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Chromosome aberrations in spermatocytes and oocytes of mice irradiated prenatally
Mutation Research, 22 (1974) 277-280 © Elsevier Scientific Publishing Company, A m s t e r d a m - Printed in The Netherlands
277
CHROMOSOME A B E R R A T I O N S IN SPERMATOCYTES AND OOCYTES OF MICE I R R A D I A T E D P R E N A T A L L Y *
W. S. TSUCHIDA AND IRENE A. UCHIDA Department of Pediatrics, McMaster University, Hamilton, Ontario (Canada)
(Received November 6th, 1973)
SUMMARY
5 pregnant mice were exposed to a single dose of 15o R whole body v-irradiation on the I2th day of gestation. The oocytes and spermatocytes, collected from the Ft progeny at ages lO-12 weeks, were examined for chromosome aberrations in metaphase I and compared with those of the progeny of non-irradiated controls. No differences were found in the type and frequency of aberrations between irradiated and controls nor between the sexes. It appears, therefore, that either primordial germ cells of both males and females are fairly resistant to radiation or an efficient selection or repair mechanism has eliminated the aberrant cells.
INTRODUCTION It is well established that radiation induces chromosome aberrations in mouse germ cells and their precursors n and that the sensitivity of these cells varies with the stage of gestationT,~°, ~3. More aberrations were detected in mice irradiated at the pronuclear stage of the zygote than in those irradiated at later stages of development. In a recent study IVANOVet al. 5 found no translocations in the spermatocytes of 7o-day-old mice irradiated on day 13.5 of fetal life with X-ray doses as high as 300 R. These results suggest that primordial germ cells at this fetal age are fairly resistant to radiation. On the other hand the absence of chromosome aberrations may be the result of selective elimination. The purpose of the present study was to use recently developed techniques of oocyte cultures to examine the meiotic chromosomes of mature females that had been exposed to radiation during fetal life and to compare the frequencies of chromosome aberrations with those of the spermatocytes of their litter mates. MATERIALSAND METHODS 7 2-month-old Swiss/ICR females were mated with males of the same strain. On the I2th day of pregnancy 5 females were placed in a lucite container and exposed to a single dose of 15o R whole body v-irradiation from a 137Cs source at 29 R/min. * Supported by a grant from the Medical Research Council of Canada.
278
w.s.
TSUCHIDA,
I. A. U C H I D A
At this age sexual differentiation of the gonads has just begun1, 8. The 2 remaining females were handled in the same manner but were not irradiated. All were placed in individual cages and allowed to litter. The Ft progeny were separated by sex at weaning and sacrificed at 10-12 weeks. No hormones were used to induce superovulation. The procedures for oocyte collection and maturation in vitro reported by HENDERSON A N D E D W A R D S 4, and slide preparation, reported by T A R K O W S K 1 1 4 , have been modified by EVANS2 and are described briefly. The ovaries were shredded with fine hypodermic needles in a 1 : I mixture of Hanks' solution and TC I99 medium. The oocytes were then teased out, cleaned by repeated pipetting through a finely drawn pipette and cultured in fetal calf serum for 5 h to obtain cells at metaphase I. They were then exposed to i % sodium citrate for 2o min. Air-dried slides were pre pared by placing a few oocytes on a clean microslide, fixed with a tiny drop of 3:1 methanol-acetic acid and quickly blown dry under the heat of a 25-W bulb. Direct preparations were made of spermatocyte chromosomes by the method of EVANSet al.3: cells collected from the seminiferous tubules were exposed to hypotonic solution for 2o min, fixed, dropped on microslides and air-dried. All slides were stained with Giemsa. Cells with well spread metaphase chromosomes were scored for chromatid breaks, fragments and chromatid exchanges. RESULTS
