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Chromosome rearrangements induced in the mouse by embryonic X-irradiation I. Pronuclear stage
Mutation Research
689
Elsevier P u b l i s h i n g C o m p a n y , A m s t e r d a m P r i n t e d in T h e N e t h e r l a n d s
CHROMOSOME REARRANGEMENTS INDUCED IN T H E MOUSE BY EMBRYONIC X-IRRADIATION I. PRONUCLEAR STAGE A. L E O N A R D AND Ga. D E K N U D T
Laboratory of Genetics, Department of Radiobiology, Centre for Studies on Nuclear Energy, Mol (Belgium) (Received M a r c h I3th, 1967)
SUMMARY
BALB/c females with vaginal plugs (day o.5 after copulation) received X-irradiation to the whole body. IOO R killed about 45% of the embryos. 38 males and 24 females irradiated at the pronuclear stage were mated with control mice from the same strain, and the testes of their sons were examined for the presence of chromosome rearrangements. Three males irradiated at day o.5 were found to be heterozygous for different reciprocal translocations. The overall frequency of induction of translocations per genome was 2.53%. Mitotic preparations made from bone-marrow of the 3 translocated animals showed no recognizably abnormal chromosomes.
INTRODUCTION
Experiments performed on mammals have shown that the effects of X-irradiation on embryos are closely related to the developmental stage at the time of exposure. At dose levels sufficient to kill some of the developing embryos the lethality is greatest when the X-irradiation is given during the preimplantation period, but according to the first reports almost all the survivors are normal with respect to their external characters2,8, xz-ls. However, RUGH AND G R U P P n have found that irradiation before implantation might also induce severe congenital anomalies. The irradiation of postimplantation stages causes considerably less prenatal death, but congenital anomalies occur more frequently 1.-14. Our experiments were performed to study the possibility of inducing some viable and transmissible chromosome rearrangements by irradiation during the preimplantation period. The results reported here concern the X-irradiation of precleavage but fertilized mouse eggs at day 0.5 after conception. MATERIAL AND METHODS
7o-day-old BALB/c mice were allowed to mate from 5 p.m. to 9 a.m. the next morning. Females with a vaginal plug were irradiated at IO a.m. with IooR to the Mutation Res., 4 (1967) 689-697
690
A. L E O N A R D ,
GH. DEKNUDT
whole body. For X-irradiation,(300 kV, 20 mA, filtration 2 mm Cu, dose rate IOO R/miD) the pregnant females were placed in the compartments of a plastic wheel. Litter size was recorded at birth, and the offspring were sexed at weaning 20 days later. The animals derived from the irradiated eggs were mated at 7 ° days of age with control animals from the same strain and tested for fertility b y examination of the first and second litters. The male offspring were killed when mature, their testes were removed, and meiotic preparations were made by the technique of EVANS et al. 3. Bone-marrow preparations were made from males irradiated in utero, whose male offspring showed multivalent configurations, b y an air-drying method based on the procedure of FORD AND HAMERTON5. The preparations were stained with toluidine blue. RESULTS
Table I shows that when females were given a whole-body dose of i o o R on day 0.5 of gestation, the litter size was reduced b y about 450/0 . 38 males and 24 females irradiated at the pronuclear stage survived and were m a t e d with control mice from the s a m e strain. No sterile animal was recorded and, as shown in Table II, the m e a n litter size of the survivors was not altered. The testes of 141 sons of the 38 males and of IOO sons of the 24 females were e x a m i n e d for the presence of c h r o m o s o m e rearrangements. Table I I I gives the distriTABLE
I
LITTER SIZE AND SEX RATIO AFTER IOO R GIVEN AT 0. 5 DAY OF GESTATION
Treatment
Pregnaneies
Number delivered
Litter average
Dead in 2o days Number Percentage
Sex ratio at weaning Number Percentage of males
Controls ioo R
21 18
148 7°
7.05 3.89
2 8
7° 38
TABLE
1.35 11. 4
47.9 61. 3
II
FERTILITY OF THE SURVIVORS
Obserration
Treatment
Number Number Litter of delivered average animals
Dead in 2o days Number Percentage
Sex ratio at weaning Number Percenof males rage
First and second litters
Controls Irradiated males Irradiated females
26
369
14.2 -k 0 . 6
44
11.9
186
57-2
38
540
14.2 k
0. 7
38
7. o
262
52.2
24
362
15.1 - - o . 9
35
9.7
16o
48.9
TABLE
III
DISTRIBUTION OF THE IRRADIATED MALES AND FEMALES ACCORDING TO THE NUMBER OF EXAMINED SONS
Sex of the irradiated parent
Number of examined sons I 2 3
4
5
6
7
8
z5
Males Females
I -
8 3
4 6
2 3
3 -
i -
i
Io 8
Mutation Res., 4 ( 1 9 6 7 ) 6 8 9 - 6 9 7
9 3
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X-IRRADIATION
691
t q
b
J
b Fig. i. Mitosis from T2 ALD.
