301
Mutation Research, 249 (1991) 301-310 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100152D
MUT 00103
Comparison of two stocks of mice in spermatogonial response to different conditions of radiation exposure * W . M . G e n e r o s o a, K . T . C a i n a, C.V. C o r n e t t
a
a n d E.L. F r o m e b
a Biology Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37731-8077 and b Engineering Physics and Mathematics Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37731-8077 (U.S.A.) (Received 5 January 1991) (Accepted 27 February 1991)
Keywords: Mouse; Spermatogonia; Reciprocal translocations; Embryo lethals; Radiation exposure
Summary In a previous report (Generoso et al., 1985) it was shown that the two hybrid stocks of mice, (C3H/R1 x 101/R1)F t and (SEC/R1 x C57BL/6)F1, differed in their responses to induction of chromosomal aberrations following exposure of the stem-cell spermatogonia to 500 R x 4 (4-week intervals) acute X-rays. The levels of response in the two stocks were paralleled by the effects on the length of the sterile period, which presumably results from stem-cell killing and repopulation. The present study was conducted in order to determine whether the differences between the two stocks in these parameter s hold true also for other conditions of radiation exposure. Thus, comparative experiments were conducted using the following acute exposure regimens: 500 R single dose, 500 R + 500 R (24-h interval), 100 R + 900 R (24-h interval), and 500 R X 4 (8-week intervals). The endpoints measured were chromosome rearrangements in diakinesis/metaphase-I meiocytes, embryonic lethality in conceptuses, length of sterile period and testis weight. Trend analysis indicated that higher frequencies of chromosome rearrangements and embryonic lethality were recovered from (C3H/R1 x 101/R1)F 1 than from (SEC/R1 x C57BL/6)F1 males, that there were no significant differences between stocks in testis weight reductions, and that there was no consistency in the direction of the significant differences that occurred in the length of the sterile period. A definitive conclusion regarding the possible association between induction of chromosomal aberrations and induction of cell killing awaits direct histological analysis of the stem-cell population.
* To Bill Russell, my esteemed mentor, colleague, and friend whose values and standards greatly influenced my career. The submitted manuscript has been authored by a contractor of the U.S. Government under contract No. DE-AC0584OR21400. Accordingly, the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes.
Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy under contract DEAC05-84OR21400 with Martin Marietta Energy Systems, Inc.
Correspondence: Dr. W.M. Generoso, Biology Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37731-8077 (U.S.A.).
302
Response of mouse stem-cell spermatogonia to the induction of chromosomal aberrations by ionizing radiation is influenced not only by the various conditions of exposure such as dose, dose-rate, and dose-fractionation, but also by the stock derivation of the males (Generoso et al., 1985; Cattanach and Kirk, 1987; Cattanach et al., 1990). Effects of each of these factors have been interpreted on the basis of the perceived nature and properties of the spermatogonial stem-cell population. However, while there is agreement on the cytological description of the spermatogonia stem cell, there is lack of consensus with respect to the kinetics of stem-cell division and renewal. Most investigators subscribe to one of the following two concepts (or to a modification). One concept holds that the single, isolated spermatogonia ( A s - A s t e m , Asingle) are composed of two populations of cells (Huckins, 1971, 1978; Huckins and Oakberg, 1978; Oakberg, 1971, 1984; Oakberg and Huckins, 1976). One type, the long-cycling A,, is thought to be the true stem cell responsible for regeneration of the seminiferous epithelium after exposure to radiation. The other type, the short-cycling A s, is thought to be the initial stage committed to differentiation after descending from the long-cycling type. The other concept holds that there is only one population of A s spermatogonia all of which are stem cells capable of regenerating the seminiferous epithelium (Lok et al., 1983; de Rooij, 1988; de Rooij et al., 1990). The
former concept contends that the stem cells divide only once during each epithelial cycle, the latter contends that they divide several times. A related issue is whether or not there is a true association between cell killing and chromosomal aberrations induced by radiation in stem-cell spermatogonia. While there are numerous cases of parallel response in the two endpoints, there are also cases of contradicting results (see reviews by Van Buul, 1983, and de Rooij, 1990). Since it is not unreasonable to assume that there are multiple targets associated with cell death, the possibility exists that the correlation that has sometimes been observed between cell killing and chromosomal aberrations results from a c o m m o n target, in combination with independent targets whose radiation-response kinetics have points of similarities as well as differences. We have attempted to obtain evidence bearing on some of these questions by using various conditions of radiation exposure and different strains of mice. While any one study may not provide definitive evidence, continuous accumulation of results in a multiplicity of experiments should help to narrow down the possibilities and eventually to resolve the issues. Accordingly, we have followed up on our observation (Generoso et al., 1985) that the two hybrid stocks ( C 3 H / R 1 × 101/R1)F~ and ( S E C / R 1 x C 5 7 B L / 6 ) F 1 differ with respect to the frequencies of aberrations scored either in spermatocytes or in the progeny, following X-ray ex-
TABLE 1 EXPOSURE CONDITIONS Exposure
500 500 100 500 500 a b c d e
R Rx2 c RX900 c Rx4 a R x 4 e
AND EFFECTS MEASURED
N u m b e r of males exposed b
12 48 48 48 48
Effects m e a s u r e d " L e n g t h of sterile period
Testis weight
Chromosome aberrations in s p e r m a t o c y t e s
+ + + + +
+ + +
+ + +
+
+
+ i n d i c a t e s d a t a w e r e collected. T h e n u m b e r s of m a l e s i n d i c a t e d are for each e x p e r i m e n t a l or c o n t r o l g r o u p in each stock. 24-h interval b e t w e e n doses. 4 - w e e k interval b e t w e e n doses. 8-week interval b e t w e e n doses.
Embryonic lethality in p r o g e n y
+ + + +
303
( S E C / R 1 x C 5 7 B L / 6 ) F 1 or C 3 H / R 1 × 1 0 1 / R1)F 1 females were used in different experiments, with all males in the experiment (regardless of stock) mated to a single type of females. At various times after the irradiated males regained fertility, they were mated to virgin ( S E C / R 1 × C57BL/6)F1 females which were killed for uterine analysis 12-15 days after observation of the vaginal plug. Also 6 fertile males from each group were killed and their testes processed individually by the air-drying technique of Evans et al. (1964). Slides were coded and 25 diakinesis/metaphase-I spermatocytes were scored from each testis.
posure of stem-cell spermatogonia. The treatment used in that study was 500 R x 4 (4-week intervals). We are now reporting the responses of these two stocks to four other conditions of radiation exposure. In all experiments, acute X-irradiation was used. Materials and methods
Two stocks of males, ( C 3 H / R 1 x 101/R1)F t and ( S E C / R 1 x C 5 7 B L / 6 ) F 1, were used in all experiments. The males were about 12 weeks old at the start of each experiment. The 5 different irradiation regimens are shown in Table 1. Irradiation was performed with a Norelco (MG) 300 X-ray machine operated at 250 kVp, and 10 mA. All doses were given as partial body irradiation at the rate of 80-88 R / m i n . Control males, which were randomly separated from the experimental ones, were not sham-exposed. Each irradiated and control male was caged individually with a single, young, sexually mature female 41-56 days after the last exposure in order to determine the length of the sterile period. Either
Results
(1) Length of sterile period and testis weight The sterile period attributed to exposure of the stem-cell spermatogonia was determined by subtracting the period it takes for the stem cell to become sperm in the ejaculate (49 days) and the length of the gestation period (19 days) from the interval between the last exposure and the appearance of first litter (Table 2). With respect to
TABLE 2 L E N G T H OF STERILE PERIOD A N D TESTIS W E I G H T OF I R R A D I A T E D MALES Exposure
Stock of males
Length of sterile period (days) a
Testis weight reduction Posttreatment interval (days)
Percent of control b
500 R
( C 3 H / R 1 × 101/R1)F 1 (SEC/R1 x C57BL/6)F1
7 7
119-122 119-122
90 84
500 R x 2 c
( C 3 H / R 1 x 101/R1)F~ (SEC/R1 x C57BL/6)F 1
52 62
162-167 162-167
60 56
100 R × 900 c
( C 3 H / R 1 × 101/R1)F1 (SEC/R1 x C57BL/6)F~
70 59
259-266 259-266
56 62
500 R x 4 d
( C 3 H / R 1 X 101/R1)F~ (SEC/R1 X C57BL/6)F x
91 69
159-161 159-161
49 47
500 R X 4 ~
( C 3 H / R 1 X 101/R1)F~ (SEC/R1 X C57BL/6)F~
38 48
172-197 172-197
58 52
a Calculated by subtracting 68 days (49 for the duration of spermatogensis and 19 for gestation period) from the interval between exposure and appearance of the first poststerile litter. Values are medians. Differences between the two stocks are significant (at 0.01 level) for all groups except the first. b Based on mean testis weight at the time of the first exposure (about 12 weeks of age) and at the time of sampling. 24-h interval between doses. d 4-week interval between doses. e 8-week interval between doses.