The 5 irradiated females produced 45 offspring (21 females and 24 males) while the 2 controls produced 21 (io females and I I males). All F1 females were used but 17 F1 males were sufficient to obtain enough analyzable cells. The numbers of oocytes collected from the experimental and control mice are shown in Table I. There were no significant differences between the two groups in the mean number of oocytes recovered per mouse nor in the number that matured in vitro TABLE
I
OOCYTES FROM MICE EXPOSED TO I 5 0 i ON THE I 2 t h DAY OF GESTATION AND COLLECTED POSTNATALLY AT AGE I O - - I 2 WEEKS COMPARED WITH NON-IRRADIATED CONTROLS
Irradiated Non-irradiated
TABLE
Number Total oocytes of mice collected
Number of oocytes /mouse
Matured in vitro Number %
Analyzable metaphases
21 IO
67 75
777 388
5 lo 237
i4o5 75o
55.3 51.7
II
FREQUENCIES OF STRUCTURAL CHROMOSOME ABERRATIONS IN OOCYTES AND SPERMATOCYTES OF MICE EXPOSED TO 1 5 0 t { ~-RADIATION ON THE I 2 t h DAY" OF GESTATION AND U N I R R A D I A T E D CONTROLS
Irradiated: Controls :
Females Males Females Males
Cells analyzed
$ bnormal cells Number Percent
Number of aberrations Chromatid Fragments Multivalents breaks
51o 728 237 819
14 18 3 23
7 io i 9
2.8 2. 5 i. 2 2.8
7 6 i 13
i 2 i i
CHROMOSOME ABERRATIONS IN PRENATALLY IRRADIATED MICE
279
i n d i c a t i n g t h a t e x p o s u r e to 15o R on t h e I 2 t h d a y of g e s t a t i o n affected n e i t h e r t h e v i a b i l i t y of t h e oocytes nor t h e a b i l i t y of d i c t y a t e oocytes to resume meiosis in vitro. Of t h e oocytes collected from t h e females i r r a d i a t e d in utero, 2.8% h a d s t r u c t u r a l a b e r r a t i o n s c o m p a r e d w i t h 1.8% in the controls (Table II). One m u l t i v a l e n t (chainof-four) was o b s e r v e d in b o t h i r r a d i a t e d a n d control cells. The rest were single c h r o m a t i d or i s o c h r o m a t i d breaks. A n a l y s e s of t h e s p e r m a t o c y t e s p r o d u c e d similar results. There were no significant differences between a n y of the frequencies. P r e n a t a l e x p o s u r e to r a d i a t i o n , therefore, d i d n o t increase the frequency of c h r o m o s o m a l a b e r r a t i o n s in the meiotic cells recovered from males a n d females p o s t - n a t a l l y nor was t h e r e a n y s e x - r e l a t e d difference in r a d i o s e n s i t i v i t y . DISCUSSION
To s t u d y t h e effect of r a d i a t i o n on oocytes the only m e t h o d a v a i l a b l e u n t i l r e c e n t l y has been t h e e x a m i n a t i o n of p r o g e n y with t h e use of genetic m a r k e r s or k a r y o t y p e s of fibroblasts. RUSSELL TM f o u n d t h a t sex h y p o p l o i d y can be increased b y i r r a d i a t i n g oocytes in p r e - l e p t o t e n e t h r o u g h diplotene stages b u t the increase is g r e a t e r when the d i c t y o t e n e oocytes of m a t u r e mice are i r r a d i a t e d . The m a j o r i t y of s t r u c t u r a l a b e r r a t i o n s c a n n o t be identified with the p r o g e n y m e t h o d , b u t if it can be assumed t h a t c h r o m o s o m e loss results m a i n l y from c h r o m o s o m e breakage, then our results can be c o m p a r e d . I t a p p e a r s t h e n t h a t p r i m o r d i a l g e r m cells are even more r e s i s t a n t to r a d i a t i o n t h a n p r e - d i c t y a t e oocytes. Similar results h a v e been o b s e r v e d in males. SEARLE a n d his colleagues TM,18 f o u n d t h a t p r i m o r d i a l g e r m cells are more r a d i o r e s i s t a n t t h a n s p e r m a t o g o n i a . Their o b s e r v a t i o n s are given f u r t h e r s u p p o r t b y our results a n d those of IVANOV et al. 5. N e i t h e r males nor females showed a n y d e t e c t a b l e increase in c h r o m o s o m e d a m a g e from e x p o s u r e of p r i m o r d i a l g e r m cells to 15o R ~-radiation. I t appears, therefore, t h a t these cells are r e l a t i v e l y insensitive to this dose of r a d i a t i o n or an efficient selection or r e p a i r m e c h a n i s m has m a s k e d the chromosome b r e a k s induced. These results are in m a r k e d c o n t r a s t to the increased f r e q u e n c y of chromosome a b e r r a tions o b s e r v e d in t h e oocytes of i r r a d i a t e d m a t u r e females15,1~. ACKNOWLEDGEMENTS
W e are grateful to Dr. C. C. LIN for m a n y helpful discussions a n d to ELIZABETH BYRNES a n d VIOLA LEE for their technical assistance. REFERENCES I BORUM, K., Oogenesis in the mouse: A study of the meiotic prophase, Exptl. Cell Res., 24
(1961) 495-507 . 2 EVANS, E. P., Personal communication. 3 EVANS, E. P., G. BRECKON AND C. E. FORD, An air drying method for meiotic preparations from m a m m a l i a n testes, Cytogenetics, 3 (1964) 289-294. 4 HENDERSON, S. A., AND R. G. EDWARDS, Chiasma frequency and m a t e r n a l age in m a m m a l s ,
Nature, 218 (1968) 22-28. 5 IVANOV, B., A. L1~ONARD AND GH. DEKNUDT, Chromosome rearrangements induced in the mouse by embryonic X-irradiation, II. Irradiation of male fetuses at the ID.5th day of gestation, Mutation Res., 18 (1973) 89-92. 6 LI~ONARD, A., AND GH. OEKNUDT, Persistence of chromosome rearrangements induced in male mice by X-irradiation of premeiotic germ cells, Mutation Res., 9 (197o) 127-133.
280
W . S . TSUCHIDA, I. A. UCHIDA
7 L~ONARD, A., AND GH. DEKNUDT, C h r o m o s o m e r e a r r a n g e m e n t s induced in the mouse by embryonic X-irradiation, I. Pronuclear stage, Mutation Res., 4 (1967) 689-697. 8 LIMA-DE FARIA, A., AND K. BORUM, The period of D N A synthesis prior to meiosis in the mouse, J. Cell Biol., 14 (1962) 381-388. 9 RUSSELL. L. 13, in A. G. STEINBERG AND A. G. BEARN (Eds.), Progress in Medical Genetics, Grune & Stratton, New York, 1962, pp. 230 294. IO RUSSELL, L. B., in Effects of Radiation of Meiotic Systems, Intern. Atomic E n e r g y _Agency, Vienna, 1968, pp. 27-41. i i RUSSELL, W. L., in A. I-IOLLAENDER (Ed.), Genetic Effects of Radiation in Mammals, McGrawHill, New York, 1954, pp. 825-859 • 12 SEARLE, ,A_. G., E. 1°. EVANS AND B. J. WEST, Studies on the induction of translocations in mouse spermatogonia, II. Effects of fast n e u t r o n irradiation, Mutation Res., 7 (1969) 235 240. 13 SEARLE, A. G., AND R. J. S. PHILLIPS, The mutagenic effectiveness of fast n e u t r o n s in male and female mice, Mutation Res., I i (1971) 97-1o5. 14 TARKOWSKI, A. K., An air-drying m e t h o d for c h r o m o s o m e p r e p a r a t i o n s from mouse eggs, Cytogenetics, 5 (1966) 394-400. 15 GILLIAVOD, N., AND A. L~ONARD, Sensitivity of the mouse oocytes to the induction of translocations b y ionizing radiations, Can. J. Genet. Cytol., 15 (1973) 363-366. 16 TSUCHIDA, W. S., AND I. A. UCHIDA, Radiation-induced c h r o m o s o m e aberrations in mouse s p e r m a t o c y t e s and oocytes, submitted.