O
O O
,I
O
O
Fig. 2. Ring quadrivalent and 18 bivalents at first meiotic metaphase in T2 ALD. b u t i o n of t h e females a n d m a l e s i r r a d i a t e d a t t h e p r o n u c l e a r stage, according to t h e n u m b e r of e x a m i n e d sons. 50 s p e r m a t o c y t e s a t diakinesis-first m e t a p h a s e were scored for each son. W h e r e a s no c h r o m o s o m e r e a r r a n g e m e n t was f o u n d in t h e s p e r m a t o c y t e s of t h e sons of i r r a d i a t e d females, some sons of 3 i r r a d i a t e d males showed s p e r m a t o c y t e s m o s t l y h a v i n g t r a n s l o c a t i o n configurations. The t r a n s l o c a t i o n h e t e r o z y g o t e Mutation Res., 4 (r967) 689-697
692
A. LEONARD, GH. DEKNUDT
|
Fig. 3. Mitosis from T 3 A L D .
~i ~i~i~i~!~~~!iii!i~i~!~!ii~i~!~i~!~ili@ i'i~!~i ~ii~i~i~!~i~i~~ ~!i~!!~i~ i ~~ ii Fig. 4. Ring quadrivalent and 18 bivalents at first meiotic metaphase in T 3 ALD.
males originated from 3 different pregnant females irradiated at d a y 0.5 of gestation after copulation with 3 different control males. The translocatiQns were n u m b e r e d T2 ALD, T3 ALD, T4 ALD. 5 male offspring from T2 A L D were analyzed, and 3 were found carrying the translocation; the translocation was present in the 2 scored males from T 3 ALD, and 2/4 male offspring from T 4 A L D showed chromosome rearrangements. Mutation Res., 4 (1967) 689-697
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X-IRRADIATION
693
Ill
Q6
Pq
Q I w
I
Fig. 5. Mitosis from T 4 ,~kLl).
Fig. 6. Ring quadrivalent and 18 bivalents at first meiotic metaphase in T 4 ALD. The o b s e r v a t i o n s at meiosis in m a l e h e t e r o z y g o t e s from all 3 t r a n s l o c a t i o n lines are s u m m a r i z e d in T a b l e IV. Ring q u a d r i v a l e n t s (R IV) were t h e p r i n c i p a l a b n o r m a l configurations o b s e r v e d i n each line. Chain q u a d r i v a l e n t s (Ch IV) a n d cells with 20 b i v a l e n t s (20 II) or with a t r i v a l e n t + u n i v a l e n t (III + I) were also observed. As shown in t h e s a m e table, the p r o p o r t i o n of t h e different t y p e s of configuration in t h e i r r a d i a t e d males, c o m p a r e d w i t h t h e p r o p o r t i o n s in t h e i r t r a n s l o c a t i o n - c a r r y i n g Mutation Res., 4 (1967) 689-697
694
A. L E O N A R D , GH. D E K N U D T
F i g . 7. C h a i n q u a d r i v a l e n t a n d 18 b i v a l e n t s a t f i r s t m e i o t i c m e t a p h a s e i n T 4 A L D .
5 4 .Q
E
3 ¸
2¸ 1
0
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7¸
4
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3 E
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,
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m
m
Mean
, t
litter
o
size
F i g . 8. F r e q u e n c y d i s t r i b u t i o n of t h e l i t t e r sizes a t b i r t h of first a n d s e c o n d l i t t e r s .
Mutation Res., 4 (1967) 689-4597
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X - I R R A D I A T I O N TABLE
IV
ANALYSIS
OF THE
Translocatio~
Animal
T2 A L D
Irradiated males T r a n s l o c a t e d sons Irradiated males T r a n s l o c a t e d sons Irradiated males T r a n s l o c a t e d sons
T 3 ALD T 4 ALI)
695
SPERMATOCYTES
OF THE
TRANSLOCATED
MALES
Total number of cells
Configurations 20 I I 18 I I + R I V
40o 400 400 400 400 400
39 34 147 119 6I 86
(9-8°0) (8.5°o) (36.7%) (29.7%) (15. 3 o!o)
(21.5°o)
333 333 232 252 227 173
(83-2%) (83.2%) (58.0%) (63.0o/o) (56.7%) (43'20o)
I8II+ChlV
28 33 21 28 ili 137
(7.o%) (8.3%) o/ (5.3/o) (7.0%) (27.7%) o/ (34'3/0)
I8II+ Ch I i 1 -+-I
1 (0.3%) i (0.3%) 4 (I'°°/O)
sons, was almost the same. 50 meiotic metaphases of irradiated males with normal progeny were examined; no chromosome rearrangement was detected. Mitotic preparations made from bone-marrow showed no recognizably abnormal chromosome in any of the 3 translocations, and it was therefore impossible to determine which chromosomes were involved in the translocation configurations. The first results show that the fertility of the translocated males appears to be lower than the normal. The frequency distribution of the litter sizes at birth of first and second litters, either in the irradiated males or in the irradiated females, was not clearly bimodal probably owing to the low incidence of semisterility. DISCUSSION