304
this endpoint, there is no difference between the two stocks at the single 500-R exposure, but statistically significant differences were observed with
the other exposure conditions. Interestingly, the direction of the differences was not always the same. With 500 R × 2 and 500 R x 4 (8-week
TABLE 3 E M B R Y O N I C L E T H A L I T Y F R O M EXPOSED S P E R M A T O G O N I A L STEM CELLS Exposure
Stocks of males
Treatment to mating interval (days) a
Group N umbe r of N umbe r Code b pregnant of implanfemales tations per pregnant female
N umbe r of living Dead embryos per implants pregnant (%) female
Induced embryonic lethality (%) ~
500 R × 2 a
(C3H/R1 × 101/R1)F1 (SEC/R1 xC57BL/6)F 1 (C3H/R1 ×101/R1)F 1 (SEC/R1×C57BL/6)F 1 (C3H/R1 × 101/R1)F 1 ( S E C / R 1 X C57BL/6)F~ (C3H/R1 × 101/R1)F a ( S E C / R I x C57BL/6)F~
151-155 151-155 151-155 151 155 396-403 396-403 396 403 396-403
C-I-1 S-I-1 C-1I-1 S-II-1 C-I-2 S-I-2 C-II-2 S-II-2
60 71 89 79 72 77 76 81
10.2 10.2 10.1 10.0 9.4 9.7 9.7 9.7
8.5 8.8 9.7 9.6 7.7 8.6 9.5 9.3
17 14 4 4 18 12 3 4
12 8
100 R + 9 0 0 R d ( C 3 H / R I X101/R1)F~ (SEC/R1 ×C57BL/6)F 1 Control (C3H/R1 x 101/R1)F I ( S E C / R 1 ×C57BL/6)F~ 100 R + 9 0 0 R a ( C 3 H / R I × 101/R1)F~ (SEC/R1 xC57BL/6)F 1 Control (C3H/RIx101/R1)F 1 (SEC/R1 xC67BL/6)F~
201-206 201-206 201-206 201-206 404-411 404-411 404 411 404 411
C-I-3 S-I-3 C-II-3 S-II-3 C-1-4 S-1-4 C-II-4 S-II-4
56 59 68 66 58 55 54 55
9.7 9.2 9.9 10.2 8.8 9.7 9.6 10.0
8.1 8.4 9.6 9.7 7.7 8.9 9.t 9.6
17 8 3 5 13 9 4 3
500 R x 4 e
Control 500 R x 2 d Control
Control 500 R x 4 e Control
500 R × 4 r Control 500 R × 4 r Control
16 13
15 7
(C3H/RI (SEC/R1 (C3H/R1 (SEC/R1 (C3H/R1 (SEC/R1 (C3H/R1 (SEC/R1
x 101/R1)F 1 xC57BL/6)F 1 × 101/RI)F1 x C57BL/6)Fa x 101/R1)F~ x C57BL/6)F1 × 101/R1)F 1 × C57BL/6)F~
202-207 202 207 202-207 202-207 362-371 362-371 362-371 362-371
C-I-5 S-I-5 C-II-5 S-II-5 C-I-6 S-I-6 C-II-6 S-II-6
80 86 77 92 69 91 99 102
9.7 10.0 10.0 10.0 10.0 9.8 10.3 10.5
7.5 8.7 9.7 9.6 7.7 8.6 9.6 10.1
23 13 3 4 23 12 6 4
23 9
(C3H/R1 (SEC/RI (C3H/R1 (SEC/R1 (C3H/R1 (SEC/RI (C3H/R1 (SEC/R1
x 101/R1)F~ xC57BL/6)F 1 x 101/R1)F1 xC57BL/6)F~ x 101/R1)F 1 x C57BL/6)F1 x 101/R1)F l xC57BL/6)F 1
174-181 174-181 174-181 174-181 293-304 293-304 293-304 293 304
C-1-7 S-I-7 C-II-7 S-II-7 C-I-8 S-I-8 C-II-8 S-II-8
81 78 75 81 70 78 76 96
9.0 9.3 9.9 9.5 8.8 9.4 10.1 10.1
6.7 7.6 9.6 9.1 6.7 7.8 9.8 9.8
25 18 3 4 24 17 3 3
30 16
a After last X-ray exposure. h Refer to Table 5 for statistical comparisons. Induced embryonic lethality calculated by the formula:
1
19 8
average living embryos (experimental) average living embryos (control) x 100
a 24-h interval between doses. e 4-week interval between doses. r 8-week interval between doses.
20 15
32 20
305 i n t e r v a l s ) e x p o s u r e s , l o n g e r sterile p e r i o d s w e r e o b s e r v e d for ( S E C / R 1 x C 5 7 B L / 6 ) F 1 t h a n for ( C 3 H / R 1 × 1 0 1 / R 1 ) F 1 males, b u t the r e v e r s e was t r u e in the c a s e of 100 R + 900 R a n d 500 R x 4 (4-week intervals) exposures. All exposures caused testis-weight reductions ( T a b l e 2). T h e s e r e d u c t i o n s w e r e g r e a t e s t a f t e r the 500 R x 4 ( 4 - w e e k i n t e r v a l s ) t r e a t m e n t , a n d s m a l lest a f t e r t h e single 5 0 0 - R e x p o s u r e . T h e y w e r e i n t e r m e d i a t e , a n d r o u g h l y similar, for the o t h e r t h r e e t r e a t m e n t r e g i m e n s . T h e r e are n o statistic a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n the t w o stocks for t e s t i s - w e i g h t c o m p a r i s o n s at a n y of the treatment regimens.
(2) Embryonic mortality E m b r y o n i c m o r t a l i t y a m o n g p r o g e n y sired b y males whose stem-cell spermatogonia had been i r r a d i a t e d was m e a s u r e d at t w o p o s t s t e r i l e p e r i o d s for e a c h e x p o s u r e c o n d i t i o n ( T a b l e 3). T h e e a r l i e r s a m p l e was g e n e r a l l y c o l l e c t e d w h e n at least t w o t h i r d s of the m a l e s h a d sired a litter. T h e t i m e at w h i c h the l a t e r s a m p l e was c o l l e c t e d was c h o s e n a r b i t r a r i l y a n d was to a c e r t a i n e x t e n t i n f l u e n c e d b y t h e a v a i l a b i l i t y of females. T h e single 5 0 0 - R e x p o s u r e was n o t t e s t e d b e c a u s e of the a n t i c i p a t e d d i f f i c u l t y in q u a n t i f y i n g the l o w r e s p o n s e exp e c t e d . T h e o t h e r f o u r e x p o s u r e c o n d i t i o n s ind u c e d s i g n i f i c a n t i n c r e a s e s in e m b r y o n i c m o r t a l i t y a m o n g c o n c e p t u s e s sired b y m a l e s f r o m e i t h e r stock, as i n d i c a t e d b y the s i g n i f i c a n t l y h i g h e r incid e n c e s of d e a d i m p l a n t a t i o n s , a l o n g w i t h signific a n t l y l o w e r n u m b e r s of living e m b r y o s ( T a b l e 4). T h e d i f f e r e n c e s are n o t a l w a y s significant, b u t the t r e n d (Fig. l a - c ) , i n d i c a t e s t h a t at all e x p o s u r e c o n d i t i o n s h i g h e r rates of e m b r y o n i c m o r t a l i t y w e r e i n d u c e d b y i r r a d i a t i o n o f the ( C 3 H / R 1 × 1 0 1 / R 1 ) F 1 s t o c k t h a n of t h e o t h e r stock. R e s u l t s o f t h e e x p e r i m e n t s in the first s a m p l i n g p e r i o d are g e n e r a l l y c o n s i s t e n t w i t h the results for the s e c o n d period.
