocker (1980) Modified genetic response to X-irradiation of mouse spermatogonial stem cells surving treatment with TEM, Mutation Res., 70, 211-220. Cattanach, B.M., and M.J. Kirk (1987) Enhanced spermatogonial stem cell killing and reduced translocation yield from X-irradiated 101/H mice, Mutation Res., 176, 69-79. Cattanach, B.M., and H. Moseley (1974) Sterile period, translocation and specific locus mutation in the mouse following fractionated X-ray treatments with different frac-
tionation intervals, Mutation Res., 25, 63-72. Cattanach, B.M., C.M. Heath and J.M. Tracey (1976) Translocation yield from the mouse spermatogonial stem cell following fractionated X-ray treatments: influence of unequal fraction size and of increasing fractionation interval, Mutation Res., 35,257-268. Cattanach, B.M., J. Peters and C. Rasberry (1989) Induction of specific locus mutations in mouse spermatogonial stem cells by combined chemical X-ray treatments, Mutation Res., 212, 91-101. Dym, M., and Y. Clermont (1970) Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of t h e r a t testis, Am. J. Anat., 128, 265-282. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3, 289-294. Favor, J., A. Neuh~iuser-Klaus and U.H. Ehling (1987) Radiation-induced forward and reverse specific locus mutations and dominant cataract mutations in treated strain BALB/c and DBA/2 male mice, Mutation Res., 177, 161-169. Gerber, G.B., and A. L6onard (1971) Influence of selection, non-uniform cell population and repair on dose-effect curves of genetic effects, Mutation Res., 12, 175-182. Joshi, G.P., W.J. Nelson, S.H. Revell and C.A. Shaw (1982) Xray-induced chromosome damage in live mammalian cells, and improved measurements of its effects on their colonyforming ability, Int. J. Radiat. Biol., 41, 161-181. Leenhouts, H.P., and K.H. Chadwick (1981) An analytical approach to the induction of translocations in spermatogonia of the mouse, Mutation Res., 82, 305-321. Lu, C.C., and M.L. Meistrich (1979) Cytotoxic effects of chemotherapeutic drugs on mouse testis cells, Cancer Res., 39, 3575-3582. Lu, C.C., M.L. Meistrich and H.D. Thames (1980) Survival of mouse testicular stem cells after y or neutron irradiation, Radiat. Res., 81,402-415. Meistrich, M.L., N.R. Hunter, N. Suzuki, P.K. Trostle and H.R. Withers (1978) Gradual regeneration of mouse testicular stem cells after exposure to ionizing radiation, Radiat. Res., 74, 349-362. Meistrich, M.L., M. Finch, C.C. Lu, A.L. de Ruiter-Bootsma and D.G. de Rooy (1984) Strain differences in the response of mouse testicular stem cells to fractionated radiation, Radiat. Res., 97, 478-487. Oakberg, E.F. (1978) Differential spermatogonial stem cell survival and mutation frequency, Mutation Res., 50, 327-340. Oftedal, P. (1968) A theoretical study of mutant yield and cell killing after treatment of heterogeneous cell populations, Hereditas, 60, 177-210. Preston, R.J., and J.G. Brewen (1976) X-ray-induced translocations in spermatogonia. II. Fractionation responses in mice, Mutation Res., 36, 333-344. Ruiter-Bootsma, A.L. de, and J.A.G. Davids (1981) Survival of spermatogonial stem cells in the CBA mouse after combined
266
exposure to I-MeV fission neutrons and hydroxyurea, Radiat. Res., 85, 38-46. Ruiter-Bootsma, A.L. de, M.F. Kramer, D.G. de Rooy and J . A . G . Davids (.1976) Response of stem cells in the mouse testis to fission neutron of I MeV mean energy and 300 kV Xrays. Methodology, dose-response studies, relative biological effectiveness, Radiat. Res., 67, 56-68. Ruiter-Bootsma, A.L. de, M.F. Kramer, D.G. de Rooy and J .A.G. Davids (1977) Survival of spermatogonial stem cells in the m o u s e after split-dose irradiation with fission neutrons of 1 MeV mean energy or 300 kV X-rays, Radiat. Res., 71, 579-592. Russell, W.L. (1954) Genetic effects of radiation in m a m m a l s , in: A. Hollaender (Ed.), Radiation Biology, Vol. 1, McGrawHill, New York, pp. 825-859. Russell, W.L. (1956) Lack of linearity between mutation rate a n d dose for X-ray induced mutations in mice, Genetics, 41, 658-659. Rutledge, J.C., K.T. Cain, L.A. Hughes, P . W . Braden and W . M . Generoso (1986) Difference between two hybrid stocks of mice in the incidence of congenital abnormalities following X-ray exposure of stem-cell spermatogonia, Mutation Res., 163,299-302. Scott, D., and F. Zampetti-Bosseler (1987) Radiation-induced c h r o m o s o m e damage and cell death in h u m a n and hamster cells, in: Radiation Research, Proc. 8th ICRR Edinburgh, Vol. 1, Taylor and Francis, London, p. 229.
Searle, A.G., E.P. Evans, C.E. l:ord and B.J. West, with ap pendix by D.G. Papworth (1968) Studies on the induction of translocations in mouse spermatogonia. 1. The effect of doserate, Mutation Res., 6, 427-436. Searle, A.G., E.P. Evans and B.J. West (1969) Studies on te induction of translocations in mouse spermatogonia. 11. Effects of fast neutron irradiation, Mutation Res., 7,235-240. Searle, A.G., C.E. Ford, E.P. Evans, C.V. Beechey, M.D. Burtenshaw and H.M. Glegg (1974) The induction of translocations in mouse spermatozoa. 1. Kinetics of doseresponse with acute X-irradiation, Mutation Res., 22, 157-174. Sheridan, W. (1971) The effects of the time interval in fractionated X-ray treatment of mouse spermatogonia, Mutation Res., 13, 163-169. Sumner, A.T. (1972) A simple technique for demonstrating centromeric heterochromatin, Exp. Cell. Res., 75, 304-306. Withers, H.R., N. Hunter, H.T. Barkley Jr. and B.O. Reid (1974) Radiation survival and regeneration characteristics of spermatogenic stem cells of mouse testis, Radiat. Res., 57, 88-103. Zwanenburg, T.S.B., P. de Boer and P. Stam (1981) Clonal analysis of radiation-induced translocations in stem-cell sperm a t o g o n i a of normal and T70H translocation heterozygous mice, Mutation Res., 83, 207-219. C o m m u n i c a t e d by F.H. Sobels