The female mouse does not generally allow mating unless she is in oestrus and mature ova are available; hence the presence of a vaginal plug indicates successful copulationl*, TM. There could be a disparity of several hours in fertilization but, since the first cleavage of the fertilized eggs occurs generally about 24 h after copulation and the female and male pronuclei remain distinct until about I h before the formation of the first cleavage spindle~,~°,x7, TM, the X-irradiation was given at the pronuclear stages. RUGtI AND WOHLFROMM12 report 46% of dead embryos after 250 R of X-irradiation given at day 0. 5 of pregnancy, and RUGH et al2 15.6% after IOO R. After 200 R at day o.5, RUSSELL AND RUSSELLle found the average number of living embryos per treated female was only about 20% of the controls. From those data, the BALB/c strain with 44.9% dead after ioo R shows a high radiosensitivity. In order to detect relatively great effects on the sex ratio it is necessary, as pointed out by RUSSELL AND R U S S E L L 16, that experiments be done on a large scale. The variation in the sex ratio of the controls shows (Tables I and II) that the material was too limited to draw any conclusion in that field. However, after 25 ° R at day 0.5 of pregnancy, R U G H AND WOHFROMM 12 found among the survivors that the sex ratio was considerably upset in favour of the females. The same observation is reported by RUGH et al2 after IOO R. As stated by FALCONER 4, the mutation rate expressed as the number of mutations that have taken place per gamete has precise meaning only when the sample of gametes on which it is based has been completely analysed. Since one would only expect half the surviving progeny of a translocation heterozygote to be heterozygous, the number of fully tested gametes depends on the distribution of I, 2, 3... tested Mutation Res., 4 (I967) 689-697
696
A. L E O N A R D , GH. D E K N U D T
sons and the probabilities of parental translocation heterozygosis when i, 2, 3... sons are shown not to be heterozygous. The probability that an FI family containing n males will reveal a visible translocation present in the irradiated parent will be 0. 5 for one analysed son, 0.75 for two, 0.875 for three, etc... The testes of the males irradiated at day 0.5 of gestation were examined for the presence of translocation and their gametes were completely analysed. Therefore, the numbers of fully-tested gametes to which our samples were equivalent were 38 for the males and 21.2 for the females out of a sample of 24 after estimation by the method of FALCONERa. Since male and female pronuclei were irradiated, the frequency of translocation per genome was estimated by the number of translocations found divided by twice the equivalent number of fully-tested gametes. The frequency of translocation per genome in the males 3/(2 x 38) and in the females 0/(2 x 21.2) was not significantly different (t = 1.o6; P < o.3), and the overall frequency of translocations per genome is 3/(2 x (38+21.2)) = 2.53%. From mating during the pre-sterile period, RUSSELL19 obtained 25% of semisterile after 500 R; 29.4% after 75 ° R and 25% after iooo R. From the results of SNELL AND AMES 21, the incidence of semi-sterility in the offspring of females irradiated with X-rays appears to be lower (2.0% after 240-280 R) than would be expected from males irradiated with the same dose; and by comparison with the results on Drosophila, RUSSELL*° concludes that translocations induced in females m a y be much less frequent than in the males. However, even though the male mature germ-cells appear to be more radiosensitive to the induction of chromosome translocations than the female ones,some chromosome anomalies induced in the female pronucleus are reported by RussELL AND SAYLORS 18, who estimate that with IOO R at i i . o o a.m. after discovery of a vaginal plug, 3.3% of paternal X-chromosomes (~ pronucleus) and 1.9% of maternal X-chromosomes (~ pronucleus) were lost. LYON et al.' found an X-autosome translocation induced in the female pronucleus after IOO R at the pronuclear stage. Therefore, either pronucleus m a y have been involved. Since previous work on semi-sterility induced by irradiation did not include cytological examination, and since it is possible for some animals to be semi-sterile without formation of quadrivalent configurations, it is difficult to establish a quantitative comparison between our results and previous experiments. However, trom our results, the radiosensitivity of the pronuclear stage to the induction of translocation appears to be very high. ACKNOWLEDGEMENT