TABLE 4 STATISTICAL COMPARISONS FOR NUMBER OF LIVING EMBRYOS AND INCIDENCE OF DEAD IMPLANTATIONS Group comparisons a
Decrease in the average Increase in the pernumber of living cent dead implants embryos per pregnant female
C-I-1 vs. C-II-1 1,17 * +0.50 b S-I-1 vs. S-II-1 0.82 * +0.50 C-I-1 vs. S-I-1 0.28+0.52
12.6 * +1.9 c 9.7 * ___1.7 3.4+2.3
C-I-2 vs. C-II-2 1.74 * ___0.49 S-I-2 vs. S-II-2 0.70 * ±0.48 C-I-2 vs. S-I-2 0.85 * + 0.47
15.4 * +1.8 7.2 * + 1.6 6.5 "t + 2.1
C-I-3 vs. C-II-3 1.56 * +0.53 S-I-3 vs. S-II-3 1.24 * +0.54 C-I-3 vs. S-I-3 0.39 + 0.54
14.6 * +2.0 3.8 * + 1.6 8.6 * +_2.3
C-I-4 vs. C-II-4 1.42 * + 0.55 S-I-4 vs. S-II-4 0.73 + 0.58 C-I-4 vs. S-I-4 1.20 * -+0.54
8.2 * _+2.0 5.3 * + 1.6 4.1 + 2.2
C-I-5 vs. C-II-5 2.19 * +0.47 S-I-5 vs. S-II-5 0.86 * +0.45 C-I-5 vs. S-I-5 1.20 t +0.44
19.3 * _+1.9 9.3 * -+ 1.5 9.3 i" -+2.2
C-I-6 vs. C-II-6 1.89 * +0.46 S-I-6 vs. S-II-6 1.47 * -+0.44 C-I-6 vs. S-I-6 0.85-+0.45
16.1 * -+2.0 8.3 * 5:1.4 10.2 t 5:2.2
C-I-7 vs. C-II-7 2.73 * _+0.46 S-I-7 vs. S-II-7 1.55 * -+0.46 C-I-7 vs. S-1-7 0.70-+0.43
22.4 * _+2.0 14.5 * _+1.8 7.5 * _+2.5
C-I-8 vs. C-II-8 3.09 * -+0.47 S-I-8 vs. S-II-8 2.10 * -+0.45 C-I-8 vs. S-I-8 0.99 * -+0.44
22.5 * 5:2.1 14.6 * 5:1.7 8.0 t 5:2.6
a Refer to Table 3 for stock of males and treatment. b Standard deviation of estimate based on assumption of Poisson variation. c Standard deviation of estimate based on assumption of binomial variation. * Significant at the 0.05 level based on a one-sided test of the null hypothesis of no treatment effect. t Significant at the 0.05 level based on a two-sided test of the null hypothesis of no difference in treatment effects between the two stocks of mice.
(3) Cytogenetic analysis of spermatocytes T h e m a l e s w e r e killed for c y t o g e n e t i c a n a l y s i s of t h e i r m e i o c y t e s s h o r t l y a f t e r the c o m p l e t i o n of the f i r s t - p e r i o d e m b r y o n i c m o r t a l i t y analysis, w i t h the e x c e p t i o n of t h o s e in the 100 R + 900 R e x p o s u r e w h i c h w e r e p r e p a r e d m u c h later. T h e 500 R × 4 ( 4 - w e e k i n t e r v a l s ) e x p o s u r e was n o t
a n a l y z e d this time. D a t a r e p o r t e d e a r l i e r ( G e n e r o s o et al., 1985) w e r e u s e d for c o m p a r i s o n b e t w e e n e x p o s u r e c o n d i t i o n s . A g a i n , as in t h e c a s e o f e m b r y o n i c m o r t a l i t y , h i g h e r t r a n s l o c a t i o n freq u e n c i e s w e r e f o u n d at all e x p o s u r e c o n d i t i o n s in t h e ( C 3 H / R 1 x 1 0 1 / R 1 ) F t s t o c k t h a n in t h e
306 ( S E C / R 1 x C 5 7 B L / 6 ) F 1 stock, a l t h o u g h the difference is n o t always significant ( T a b l e 5).
1985; R u t l e d g e et al., 1986). These e n d p o i n t s were embryonic mortality and malformations among c o n c e p t u s e s sired, frequency of t r a n s l o c a t i o n s in s p e r m a t o c y t e s of e x p o s e d males, a n d length of sterile period. C y t o l o g i c a l evidence (Preston a n d Brewen, 1976) suggested that the stem-cell p o p u l a tion has n o t r e t u r n e d to ' n o r m a l ' by 4 weeks after the first d o s e of 500 R. In a d d i t i o n , the lengths of the sterile p e r i o d s f o u n d in the s t u d y of G e n e r o s o et al. (1985) i n d i c a t e d that the length of time n e e d e d for the c o m p l e t i o n of stem-cell r e p o p u l a tion increases with each a d d i t i o n a l 500-R dose.
Discussion
In the earlier studies that h a d revealed differences between the two stocks, ( C 3 H / R 1 x 1 0 1 / R 1 ) F t a n d ( S E C / R 1 x C 5 7 B L / 6 ) F t, the f o r m e r stock e x h i b i t e d higher responses than the l a t t e r in all e n d p o i n t s m e a s u r e d to i n d i c a t e genetic a n d cell killing effects of 500 R x 4 (4-week intervals) in stem-cell s p e r m a t o g o n i a ( G e n e r o s o et al.,
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Fig. 1. (a) Boxplots of the number of implants per pregnant female. (b) Boxplots of the percentage of dead implants per pregnant female. (c) Boxplots of the number of living implants per pregnant female. Rectangular bar contains 50% of the sample in the distribution. Line inside the bar represent the median of the distribution. Numbers 1-8 correspond to the last digit of the group codes in Table 3. (For details of boxplots, see Becker et al., 1988)
307
was included on the assumption (Cattanach et al., 1985) that following the first dose, the surviving stem-cell population is a mixture of formerly radiosensitive and formerly radioresistant cells that had been triggered into a similar degree of sensitivity to translocation induction and cell killing. The remaining exposure condition (500 R x 4, 8week intervals) was included assuming that stemcell repopulation is already completed by the time the second or the subsequent doses are given. The following are the basic findings of the present study. (1) The hybrid stock ( C 3 H / R 1 × 101/R1)F 1 exhibited higher levels of early embryonic mortality among progeny sired at all exposure conditions tested than the hybrid
In the present study, 5 exposure conditions were used, including a repeat of the 500 R × 4 (4-week intervals) regimen. The lowest exposure, a single 500-R dose, was used because it is in the ascending portion of the dose-response curve for induced chromosomal aberrations (Preston and Brewen, 1973), it kills all gonial cells except the resistant isolated A s (Oakberg and Huckins, 1976), and it produces a short sterile period indicating a minimal effect on stem-cell repopulation. The 500 R × 2 (24-h interval) exposure was included on the assumption that the A s cells surviving the first dose are a homogeneous population at the time the second dose is given (Russell, 1962; Oakberg, 1978). The 100 R + 900 R (24-h interval) exposure
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C57BL)F 1
308
( S E C / R 1 × C 5 7 B L / 6 ) F 1. (2) The cytogenetic resuits paralleled those of embryonic mortality at all exposure conditions in which both endpoints were studied. In the case of 500-R single exposure, also, ( C 3 H / R 1 x 101/R1)F~ yields a higher frequency of chromosomal aberrations than does ( S E C / R 1 x C57BL/6)F~ but the difference is not statistically significant. (3) In terms of the length of sterile period, the response of the two stocks relative to each other was not consistent for all exposure conditions tested. (4) No significant differences in testes weight reduction were observed between the two stocks. Thus, both embryonic mortality and cytogenetic data indicate higher sensitivity of the gonial stem cells of ( C 3 H / R 1 x
1 0 1 / R 1 ) F 1 males to induction of chromosomal aberrations with X-rays than of the stem cells of ( S E C / R 1 x C57BL/6)F1 males. On the other hand, the meaning conveyed by the results on testis weight and length of sterile period is unclear. Testis weight as measured in the present study is perhaps the least reliable measure of the degree of gonial stem-cell killing. The total contribution of all stem cells to the weight of the testis is negligible. It seems reasonable to assume that the important factors affecting testis-weight change are the degree of radiation damage to other testicular components, and the status of repopulation from the spermatogonial stem-cell population. The
Dead Implants Per Pregnant Female g_
g_
g_
8
1
I
0.
o_
1
I I I
I I I
Z
I
1
o_
" 1
2
3
4
5
6
7
2
8
(C3H/RI
3 4
5 6 7
8
1 2 3 4 5 6 7 8 1 1
IRRADIATED
CONTROL X
t
101/RI)F I
Fig. 1 ( c o n t i n u e d ) .
_J
2
3
4
5
6
IRRADIATED
CONTROL (SEC
X
C57BL)F 1
7
8
309
questions, therefore, are (a) to what extent do gonial stem cells approximate the average response of all testicular components, (b) is the response of each component relative to the rest similar in the two stocks, and (c) does the manner and speed of repopulation differ in the two stocks? Thus, the length of the sterile period is not solely a function of the number of stem cells killed. Conceivably, it could also be a function of the rate of repopulation and the baseline size of the stem-cell population in unexposed males. The lack of parallelism between testis-weight and sterile-period data makes it even more difficult to determine whether there is relationship between stem-cell killing and aberration induction. In order to study this issue definitively it will be essential to perform a direct histological analysis of the A s spermatogonia in the two stocks of mice used in the present study. In our earlier study (Generoso et al., 1985) the question was raised as to whether the basis for the stock difference in the incidence of induced embryonic lethality is the property of the stem cells or of processes subsequent to the formation of aberrations in the exposed stem cell. By estimating the incidences of embryonic lethality expected from the cytological data in the two stocks, and comparing them with those actually observed, we concluded at that time that not only were there
fewer aberrations induced in stem-cell gonia of ( S E C / R 1 x C 5 7 B L / 6 ) F 1 males, but that meiocytes with aberrations gave rise to a lower proportion of unbalanced sperm in ( S E C / R 1 x C 5 7 B L / 6 ) F 1 than in ( C 3 H / R 1 x 1 0 1 / R 1 ) F r Similar comparisons between expected and observed embryonic lethality for three of the exposures in the present study failed to confirm this earlier conclusion. Thus, it appears that the difference between the two stocks in terms of transmitted chromosomal aberrations is attributable solely to the responses of the respective gonial stem cells. In the Introduction, two related issues that are important in evaluating the response of stem-cell spermatogonia to ionizing radiations were discussed in relation to the difference between two stocks in the induction of chromosomal aberrations. The first is the true nature of the stem cell, the second is whether there is any real association between aberration induction and stem-cell killing. While no additional information from the present study contributed directly to the clarification of these issues, the results have made it clear that it will be necessary to perform a detailed, direct cytological analysis of the stem-cell population for this purpose in experiments involving the two stocks of mice and the exposure conditions used in the present report.
TABLE 5 R E C I P R O C A L T R A N S L O C A T I O N S SCORED IN D E S C E N D A N T M E I O C Y T E S O F I R R A D I A T E D S P E R M A T O G O N I A L STEM CELLS Exposure
Stock of mice a
Number of cells with translocations b
N umbe r of trans-
0
1
> 2
locations per cell
2
500 R
( C 3 H / R 1 X 101/R1)F~ ( S E C / R 1 x C57BL/6)F~
257 267
38 30
4 2
1 1
0.16 0.12
500 R × 2 c
( C 3 H / R 1 × 101/R1)F~ ( S E C / R 1 x C57BL/6)F~
199 229
89 61
12 7
0 3
0.38 0.28
100 R × 9 0 0 ¢
( C 3 H / R 1 × 101/R1)F~ (SEC/R1 x C57BL/6)F1
212 255
69 35
14 8
5 2
0.38 e 0.19
500 R x 4 d
( C 3 H / R 1 × 101/R1)F~ (SEC/R1 × C57BL/6)F1
186 206
86 75
22 16
6 3
0.49 e 0.38
a b c d e
e
The posttreatment intervals when testis weight was measured and chromosome preparations were made are shown in Table 2. 300 cells were scored from 6 males (25 cells per testis). 24-h interval between doses. 8-week interval between doses. Significantly different from the value for the other stock ( P < 0.05, two-sided Student's t-test).
310
Acknowledgements We are grateful to Drs. L.B. Russell, G.A. Sega and P.B. Selby for critical review of the manuscript.
References Becker, R.A., J.M. Chambers and A.R. Wilks (1988) The New S Language, Wadsworth and Brooks/Cole, Pacific Grove, CA. 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., C. Jones and D.G. Papworth (1985) Specificlocus mutation response to unequal, 1 + 9 Gy X-ray fractionations at 24-h and 4-day fraction intervals, Mutation Res., 149, 105-118. Cattanach, B.M., C. Rasberry and C. Beechy (1990) Factors affecting mutation induction by X-rays in the spermatogonial stem cells of mice of strain 101/H, in: Biology of Mammalian Germ Cell Mutagenesis, Banbury Report 34, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 209-220. de Rooij, D.G. (1988) Regulation of the proliferation of spermatogonial stem cells, J. Cell Sci., Suppl. 10, 181 194. de Rooij, D.G., Y. van der Meer, A.M.M. van Pelt and P.P.W. van Buul (1990) Correlation between proliferative activity of mouse spermatogonial stem cells and their sensitivity for cell killing and induction of reciprocal translocations by irradiation, in: Biology of Mammalian Germ Cell Mutagenesis, Banbury Report 34, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 35-49. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3, 289-294. Generoso, W.M., K.T. Cain, C.V. Cornett, N.L.A. Cacheiro, L.A. Hughes and P.W. Braden (1985) Difference in the response of two hybrid stocks of mice to X-ray induction of chromosome aberrations in spermatogonial stem cells, Mutation Res., 152, 217-223. Huckins, C. (1971) The spermatogonial stem cell population in
adult rats, III. Evidence for a long-cycling population, ('ell Tissue Kinet., 4, 335-349. Huckins, C. (1978) Behavior of stem-cell spermatogonia in the adult rat irradiated testis, Biol. Reprod., 19, 747-760. Huckins, C., and E.F. Oakberg (1978) Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tabules, II. The irradiated testis, Anat. Rec., 192, 529-542. Lok, D., M.T. Jansen and D.G. de Rooij (1983) Spermatogonial multiplication in the Chinese hamster, II. Cell cycle properties of undifferentiated spermatogonia, Cell Tissue Kinet., 16, 19-29. Oakberg, E.F. (1971) Spermatogonial stem cell renewal in the mouse, Anat. Rec., 169, 515-532. Oakberg, E.F. (1978) Differential spermatogonial stem-cell survival and mutation frequency, Mutation Res., 50, 327340. Oakberg, E.F. (1984) Germ cell toxicity: Significance in genetic and fertility effects of radiation and chemicals, in: E.H.Y. Chu and W.M. Generoso (Eds.), Mutation, Cancer, and Malformation, Plenum, New York, pp. 549-590. Oakberg, E.F., and C. Huckins (1976) Spermatogonial stem cell renewal in the mouse, as revealed by 3H-thymidine labeling and irradiation, in: A.B. Cairnie, P.K. Lala, and D.G. Osmond (Eds.), Stem Cells of Renewing Cell Populations, Academic Press, New York, pp. 287-302. Preston, R.J., and J.G. Brewen (1973) X-ray-induced translocations in spermatogonia, 1. Dose and fractionation responses in mice, Mutation Res., 19, 215-223. Preston, R.J., and J.G. Brewen (1976) X-Ray-induced translocations in spermatogonia, il. Fractionation responses in mice, Mutation Res., 36, 333 344. Russell, W.L. (1962) An augmenting effect of dose fractionation on radiation-induced mutation rate in mice, Proc. Natl. Acad. Sci. (U.S.A), 48, 1724-1727. 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. van Buul, P.P.W. (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, Liss, New York, pp. 369-400.