We are indebted to Dr. A. G. SEARLE and to Dr. C. E. FORD for drawing our attention to the need for this type of experiment and for their helpful criticisms during the preparation of this paper, and to Professor F. H. SOBELS and Dr. R. K. APPLEYARD for their interest. This work was supported in part b y Research Contract No. o53-64-3-BIOB Euratom-C.E.N. and b y grants from the Fonds de la Recherche Scientifique Fondamentale Collective. Mutation Res., 4 (1967) 689-697
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X - I R R A D I A T I O N
697
REFERENCES I AUSTIN, C. R., T h e f o r m a t i o n , g r o w t h a n d c o n j u g a t i o n of t h e pronuclei in t h e r a t egg, J. Roy. Microscop. Sue., 71 (1951) 295-3o6. 2 BURCKHARD, G., ~)ber der Einfluss der R 6 n t g e n s t r a h l e n a u f d e n tierisehen O r g a n i s m u s , insbes o n d e r e a u f die GraviditXt, Volkmanns Sarnml. Klin. Vortrage, 40o (19o5) 469-480. 3 EVANS, E. P., G. BRECKON AND C. E. FORD, A n a i r - d r y i n g m e t h o d for meiotic p r e p a r a t i o n s f r o m m a m m a l i a n testes, Cytogenetics, 3 (1964) 289-294. 4 FALCONER, D. S., T h e e s t i m a t i o n of m u t a t i o n r a t e s f r o m i n c o m p l e t e l y t e s t e d g a m e t e s , a n d t h e d e t e c t i o n of m u t a t i o n s in m a m m a l s , J. Genet., 49 (1949) 226-234. 5 FORD, C. E., AND J. L. HAMERTON, A colchicine h y p o t o n i c citrate, s q u a s h s e q u e n c e for m a m maliafi c h r o m o s o m e s , Stain Technol., 31 (1956) 247-251. 6 LYON, M. F., R. J. S. PHILLIPS AND A. G. SEARLE, T h e overall r a t e s of d o m i n a n t a n d recessive lethal a n d visible m u t a t i o n i n d u c e d b y s p e r m a t o g o n i a l X - i r r a d i a t i o n of mice, Genet. Res., 5 (1964) 448-467 • 7 L Y o n , M. F., A. G. SEARLE, C. E. FORD AND S. OHNO, A 1DOUSet r a n s l o c a t i o n s u p p r e s s i n g sexlinked variegation, Cytogenetics, 3 (1964) 306-323 • 8 PARKES, A. S., O n t h e occurrence of t h e oestrous-cycle after X - r a y sterilization, II. I r r a d i a t i o n a t or before birth, Proc. Roy. Soc. (Lond.), IOi (1927) 71-95 . 9 RUGH, R., DUHAMEL, L., A. W. OSBORNE AND A. VARNA, P e r s i s t e n t s t u n t i n g following X-irradia-tion of t h e foetus, Amer. J. Anat., 115 (1964) 185-198. io RUGH, R., AND E. GRUPP, Effect of low level-X-irrad i a t i o n o n t h e fertilized egg of t h e m a m m a l , Exptl. Cell Res., 25 (1961) 3o2-31o. I I RUGH, R., AND E. GRUPP, Low levels of X - i r r a d i a t i o n a n d t h e e a r l y m a m m a l i a n e m b r y o , Am. J. Roentgenol. Radium Therwpy Nucl. Med., 87 (1962) 559-566. 12 RUGH, R., AND M. WOHLFROMM, Can t h e m a m m a l i a n e m b r y o be killed b y X - i r r a d i a t i o n ? J. Exptl. Zool., 151 (1962) 227-243. 13 RUSSELL, L. B., X - r a y i n d u c e d d e v e l o p m e n t a l a b n o r m a l i t i e s in t h e m o u s e a n d t h e i r u s e in t h e a n a l y s i s of e m b r y o l o g i c a l p a t t e r n s , I. E x t e r n a l a n d gross visceral changes, J. Exptl. Zool., 114 (195 ° ) 545-602. 14 RUSSELL, L. B., Effects of r a d i a t i o n on m a m m a l i a n p r e n a t a l d e v e l o p m e n t , in A. HOLLAENDER (Ed.), Radiation Biology, McGraw-Hill, N e w York, 1954, P. 861-918. 15 RUSSELL, L. B., AND W. L. RUSSELL, T h e effects of r a d i a t i o n on t h e p r e i m p l a n t a t i o n s t a g e s of t h e m o u s e e m b r y o , Anat. Rec., lO8 (195 o) 521. 16 RUSSELL, L. B., AND Vq. L. RUSSELL, A n a n a l y s i s of t h e c h a n g i n g r a d i a t i o n response of t h e d e v e l o p i n g m o u s e e m b r y o , J. Cellular Cutup. Physiol., 43, Suppl. i (1954) lO3-149. 17 RUSSELL, L. B., AND W. L. RUSSELL, P a t h w a y s of r a d i a t i o n effects in t h e m o t h e r a n d t h e e m b r y o , Cold Spring Harbor Syrup. Quant. Biol., 19 (1954) 5o-59 • I8 RUSSELL, L. B., AND C. L. SAYLORS, T h e relative s e n s i t i v i t y of v a r i o u s germ-cell s t a g e s of t h e m o u s e to r a d i a t i o n - i n d u c e d n o n disfunction, c h r o m o s o m e losses a n d deficiencies, in F. H. SOBELS (Ed.), Repair from Genetic Radiation Damage and Dil~erential Radiosensitivity in Germ-Cells, Oxford, 1963, p. 313-34 o. 19 RUSSELL, W. L., T h e incidence of sterility a n d p a r t i a l sterility in t h e d e s c e n d a n t s of X - i r r a d i a t e d mice, Genetics, 35 (195 o) 689. 20 RUSSELL, W. L., Genetic effects of r a d i a t i o n in m a m m a l s , in A. HOLLAENDER (Ed.), Radiation Biology, McGraw-Hill, N e w York, 1954, p. 825-859. 2I SNELL, G. D., AND F. B. AMES, H e r e d i t a r y c h a n g e s in t h e d e s c e n d a n t s of female mice e x p o s e d to r o e n t g e n rays, Am. J. Roentgenol. Radium Therapy Nucl. Med., 41 (1939) 248-255.