Mutation Research, 249 (1991) 301-310 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100152D
MUT 00103
Comparison of two stocks of mice in spermatogonial response to different conditions of radiation exposure * W . M . G e n e r o s o a, K . T . C a i n a, C.V. C o r n e t t
a
a n d E.L. F r o m e b
a Biology Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37731-8077 and b Engineering Physics and Mathematics Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37731-8077 (U.S.A.) (Received 5 January 1991) (Accepted 27 February 1991)
Keywords: Mouse; Spermatogonia; Reciprocal translocations; Embryo lethals; Radiation exposure
Summary In a previous report (Generoso et al., 1985) it was shown that the two hybrid stocks of mice, (C3H/R1 x 101/R1)F t and (SEC/R1 x C57BL/6)F1, differed in their responses to induction of chromosomal aberrations following exposure of the stem-cell spermatogonia to 500 R x 4 (4-week intervals) acute X-rays. The levels of response in the two stocks were paralleled by the effects on the length of the sterile period, which presumably results from stem-cell killing and repopulation. The present study was conducted in order to determine whether the differences between the two stocks in these parameter s hold true also for other conditions of radiation exposure. Thus, comparative experiments were conducted using the following acute exposure regimens: 500 R single dose, 500 R + 500 R (24-h interval), 100 R + 900 R (24-h interval), and 500 R X 4 (8-week intervals). The endpoints measured were chromosome rearrangements in diakinesis/metaphase-I meiocytes, embryonic lethality in conceptuses, length of sterile period and testis weight. Trend analysis indicated that higher frequencies of chromosome rearrangements and embryonic lethality were recovered from (C3H/R1 x 101/R1)F 1 than from (SEC/R1 x C57BL/6)F1 males, that there were no significant differences between stocks in testis weight reductions, and that there was no consistency in the direction of the significant differences that occurred in the length of the sterile period. A definitive conclusion regarding the possible association between induction of chromosomal aberrations and induction of cell killing awaits direct histological analysis of the stem-cell population.
* To Bill Russell, my esteemed mentor, colleague, and friend whose values and standards greatly influenced my career. The submitted manuscript has been authored by a contractor of the U.S. Government under contract No. DE-AC0584OR21400. Accordingly, the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes.
Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy under contract DEAC05-84OR21400 with Martin Marietta Energy Systems, Inc.
Correspondence: Dr. W.M. Generoso, Biology Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37731-8077 (U.S.A.).
302
Response of mouse stem-cell spermatogonia to the induction of chromosomal aberrations by ionizing radiation is influenced not only by the various conditions of exposure such as dose, dose-rate, and dose-fractionation, but also by the stock derivation of the males (Generoso et al., 1985; Cattanach and Kirk, 1987; Cattanach et al., 1990). Effects of each of these factors have been interpreted on the basis of the perceived nature and properties of the spermatogonial stem-cell population. However, while there is agreement on the cytological description of the spermatogonia stem cell, there is lack of consensus with respect to the kinetics of stem-cell division and renewal. Most investigators subscribe to one of the following two concepts (or to a modification). One concept holds that the single, isolated spermatogonia ( A s - A s t e m , Asingle) are composed of two populations of cells (Huckins, 1971, 1978; Huckins and Oakberg, 1978; Oakberg, 1971, 1984; Oakberg and Huckins, 1976). One type, the long-cycling A,, is thought to be the true stem cell responsible for regeneration of the seminiferous epithelium after exposure to radiation. The other type, the short-cycling A s, is thought to be the initial stage committed to differentiation after descending from the long-cycling type. The other concept holds that there is only one population of A s spermatogonia all of which are stem cells capable of regenerating the seminiferous epithelium (Lok et al., 1983; de Rooij, 1988; de Rooij et al., 1990). The
former concept contends that the stem cells divide only once during each epithelial cycle, the latter contends that they divide several times. A related issue is whether or not there is a true association between cell killing and chromosomal aberrations induced by radiation in stem-cell spermatogonia. While there are numerous cases of parallel response in the two endpoints, there are also cases of contradicting results (see reviews by Van Buul, 1983, and de Rooij, 1990). Since it is not unreasonable to assume that there are multiple targets associated with cell death, the possibility exists that the correlation that has sometimes been observed between cell killing and chromosomal aberrations results from a c o m m o n target, in combination with independent targets whose radiation-response kinetics have points of similarities as well as differences. We have attempted to obtain evidence bearing on some of these questions by using various conditions of radiation exposure and different strains of mice. While any one study may not provide definitive evidence, continuous accumulation of results in a multiplicity of experiments should help to narrow down the possibilities and eventually to resolve the issues. Accordingly, we have followed up on our observation (Generoso et al., 1985) that the two hybrid stocks ( C 3 H / R 1 × 101/R1)F~ and ( S E C / R 1 x C 5 7 B L / 6 ) F 1 differ with respect to the frequencies of aberrations scored either in spermatocytes or in the progeny, following X-ray ex-
TABLE 1 EXPOSURE CONDITIONS Exposure
500 500 100 500 500 a b c d e
R Rx2 c RX900 c Rx4 a R x 4 e
AND EFFECTS MEASURED
N u m b e r of males exposed b
12 48 48 48 48
Effects m e a s u r e d " L e n g t h of sterile period
Testis weight
Chromosome aberrations in s p e r m a t o c y t e s
+ + + + +
+ + +
+ + +
+
+
+ i n d i c a t e s d a t a w e r e collected. T h e n u m b e r s of m a l e s i n d i c a t e d are for each e x p e r i m e n t a l or c o n t r o l g r o u p in each stock. 24-h interval b e t w e e n doses. 4 - w e e k interval b e t w e e n doses. 8-week interval b e t w e e n doses.
Embryonic lethality in p r o g e n y
+ + + +
303
( S E C / R 1 x C 5 7 B L / 6 ) F 1 or C 3 H / R 1 × 1 0 1 / R1)F 1 females were used in different experiments, with all males in the experiment (regardless of stock) mated to a single type of females. At various times after the irradiated males regained fertility, they were mated to virgin ( S E C / R 1 × C57BL/6)F1 females which were killed for uterine analysis 12-15 days after observation of the vaginal plug. Also 6 fertile males from each group were killed and their testes processed individually by the air-drying technique of Evans et al. (1964). Slides were coded and 25 diakinesis/metaphase-I spermatocytes were scored from each testis.
posure of stem-cell spermatogonia. The treatment used in that study was 500 R x 4 (4-week intervals). We are now reporting the responses of these two stocks to four other conditions of radiation exposure. In all experiments, acute X-irradiation was used. Materials and methods
Two stocks of males, ( C 3 H / R 1 x 101/R1)F t and ( S E C / R 1 x C 5 7 B L / 6 ) F 1, were used in all experiments. The males were about 12 weeks old at the start of each experiment. The 5 different irradiation regimens are shown in Table 1. Irradiation was performed with a Norelco (MG) 300 X-ray machine operated at 250 kVp, and 10 mA. All doses were given as partial body irradiation at the rate of 80-88 R / m i n . Control males, which were randomly separated from the experimental ones, were not sham-exposed. Each irradiated and control male was caged individually with a single, young, sexually mature female 41-56 days after the last exposure in order to determine the length of the sterile period. Either
Results
(1) Length of sterile period and testis weight The sterile period attributed to exposure of the stem-cell spermatogonia was determined by subtracting the period it takes for the stem cell to become sperm in the ejaculate (49 days) and the length of the gestation period (19 days) from the interval between the last exposure and the appearance of first litter (Table 2). With respect to
TABLE 2 L E N G T H OF STERILE PERIOD A N D TESTIS W E I G H T OF I R R A D I A T E D MALES Exposure
Stock of males
Length of sterile period (days) a
Testis weight reduction Posttreatment interval (days)
Percent of control b
500 R
( C 3 H / R 1 × 101/R1)F 1 (SEC/R1 x C57BL/6)F1
7 7
119-122 119-122
90 84
500 R x 2 c
( C 3 H / R 1 x 101/R1)F~ (SEC/R1 x C57BL/6)F 1
52 62
162-167 162-167
60 56
100 R × 900 c
( C 3 H / R 1 × 101/R1)F1 (SEC/R1 x C57BL/6)F~
70 59
259-266 259-266
56 62
500 R x 4 d
( C 3 H / R 1 X 101/R1)F~ (SEC/R1 X C57BL/6)F x
91 69
159-161 159-161
49 47
500 R X 4 ~
( C 3 H / R 1 X 101/R1)F~ (SEC/R1 X C57BL/6)F~
38 48
172-197 172-197
58 52
a Calculated by subtracting 68 days (49 for the duration of spermatogensis and 19 for gestation period) from the interval between exposure and appearance of the first poststerile litter. Values are medians. Differences between the two stocks are significant (at 0.01 level) for all groups except the first. b Based on mean testis weight at the time of the first exposure (about 12 weeks of age) and at the time of sampling. 24-h interval between doses. d 4-week interval between doses. e 8-week interval between doses.
304
this endpoint, there is no difference between the two stocks at the single 500-R exposure, but statistically significant differences were observed with
the other exposure conditions. Interestingly, the direction of the differences was not always the same. With 500 R × 2 and 500 R x 4 (8-week
TABLE 3 E M B R Y O N I C L E T H A L I T Y F R O M EXPOSED S P E R M A T O G O N I A L STEM CELLS Exposure
Stocks of males
Treatment to mating interval (days) a
Group N umbe r of N umbe r Code b pregnant of implanfemales tations per pregnant female
N umbe r of living Dead embryos per implants pregnant (%) female
Induced embryonic lethality (%) ~
500 R × 2 a
(C3H/R1 × 101/R1)F1 (SEC/R1 xC57BL/6)F 1 (C3H/R1 ×101/R1)F 1 (SEC/R1×C57BL/6)F 1 (C3H/R1 × 101/R1)F 1 ( S E C / R 1 X C57BL/6)F~ (C3H/R1 × 101/R1)F a ( S E C / R I x C57BL/6)F~
151-155 151-155 151-155 151 155 396-403 396-403 396 403 396-403
C-I-1 S-I-1 C-1I-1 S-II-1 C-I-2 S-I-2 C-II-2 S-II-2
60 71 89 79 72 77 76 81
10.2 10.2 10.1 10.0 9.4 9.7 9.7 9.7
8.5 8.8 9.7 9.6 7.7 8.6 9.5 9.3
17 14 4 4 18 12 3 4
12 8
100 R + 9 0 0 R d ( C 3 H / R I X101/R1)F~ (SEC/R1 ×C57BL/6)F 1 Control (C3H/R1 x 101/R1)F I ( S E C / R 1 ×C57BL/6)F~ 100 R + 9 0 0 R a ( C 3 H / R I × 101/R1)F~ (SEC/R1 xC57BL/6)F 1 Control (C3H/RIx101/R1)F 1 (SEC/R1 xC67BL/6)F~
201-206 201-206 201-206 201-206 404-411 404-411 404 411 404 411
C-I-3 S-I-3 C-II-3 S-II-3 C-1-4 S-1-4 C-II-4 S-II-4
56 59 68 66 58 55 54 55
9.7 9.2 9.9 10.2 8.8 9.7 9.6 10.0
8.1 8.4 9.6 9.7 7.7 8.9 9.t 9.6
17 8 3 5 13 9 4 3
500 R x 4 e
Control 500 R x 2 d Control
Control 500 R x 4 e Control
500 R × 4 r Control 500 R × 4 r Control
16 13
15 7
(C3H/RI (SEC/R1 (C3H/R1 (SEC/R1 (C3H/R1 (SEC/R1 (C3H/R1 (SEC/R1
x 101/R1)F 1 xC57BL/6)F 1 × 101/RI)F1 x C57BL/6)Fa x 101/R1)F~ x C57BL/6)F1 × 101/R1)F 1 × C57BL/6)F~
202-207 202 207 202-207 202-207 362-371 362-371 362-371 362-371
C-I-5 S-I-5 C-II-5 S-II-5 C-I-6 S-I-6 C-II-6 S-II-6
80 86 77 92 69 91 99 102
9.7 10.0 10.0 10.0 10.0 9.8 10.3 10.5
7.5 8.7 9.7 9.6 7.7 8.6 9.6 10.1
23 13 3 4 23 12 6 4
23 9
(C3H/R1 (SEC/RI (C3H/R1 (SEC/R1 (C3H/R1 (SEC/RI (C3H/R1 (SEC/R1
x 101/R1)F~ xC57BL/6)F 1 x 101/R1)F1 xC57BL/6)F~ x 101/R1)F 1 x C57BL/6)F1 x 101/R1)F l xC57BL/6)F 1
174-181 174-181 174-181 174-181 293-304 293-304 293-304 293 304
C-1-7 S-I-7 C-II-7 S-II-7 C-I-8 S-I-8 C-II-8 S-II-8
81 78 75 81 70 78 76 96
9.0 9.3 9.9 9.5 8.8 9.4 10.1 10.1
6.7 7.6 9.6 9.1 6.7 7.8 9.8 9.8
25 18 3 4 24 17 3 3
30 16
a After last X-ray exposure. h Refer to Table 5 for statistical comparisons. Induced embryonic lethality calculated by the formula:
1
19 8
average living embryos (experimental) average living embryos (control) x 100
a 24-h interval between doses. e 4-week interval between doses. r 8-week interval between doses.
20 15
32 20
305 i n t e r v a l s ) e x p o s u r e s , l o n g e r sterile p e r i o d s w e r e o b s e r v e d for ( S E C / R 1 x C 5 7 B L / 6 ) F 1 t h a n for ( C 3 H / R 1 × 1 0 1 / R 1 ) F 1 males, b u t the r e v e r s e was t r u e in the c a s e of 100 R + 900 R a n d 500 R x 4 (4-week intervals) exposures. All exposures caused testis-weight reductions ( T a b l e 2). T h e s e r e d u c t i o n s w e r e g r e a t e s t a f t e r the 500 R x 4 ( 4 - w e e k i n t e r v a l s ) t r e a t m e n t , a n d s m a l lest a f t e r t h e single 5 0 0 - R e x p o s u r e . T h e y w e r e i n t e r m e d i a t e , a n d r o u g h l y similar, for the o t h e r t h r e e t r e a t m e n t r e g i m e n s . T h e r e are n o statistic a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n the t w o stocks for t e s t i s - w e i g h t c o m p a r i s o n s at a n y of the treatment regimens.
(2) Embryonic mortality E m b r y o n i c m o r t a l i t y a m o n g p r o g e n y sired b y males whose stem-cell spermatogonia had been i r r a d i a t e d was m e a s u r e d at t w o p o s t s t e r i l e p e r i o d s for e a c h e x p o s u r e c o n d i t i o n ( T a b l e 3). T h e e a r l i e r s a m p l e was g e n e r a l l y c o l l e c t e d w h e n at least t w o t h i r d s of the m a l e s h a d sired a litter. T h e t i m e at w h i c h the l a t e r s a m p l e was c o l l e c t e d was c h o s e n a r b i t r a r i l y a n d was to a c e r t a i n e x t e n t i n f l u e n c e d b y t h e a v a i l a b i l i t y of females. T h e single 5 0 0 - R e x p o s u r e was n o t t e s t e d b e c a u s e of the a n t i c i p a t e d d i f f i c u l t y in q u a n t i f y i n g the l o w r e s p o n s e exp e c t e d . T h e o t h e r f o u r e x p o s u r e c o n d i t i o n s ind u c e d s i g n i f i c a n t i n c r e a s e s in e m b r y o n i c m o r t a l i t y a m o n g c o n c e p t u s e s sired b y m a l e s f r o m e i t h e r stock, as i n d i c a t e d b y the s i g n i f i c a n t l y h i g h e r incid e n c e s of d e a d i m p l a n t a t i o n s , a l o n g w i t h signific a n t l y l o w e r n u m b e r s of living e m b r y o s ( T a b l e 4). T h e d i f f e r e n c e s are n o t a l w a y s significant, b u t the t r e n d (Fig. l a - c ) , i n d i c a t e s t h a t at all e x p o s u r e c o n d i t i o n s h i g h e r rates of e m b r y o n i c m o r t a l i t y w e r e i n d u c e d b y i r r a d i a t i o n o f the ( C 3 H / R 1 × 1 0 1 / R 1 ) F 1 s t o c k t h a n of t h e o t h e r stock. R e s u l t s o f t h e e x p e r i m e n t s in the first s a m p l i n g p e r i o d are g e n e r a l l y c o n s i s t e n t w i t h the results for the s e c o n d period.