Mutation Res., 4 (1967) 689-697
689
Elsevier P u b l i s h i n g C o m p a n y , A m s t e r d a m P r i n t e d in T h e N e t h e r l a n d s
CHROMOSOME REARRANGEMENTS INDUCED IN T H E MOUSE BY EMBRYONIC X-IRRADIATION I. PRONUCLEAR STAGE A. L E O N A R D AND Ga. D E K N U D T
Laboratory of Genetics, Department of Radiobiology, Centre for Studies on Nuclear Energy, Mol (Belgium) (Received M a r c h I3th, 1967)
SUMMARY
BALB/c females with vaginal plugs (day o.5 after copulation) received X-irradiation to the whole body. IOO R killed about 45% of the embryos. 38 males and 24 females irradiated at the pronuclear stage were mated with control mice from the same strain, and the testes of their sons were examined for the presence of chromosome rearrangements. Three males irradiated at day o.5 were found to be heterozygous for different reciprocal translocations. The overall frequency of induction of translocations per genome was 2.53%. Mitotic preparations made from bone-marrow of the 3 translocated animals showed no recognizably abnormal chromosomes.
INTRODUCTION
Experiments performed on mammals have shown that the effects of X-irradiation on embryos are closely related to the developmental stage at the time of exposure. At dose levels sufficient to kill some of the developing embryos the lethality is greatest when the X-irradiation is given during the preimplantation period, but according to the first reports almost all the survivors are normal with respect to their external characters2,8, xz-ls. However, RUGH AND G R U P P n have found that irradiation before implantation might also induce severe congenital anomalies. The irradiation of postimplantation stages causes considerably less prenatal death, but congenital anomalies occur more frequently 1.-14. Our experiments were performed to study the possibility of inducing some viable and transmissible chromosome rearrangements by irradiation during the preimplantation period. The results reported here concern the X-irradiation of precleavage but fertilized mouse eggs at day 0.5 after conception. MATERIAL AND METHODS
7o-day-old BALB/c mice were allowed to mate from 5 p.m. to 9 a.m. the next morning. Females with a vaginal plug were irradiated at IO a.m. with IooR to the Mutation Res., 4 (1967) 689-697
690
A. L E O N A R D ,
GH. DEKNUDT
whole body. For X-irradiation,(300 kV, 20 mA, filtration 2 mm Cu, dose rate IOO R/miD) the pregnant females were placed in the compartments of a plastic wheel. Litter size was recorded at birth, and the offspring were sexed at weaning 20 days later. The animals derived from the irradiated eggs were mated at 7 ° days of age with control animals from the same strain and tested for fertility b y examination of the first and second litters. The male offspring were killed when mature, their testes were removed, and meiotic preparations were made by the technique of EVANS et al. 3. Bone-marrow preparations were made from males irradiated in utero, whose male offspring showed multivalent configurations, b y an air-drying method based on the procedure of FORD AND HAMERTON5. The preparations were stained with toluidine blue. RESULTS
Table I shows that when females were given a whole-body dose of i o o R on day 0.5 of gestation, the litter size was reduced b y about 450/0 . 38 males and 24 females irradiated at the pronuclear stage survived and were m a t e d with control mice from the s a m e strain. No sterile animal was recorded and, as shown in Table II, the m e a n litter size of the survivors was not altered. The testes of 141 sons of the 38 males and of IOO sons of the 24 females were e x a m i n e d for the presence of c h r o m o s o m e rearrangements. Table I I I gives the distriTABLE
I
LITTER SIZE AND SEX RATIO AFTER IOO R GIVEN AT 0. 5 DAY OF GESTATION
Treatment
Pregnaneies
Number delivered
Litter average
Dead in 2o days Number Percentage
Sex ratio at weaning Number Percentage of males
Controls ioo R
21 18
148 7°
7.05 3.89
2 8
7° 38
TABLE
1.35 11. 4
47.9 61. 3
II
FERTILITY OF THE SURVIVORS
Obserration
Treatment
Number Number Litter of delivered average animals
Dead in 2o days Number Percentage
Sex ratio at weaning Number Percenof males rage
First and second litters
Controls Irradiated males Irradiated females
26
369
14.2 -k 0 . 6
44
11.9
186
57-2
38
540
14.2 k
0. 7
38
7. o
262
52.2
24
362
15.1 - - o . 9
35
9.7
16o
48.9
TABLE
III
DISTRIBUTION OF THE IRRADIATED MALES AND FEMALES ACCORDING TO THE NUMBER OF EXAMINED SONS
Sex of the irradiated parent
Number of examined sons I 2 3
4
5
6
7
8
z5
Males Females
I -
8 3
4 6
2 3
3 -
i -
i
Io 8
Mutation Res., 4 ( 1 9 6 7 ) 6 8 9 - 6 9 7
9 3
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X-IRRADIATION
691
t q
b
J
b Fig. i. Mitosis from T2 ALD.