TABLE 4 STATISTICAL COMPARISONS FOR NUMBER OF LIVING EMBRYOS AND INCIDENCE OF DEAD IMPLANTATIONS Group comparisons a
Decrease in the average Increase in the pernumber of living cent dead implants embryos per pregnant female
C-I-1 vs. C-II-1 1,17 * +0.50 b S-I-1 vs. S-II-1 0.82 * +0.50 C-I-1 vs. S-I-1 0.28+0.52
12.6 * +1.9 c 9.7 * ___1.7 3.4+2.3
C-I-2 vs. C-II-2 1.74 * ___0.49 S-I-2 vs. S-II-2 0.70 * ±0.48 C-I-2 vs. S-I-2 0.85 * + 0.47
15.4 * +1.8 7.2 * + 1.6 6.5 "t + 2.1
C-I-3 vs. C-II-3 1.56 * +0.53 S-I-3 vs. S-II-3 1.24 * +0.54 C-I-3 vs. S-I-3 0.39 + 0.54
14.6 * +2.0 3.8 * + 1.6 8.6 * +_2.3
C-I-4 vs. C-II-4 1.42 * + 0.55 S-I-4 vs. S-II-4 0.73 + 0.58 C-I-4 vs. S-I-4 1.20 * -+0.54
8.2 * _+2.0 5.3 * + 1.6 4.1 + 2.2
C-I-5 vs. C-II-5 2.19 * +0.47 S-I-5 vs. S-II-5 0.86 * +0.45 C-I-5 vs. S-I-5 1.20 t +0.44
19.3 * _+1.9 9.3 * -+ 1.5 9.3 i" -+2.2
C-I-6 vs. C-II-6 1.89 * +0.46 S-I-6 vs. S-II-6 1.47 * -+0.44 C-I-6 vs. S-I-6 0.85-+0.45
16.1 * -+2.0 8.3 * 5:1.4 10.2 t 5:2.2
C-I-7 vs. C-II-7 2.73 * _+0.46 S-I-7 vs. S-II-7 1.55 * -+0.46 C-I-7 vs. S-1-7 0.70-+0.43
22.4 * _+2.0 14.5 * _+1.8 7.5 * _+2.5
C-I-8 vs. C-II-8 3.09 * -+0.47 S-I-8 vs. S-II-8 2.10 * -+0.45 C-I-8 vs. S-I-8 0.99 * -+0.44
22.5 * 5:2.1 14.6 * 5:1.7 8.0 t 5:2.6
a Refer to Table 3 for stock of males and treatment. b Standard deviation of estimate based on assumption of Poisson variation. c Standard deviation of estimate based on assumption of binomial variation. * Significant at the 0.05 level based on a one-sided test of the null hypothesis of no treatment effect. t Significant at the 0.05 level based on a two-sided test of the null hypothesis of no difference in treatment effects between the two stocks of mice.
(3) Cytogenetic analysis of spermatocytes T h e m a l e s w e r e killed for c y t o g e n e t i c a n a l y s i s of t h e i r m e i o c y t e s s h o r t l y a f t e r the c o m p l e t i o n of the f i r s t - p e r i o d e m b r y o n i c m o r t a l i t y analysis, w i t h the e x c e p t i o n of t h o s e in the 100 R + 900 R e x p o s u r e w h i c h w e r e p r e p a r e d m u c h later. T h e 500 R × 4 ( 4 - w e e k i n t e r v a l s ) e x p o s u r e was n o t
a n a l y z e d this time. D a t a r e p o r t e d e a r l i e r ( G e n e r o s o et al., 1985) w e r e u s e d for c o m p a r i s o n b e t w e e n e x p o s u r e c o n d i t i o n s . A g a i n , as in t h e c a s e o f e m b r y o n i c m o r t a l i t y , h i g h e r t r a n s l o c a t i o n freq u e n c i e s w e r e f o u n d at all e x p o s u r e c o n d i t i o n s in t h e ( C 3 H / R 1 x 1 0 1 / R 1 ) F t s t o c k t h a n in t h e
306 ( S E C / R 1 x C 5 7 B L / 6 ) F 1 stock, a l t h o u g h the difference is n o t always significant ( T a b l e 5).
1985; R u t l e d g e et al., 1986). These e n d p o i n t s were embryonic mortality and malformations among c o n c e p t u s e s sired, frequency of t r a n s l o c a t i o n s in s p e r m a t o c y t e s of e x p o s e d males, a n d length of sterile period. C y t o l o g i c a l evidence (Preston a n d Brewen, 1976) suggested that the stem-cell p o p u l a tion has n o t r e t u r n e d to ' n o r m a l ' by 4 weeks after the first d o s e of 500 R. In a d d i t i o n , the lengths of the sterile p e r i o d s f o u n d in the s t u d y of G e n e r o s o et al. (1985) i n d i c a t e d that the length of time n e e d e d for the c o m p l e t i o n of stem-cell r e p o p u l a tion increases with each a d d i t i o n a l 500-R dose.
Discussion
In the earlier studies that h a d revealed differences between the two stocks, ( C 3 H / R 1 x 1 0 1 / R 1 ) F t a n d ( S E C / R 1 x C 5 7 B L / 6 ) F t, the f o r m e r stock e x h i b i t e d higher responses than the l a t t e r in all e n d p o i n t s m e a s u r e d to i n d i c a t e genetic a n d cell killing effects of 500 R x 4 (4-week intervals) in stem-cell s p e r m a t o g o n i a ( G e n e r o s o et al.,
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Fig. 1. (a) Boxplots of the number of implants per pregnant female. (b) Boxplots of the percentage of dead implants per pregnant female. (c) Boxplots of the number of living implants per pregnant female. Rectangular bar contains 50% of the sample in the distribution. Line inside the bar represent the median of the distribution. Numbers 1-8 correspond to the last digit of the group codes in Table 3. (For details of boxplots, see Becker et al., 1988)
307
was included on the assumption (Cattanach et al., 1985) that following the first dose, the surviving stem-cell population is a mixture of formerly radiosensitive and formerly radioresistant cells that had been triggered into a similar degree of sensitivity to translocation induction and cell killing. The remaining exposure condition (500 R x 4, 8week intervals) was included assuming that stemcell repopulation is already completed by the time the second or the subsequent doses are given. The following are the basic findings of the present study. (1) The hybrid stock ( C 3 H / R 1 × 101/R1)F 1 exhibited higher levels of early embryonic mortality among progeny sired at all exposure conditions tested than the hybrid
In the present study, 5 exposure conditions were used, including a repeat of the 500 R × 4 (4-week intervals) regimen. The lowest exposure, a single 500-R dose, was used because it is in the ascending portion of the dose-response curve for induced chromosomal aberrations (Preston and Brewen, 1973), it kills all gonial cells except the resistant isolated A s (Oakberg and Huckins, 1976), and it produces a short sterile period indicating a minimal effect on stem-cell repopulation. The 500 R × 2 (24-h interval) exposure was included on the assumption that the A s cells surviving the first dose are a homogeneous population at the time the second dose is given (Russell, 1962; Oakberg, 1978). The 100 R + 900 R (24-h interval) exposure
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308
( S E C / R 1 × C 5 7 B L / 6 ) F 1. (2) The cytogenetic resuits paralleled those of embryonic mortality at all exposure conditions in which both endpoints were studied. In the case of 500-R single exposure, also, ( C 3 H / R 1 x 101/R1)F~ yields a higher frequency of chromosomal aberrations than does ( S E C / R 1 x C57BL/6)F~ but the difference is not statistically significant. (3) In terms of the length of sterile period, the response of the two stocks relative to each other was not consistent for all exposure conditions tested. (4) No significant differences in testes weight reduction were observed between the two stocks. Thus, both embryonic mortality and cytogenetic data indicate higher sensitivity of the gonial stem cells of ( C 3 H / R 1 x
1 0 1 / R 1 ) F 1 males to induction of chromosomal aberrations with X-rays than of the stem cells of ( S E C / R 1 x C57BL/6)F1 males. On the other hand, the meaning conveyed by the results on testis weight and length of sterile period is unclear. Testis weight as measured in the present study is perhaps the least reliable measure of the degree of gonial stem-cell killing. The total contribution of all stem cells to the weight of the testis is negligible. It seems reasonable to assume that the important factors affecting testis-weight change are the degree of radiation damage to other testicular components, and the status of repopulation from the spermatogonial stem-cell population. The
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309
questions, therefore, are (a) to what extent do gonial stem cells approximate the average response of all testicular components, (b) is the response of each component relative to the rest similar in the two stocks, and (c) does the manner and speed of repopulation differ in the two stocks? Thus, the length of the sterile period is not solely a function of the number of stem cells killed. Conceivably, it could also be a function of the rate of repopulation and the baseline size of the stem-cell population in unexposed males. The lack of parallelism between testis-weight and sterile-period data makes it even more difficult to determine whether there is relationship between stem-cell killing and aberration induction. In order to study this issue definitively it will be essential to perform a direct histological analysis of the A s spermatogonia in the two stocks of mice used in the present study. In our earlier study (Generoso et al., 1985) the question was raised as to whether the basis for the stock difference in the incidence of induced embryonic lethality is the property of the stem cells or of processes subsequent to the formation of aberrations in the exposed stem cell. By estimating the incidences of embryonic lethality expected from the cytological data in the two stocks, and comparing them with those actually observed, we concluded at that time that not only were there
fewer aberrations induced in stem-cell gonia of ( S E C / R 1 x C 5 7 B L / 6 ) F 1 males, but that meiocytes with aberrations gave rise to a lower proportion of unbalanced sperm in ( S E C / R 1 x C 5 7 B L / 6 ) F 1 than in ( C 3 H / R 1 x 1 0 1 / R 1 ) F r Similar comparisons between expected and observed embryonic lethality for three of the exposures in the present study failed to confirm this earlier conclusion. Thus, it appears that the difference between the two stocks in terms of transmitted chromosomal aberrations is attributable solely to the responses of the respective gonial stem cells. In the Introduction, two related issues that are important in evaluating the response of stem-cell spermatogonia to ionizing radiations were discussed in relation to the difference between two stocks in the induction of chromosomal aberrations. The first is the true nature of the stem cell, the second is whether there is any real association between aberration induction and stem-cell killing. While no additional information from the present study contributed directly to the clarification of these issues, the results have made it clear that it will be necessary to perform a detailed, direct cytological analysis of the stem-cell population for this purpose in experiments involving the two stocks of mice and the exposure conditions used in the present report.
TABLE 5 R E C I P R O C A L T R A N S L O C A T I O N S SCORED IN D E S C E N D A N T M E I O C Y T E S O F I R R A D I A T E D S P E R M A T O G O N I A L STEM CELLS Exposure
Stock of mice a
Number of cells with translocations b
N umbe r of trans-
0
1
> 2
locations per cell
2
500 R
( C 3 H / R 1 X 101/R1)F~ ( S E C / R 1 x C57BL/6)F~
257 267
38 30
4 2
1 1
0.16 0.12
500 R × 2 c
( C 3 H / R 1 × 101/R1)F~ ( S E C / R 1 x C57BL/6)F~
199 229
89 61
12 7
0 3
0.38 0.28
100 R × 9 0 0 ¢
( C 3 H / R 1 × 101/R1)F~ (SEC/R1 x C57BL/6)F1
212 255
69 35
14 8
5 2
0.38 e 0.19
500 R x 4 d
( C 3 H / R 1 × 101/R1)F~ (SEC/R1 × C57BL/6)F1
186 206
86 75
22 16
6 3
0.49 e 0.38
a b c d e
e
The posttreatment intervals when testis weight was measured and chromosome preparations were made are shown in Table 2. 300 cells were scored from 6 males (25 cells per testis). 24-h interval between doses. 8-week interval between doses. Significantly different from the value for the other stock ( P < 0.05, two-sided Student's t-test).
310
Acknowledgements We are grateful to Drs. L.B. Russell, G.A. Sega and P.B. Selby for critical review of the manuscript.
References Becker, R.A., J.M. Chambers and A.R. Wilks (1988) The New S Language, Wadsworth and Brooks/Cole, Pacific Grove, CA. 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., C. Jones and D.G. Papworth (1985) Specificlocus mutation response to unequal, 1 + 9 Gy X-ray fractionations at 24-h and 4-day fraction intervals, Mutation Res., 149, 105-118. Cattanach, B.M., C. Rasberry and C. Beechy (1990) Factors affecting mutation induction by X-rays in the spermatogonial stem cells of mice of strain 101/H, in: Biology of Mammalian Germ Cell Mutagenesis, Banbury Report 34, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 209-220. de Rooij, D.G. (1988) Regulation of the proliferation of spermatogonial stem cells, J. Cell Sci., Suppl. 10, 181 194. de Rooij, D.G., Y. van der Meer, A.M.M. van Pelt and P.P.W. van Buul (1990) Correlation between proliferative activity of mouse spermatogonial stem cells and their sensitivity for cell killing and induction of reciprocal translocations by irradiation, in: Biology of Mammalian Germ Cell Mutagenesis, Banbury Report 34, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 35-49. Evans, E.P., G. Breckon and C.E. Ford (1964) An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3, 289-294. Generoso, W.M., K.T. Cain, C.V. Cornett, N.L.A. Cacheiro, L.A. Hughes and P.W. Braden (1985) Difference in the response of two hybrid stocks of mice to X-ray induction of chromosome aberrations in spermatogonial stem cells, Mutation Res., 152, 217-223. Huckins, C. (1971) The spermatogonial stem cell population in
adult rats, III. Evidence for a long-cycling population, ('ell Tissue Kinet., 4, 335-349. Huckins, C. (1978) Behavior of stem-cell spermatogonia in the adult rat irradiated testis, Biol. Reprod., 19, 747-760. Huckins, C., and E.F. Oakberg (1978) Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tabules, II. The irradiated testis, Anat. Rec., 192, 529-542. Lok, D., M.T. Jansen and D.G. de Rooij (1983) Spermatogonial multiplication in the Chinese hamster, II. Cell cycle properties of undifferentiated spermatogonia, Cell Tissue Kinet., 16, 19-29. Oakberg, E.F. (1971) Spermatogonial stem cell renewal in the mouse, Anat. Rec., 169, 515-532. Oakberg, E.F. (1978) Differential spermatogonial stem-cell survival and mutation frequency, Mutation Res., 50, 327340. Oakberg, E.F. (1984) Germ cell toxicity: Significance in genetic and fertility effects of radiation and chemicals, in: E.H.Y. Chu and W.M. Generoso (Eds.), Mutation, Cancer, and Malformation, Plenum, New York, pp. 549-590. Oakberg, E.F., and C. Huckins (1976) Spermatogonial stem cell renewal in the mouse, as revealed by 3H-thymidine labeling and irradiation, in: A.B. Cairnie, P.K. Lala, and D.G. Osmond (Eds.), Stem Cells of Renewing Cell Populations, Academic Press, New York, pp. 287-302. Preston, R.J., and J.G. Brewen (1973) X-ray-induced translocations in spermatogonia, 1. Dose and fractionation responses in mice, Mutation Res., 19, 215-223. Preston, R.J., and J.G. Brewen (1976) X-Ray-induced translocations in spermatogonia, il. Fractionation responses in mice, Mutation Res., 36, 333 344. Russell, W.L. (1962) An augmenting effect of dose fractionation on radiation-induced mutation rate in mice, Proc. Natl. Acad. Sci. (U.S.A), 48, 1724-1727. 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. van Buul, P.P.W. (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, Liss, New York, pp. 369-400.