O
O O
,I
O
O
Fig. 2. Ring quadrivalent and 18 bivalents at first meiotic metaphase in T2 ALD. b u t i o n of t h e females a n d m a l e s i r r a d i a t e d a t t h e p r o n u c l e a r stage, according to t h e n u m b e r of e x a m i n e d sons. 50 s p e r m a t o c y t e s a t diakinesis-first m e t a p h a s e were scored for each son. W h e r e a s no c h r o m o s o m e r e a r r a n g e m e n t was f o u n d in t h e s p e r m a t o c y t e s of t h e sons of i r r a d i a t e d females, some sons of 3 i r r a d i a t e d males showed s p e r m a t o c y t e s m o s t l y h a v i n g t r a n s l o c a t i o n configurations. The t r a n s l o c a t i o n h e t e r o z y g o t e Mutation Res., 4 (r967) 689-697
692
A. LEONARD, GH. DEKNUDT
|
Fig. 3. Mitosis from T 3 A L D .
~i ~i~i~i~!~~~!iii!i~i~!~!ii~i~!~i~!~ili@ i'i~!~i ~ii~i~i~!~i~i~~ ~!i~!!~i~ i ~~ ii Fig. 4. Ring quadrivalent and 18 bivalents at first meiotic metaphase in T 3 ALD.
males originated from 3 different pregnant females irradiated at d a y 0.5 of gestation after copulation with 3 different control males. The translocatiQns were n u m b e r e d T2 ALD, T3 ALD, T4 ALD. 5 male offspring from T2 A L D were analyzed, and 3 were found carrying the translocation; the translocation was present in the 2 scored males from T 3 ALD, and 2/4 male offspring from T 4 A L D showed chromosome rearrangements. Mutation Res., 4 (1967) 689-697
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X-IRRADIATION
693
Ill
Q6
Pq
Q I w
I
Fig. 5. Mitosis from T 4 ,~kLl).
Fig. 6. Ring quadrivalent and 18 bivalents at first meiotic metaphase in T 4 ALD. The o b s e r v a t i o n s at meiosis in m a l e h e t e r o z y g o t e s from all 3 t r a n s l o c a t i o n lines are s u m m a r i z e d in T a b l e IV. Ring q u a d r i v a l e n t s (R IV) were t h e p r i n c i p a l a b n o r m a l configurations o b s e r v e d i n each line. Chain q u a d r i v a l e n t s (Ch IV) a n d cells with 20 b i v a l e n t s (20 II) or with a t r i v a l e n t + u n i v a l e n t (III + I) were also observed. As shown in t h e s a m e table, the p r o p o r t i o n of t h e different t y p e s of configuration in t h e i r r a d i a t e d males, c o m p a r e d w i t h t h e p r o p o r t i o n s in t h e i r t r a n s l o c a t i o n - c a r r y i n g Mutation Res., 4 (1967) 689-697
694
A. L E O N A R D , GH. D E K N U D T
F i g . 7. C h a i n q u a d r i v a l e n t a n d 18 b i v a l e n t s a t f i r s t m e i o t i c m e t a p h a s e i n T 4 A L D .
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Mutation Res., 4 (1967) 689-4597
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X - I R R A D I A T I O N TABLE
IV
ANALYSIS
OF THE
Translocatio~
Animal
T2 A L D
Irradiated males T r a n s l o c a t e d sons Irradiated males T r a n s l o c a t e d sons Irradiated males T r a n s l o c a t e d sons
T 3 ALD T 4 ALI)
695
SPERMATOCYTES
OF THE
TRANSLOCATED
MALES
Total number of cells
Configurations 20 I I 18 I I + R I V
40o 400 400 400 400 400
39 34 147 119 6I 86
(9-8°0) (8.5°o) (36.7%) (29.7%) (15. 3 o!o)
(21.5°o)
333 333 232 252 227 173
(83-2%) (83.2%) (58.0%) (63.0o/o) (56.7%) (43'20o)
I8II+ChlV
28 33 21 28 ili 137
(7.o%) (8.3%) o/ (5.3/o) (7.0%) (27.7%) o/ (34'3/0)
I8II+ Ch I i 1 -+-I
1 (0.3%) i (0.3%) 4 (I'°°/O)
sons, was almost the same. 50 meiotic metaphases of irradiated males with normal progeny were examined; no chromosome rearrangement was detected. Mitotic preparations made from bone-marrow showed no recognizably abnormal chromosome in any of the 3 translocations, and it was therefore impossible to determine which chromosomes were involved in the translocation configurations. The first results show that the fertility of the translocated males appears to be lower than the normal. The frequency distribution of the litter sizes at birth of first and second litters, either in the irradiated males or in the irradiated females, was not clearly bimodal probably owing to the low incidence of semisterility. DISCUSSION
The female mouse does not generally allow mating unless she is in oestrus and mature ova are available; hence the presence of a vaginal plug indicates successful copulationl*, TM. There could be a disparity of several hours in fertilization but, since the first cleavage of the fertilized eggs occurs generally about 24 h after copulation and the female and male pronuclei remain distinct until about I h before the formation of the first cleavage spindle~,~°,x7, TM, the X-irradiation was given at the pronuclear stages. RUGtI AND WOHLFROMM12 report 46% of dead embryos after 250 R of X-irradiation given at day 0. 5 of pregnancy, and RUGH et al2 15.6% after IOO R. After 200 R at day o.5, RUSSELL AND RUSSELLle found the average number of living embryos per treated female was only about 20% of the controls. From those data, the BALB/c strain with 44.9% dead after ioo R shows a high radiosensitivity. In order to detect relatively great effects on the sex ratio it is necessary, as pointed out by RUSSELL AND R U S S E L L 16, that experiments be done on a large scale. The variation in the sex ratio of the controls shows (Tables I and II) that the material was too limited to draw any conclusion in that field. However, after 25 ° R at day 0.5 of pregnancy, R U G H AND WOHFROMM 12 found among the survivors that the sex ratio was considerably upset in favour of the females. The same observation is reported by RUGH et al2 after IOO R. As stated by FALCONER 4, the mutation rate expressed as the number of mutations that have taken place per gamete has precise meaning only when the sample of gametes on which it is based has been completely analysed. Since one would only expect half the surviving progeny of a translocation heterozygote to be heterozygous, the number of fully tested gametes depends on the distribution of I, 2, 3... tested Mutation Res., 4 (I967) 689-697
696
A. L E O N A R D , GH. D E K N U D T
sons and the probabilities of parental translocation heterozygosis when i, 2, 3... sons are shown not to be heterozygous. The probability that an FI family containing n males will reveal a visible translocation present in the irradiated parent will be 0. 5 for one analysed son, 0.75 for two, 0.875 for three, etc... The testes of the males irradiated at day 0.5 of gestation were examined for the presence of translocation and their gametes were completely analysed. Therefore, the numbers of fully-tested gametes to which our samples were equivalent were 38 for the males and 21.2 for the females out of a sample of 24 after estimation by the method of FALCONERa. Since male and female pronuclei were irradiated, the frequency of translocation per genome was estimated by the number of translocations found divided by twice the equivalent number of fully-tested gametes. The frequency of translocation per genome in the males 3/(2 x 38) and in the females 0/(2 x 21.2) was not significantly different (t = 1.o6; P < o.3), and the overall frequency of translocations per genome is 3/(2 x (38+21.2)) = 2.53%. From mating during the pre-sterile period, RUSSELL19 obtained 25% of semisterile after 500 R; 29.4% after 75 ° R and 25% after iooo R. From the results of SNELL AND AMES 21, the incidence of semi-sterility in the offspring of females irradiated with X-rays appears to be lower (2.0% after 240-280 R) than would be expected from males irradiated with the same dose; and by comparison with the results on Drosophila, RUSSELL*° concludes that translocations induced in females m a y be much less frequent than in the males. However, even though the male mature germ-cells appear to be more radiosensitive to the induction of chromosome translocations than the female ones,some chromosome anomalies induced in the female pronucleus are reported by RussELL AND SAYLORS 18, who estimate that with IOO R at i i . o o a.m. after discovery of a vaginal plug, 3.3% of paternal X-chromosomes (~ pronucleus) and 1.9% of maternal X-chromosomes (~ pronucleus) were lost. LYON et al.' found an X-autosome translocation induced in the female pronucleus after IOO R at the pronuclear stage. Therefore, either pronucleus m a y have been involved. Since previous work on semi-sterility induced by irradiation did not include cytological examination, and since it is possible for some animals to be semi-sterile without formation of quadrivalent configurations, it is difficult to establish a quantitative comparison between our results and previous experiments. However, trom our results, the radiosensitivity of the pronuclear stage to the induction of translocation appears to be very high. ACKNOWLEDGEMENT
We are indebted to Dr. A. G. SEARLE and to Dr. C. E. FORD for drawing our attention to the need for this type of experiment and for their helpful criticisms during the preparation of this paper, and to Professor F. H. SOBELS and Dr. R. K. APPLEYARD for their interest. This work was supported in part b y Research Contract No. o53-64-3-BIOB Euratom-C.E.N. and b y grants from the Fonds de la Recherche Scientifique Fondamentale Collective. Mutation Res., 4 (1967) 689-697
CHROMOSOME REARRANGEMENTS AFTER EMBRYONIC X - I R R A D I A T I O N
697
REFERENCES I AUSTIN, C. R., T h e f o r m a t i o n , g r o w t h a n d c o n j u g a t i o n of t h e pronuclei in t h e r a t egg, J. Roy. Microscop. Sue., 71 (1951) 295-3o6. 2 BURCKHARD, G., ~)ber der Einfluss der R 6 n t g e n s t r a h l e n a u f d e n tierisehen O r g a n i s m u s , insbes o n d e r e a u f die GraviditXt, Volkmanns Sarnml. Klin. Vortrage, 40o (19o5) 469-480. 3 EVANS, E. P., G. BRECKON AND C. E. FORD, A n a i r - d r y i n g m e t h o d for meiotic p r e p a r a t i o n s f r o m m a m m a l i a n testes, Cytogenetics, 3 (1964) 289-294. 4 FALCONER, D. S., T h e e s t i m a t i o n of m u t a t i o n r a t e s f r o m i n c o m p l e t e l y t e s t e d g a m e t e s , a n d t h e d e t e c t i o n of m u t a t i o n s in m a m m a l s , J. Genet., 49 (1949) 226-234. 5 FORD, C. E., AND J. L. HAMERTON, A colchicine h y p o t o n i c citrate, s q u a s h s e q u e n c e for m a m maliafi c h r o m o s o m e s , Stain Technol., 31 (1956) 247-251. 6 LYON, M. F., R. J. S. PHILLIPS AND A. G. SEARLE, T h e overall r a t e s of d o m i n a n t a n d recessive lethal a n d visible m u t a t i o n i n d u c e d b y s p e r m a t o g o n i a l X - i r r a d i a t i o n of mice, Genet. Res., 5 (1964) 448-467 • 7 L Y o n , M. F., A. G. SEARLE, C. E. FORD AND S. OHNO, A 1DOUSet r a n s l o c a t i o n s u p p r e s s i n g sexlinked variegation, Cytogenetics, 3 (1964) 306-323 • 8 PARKES, A. S., O n t h e occurrence of t h e oestrous-cycle after X - r a y sterilization, II. I r r a d i a t i o n a t or before birth, Proc. Roy. Soc. (Lond.), IOi (1927) 71-95 . 9 RUGH, R., DUHAMEL, L., A. W. OSBORNE AND A. VARNA, P e r s i s t e n t s t u n t i n g following X-irradia-tion of t h e foetus, Amer. J. Anat., 115 (1964) 185-198. io RUGH, R., AND E. GRUPP, Effect of low level-X-irrad i a t i o n o n t h e fertilized egg of t h e m a m m a l , Exptl. Cell Res., 25 (1961) 3o2-31o. I I RUGH, R., AND E. GRUPP, Low levels of X - i r r a d i a t i o n a n d t h e e a r l y m a m m a l i a n e m b r y o , Am. J. Roentgenol. Radium Therwpy Nucl. Med., 87 (1962) 559-566. 12 RUGH, R., AND M. WOHLFROMM, Can t h e m a m m a l i a n e m b r y o be killed b y X - i r r a d i a t i o n ? J. Exptl. Zool., 151 (1962) 227-243. 13 RUSSELL, L. B., X - r a y i n d u c e d d e v e l o p m e n t a l a b n o r m a l i t i e s in t h e m o u s e a n d t h e i r u s e in t h e a n a l y s i s of e m b r y o l o g i c a l p a t t e r n s , I. E x t e r n a l a n d gross visceral changes, J. Exptl. Zool., 114 (195 ° ) 545-602. 14 RUSSELL, L. B., Effects of r a d i a t i o n on m a m m a l i a n p r e n a t a l d e v e l o p m e n t , in A. HOLLAENDER (Ed.), Radiation Biology, McGraw-Hill, N e w York, 1954, P. 861-918. 15 RUSSELL, L. B., AND W. L. RUSSELL, T h e effects of r a d i a t i o n on t h e p r e i m p l a n t a t i o n s t a g e s of t h e m o u s e e m b r y o , Anat. Rec., lO8 (195 o) 521. 16 RUSSELL, L. B., AND Vq. L. RUSSELL, A n a n a l y s i s of t h e c h a n g i n g r a d i a t i o n response of t h e d e v e l o p i n g m o u s e e m b r y o , J. Cellular Cutup. Physiol., 43, Suppl. i (1954) lO3-149. 17 RUSSELL, L. B., AND W. L. RUSSELL, P a t h w a y s of r a d i a t i o n effects in t h e m o t h e r a n d t h e e m b r y o , Cold Spring Harbor Syrup. Quant. Biol., 19 (1954) 5o-59 • I8 RUSSELL, L. B., AND C. L. SAYLORS, T h e relative s e n s i t i v i t y of v a r i o u s germ-cell s t a g e s of t h e m o u s e to r a d i a t i o n - i n d u c e d n o n disfunction, c h r o m o s o m e losses a n d deficiencies, in F. H. SOBELS (Ed.), Repair from Genetic Radiation Damage and Dil~erential Radiosensitivity in Germ-Cells, Oxford, 1963, p. 313-34 o. 19 RUSSELL, W. L., T h e incidence of sterility a n d p a r t i a l sterility in t h e d e s c e n d a n t s of X - i r r a d i a t e d mice, Genetics, 35 (195 o) 689. 20 RUSSELL, W. L., Genetic effects of r a d i a t i o n in m a m m a l s , in A. HOLLAENDER (Ed.), Radiation Biology, McGraw-Hill, N e w York, 1954, p. 825-859. 2I SNELL, G. D., AND F. B. AMES, H e r e d i t a r y c h a n g e s in t h e d e s c e n d a n t s of female mice e x p o s e d to r o e n t g e n rays, Am. J. Roentgenol. Radium Therapy Nucl. Med., 41 (1939) 248-255.
Mutation Res., 4 (1967) 689-697