277
CHROMOSOME A B E R R A T I O N S IN SPERMATOCYTES AND OOCYTES OF MICE I R R A D I A T E D P R E N A T A L L Y *
W. S. TSUCHIDA AND IRENE A. UCHIDA Department of Pediatrics, McMaster University, Hamilton, Ontario (Canada)
(Received November 6th, 1973)
SUMMARY
5 pregnant mice were exposed to a single dose of 15o R whole body v-irradiation on the I2th day of gestation. The oocytes and spermatocytes, collected from the Ft progeny at ages lO-12 weeks, were examined for chromosome aberrations in metaphase I and compared with those of the progeny of non-irradiated controls. No differences were found in the type and frequency of aberrations between irradiated and controls nor between the sexes. It appears, therefore, that either primordial germ cells of both males and females are fairly resistant to radiation or an efficient selection or repair mechanism has eliminated the aberrant cells.
INTRODUCTION It is well established that radiation induces chromosome aberrations in mouse germ cells and their precursors n and that the sensitivity of these cells varies with the stage of gestationT,~°, ~3. More aberrations were detected in mice irradiated at the pronuclear stage of the zygote than in those irradiated at later stages of development. In a recent study IVANOVet al. 5 found no translocations in the spermatocytes of 7o-day-old mice irradiated on day 13.5 of fetal life with X-ray doses as high as 300 R. These results suggest that primordial germ cells at this fetal age are fairly resistant to radiation. On the other hand the absence of chromosome aberrations may be the result of selective elimination. The purpose of the present study was to use recently developed techniques of oocyte cultures to examine the meiotic chromosomes of mature females that had been exposed to radiation during fetal life and to compare the frequencies of chromosome aberrations with those of the spermatocytes of their litter mates. MATERIALSAND METHODS 7 2-month-old Swiss/ICR females were mated with males of the same strain. On the I2th day of pregnancy 5 females were placed in a lucite container and exposed to a single dose of 15o R whole body v-irradiation from a 137Cs source at 29 R/min. * Supported by a grant from the Medical Research Council of Canada.
278
w.s.
TSUCHIDA,
I. A. U C H I D A
At this age sexual differentiation of the gonads has just begun1, 8. The 2 remaining females were handled in the same manner but were not irradiated. All were placed in individual cages and allowed to litter. The Ft progeny were separated by sex at weaning and sacrificed at 10-12 weeks. No hormones were used to induce superovulation. The procedures for oocyte collection and maturation in vitro reported by HENDERSON A N D E D W A R D S 4, and slide preparation, reported by T A R K O W S K 1 1 4 , have been modified by EVANS2 and are described briefly. The ovaries were shredded with fine hypodermic needles in a 1 : I mixture of Hanks' solution and TC I99 medium. The oocytes were then teased out, cleaned by repeated pipetting through a finely drawn pipette and cultured in fetal calf serum for 5 h to obtain cells at metaphase I. They were then exposed to i % sodium citrate for 2o min. Air-dried slides were pre pared by placing a few oocytes on a clean microslide, fixed with a tiny drop of 3:1 methanol-acetic acid and quickly blown dry under the heat of a 25-W bulb. Direct preparations were made of spermatocyte chromosomes by the method of EVANSet al.3: cells collected from the seminiferous tubules were exposed to hypotonic solution for 2o min, fixed, dropped on microslides and air-dried. All slides were stained with Giemsa. Cells with well spread metaphase chromosomes were scored for chromatid breaks, fragments and chromatid exchanges. RESULTS
The 5 irradiated females produced 45 offspring (21 females and 24 males) while the 2 controls produced 21 (io females and I I males). All F1 females were used but 17 F1 males were sufficient to obtain enough analyzable cells. The numbers of oocytes collected from the experimental and control mice are shown in Table I. There were no significant differences between the two groups in the mean number of oocytes recovered per mouse nor in the number that matured in vitro TABLE
I
OOCYTES FROM MICE EXPOSED TO I 5 0 i ON THE I 2 t h DAY OF GESTATION AND COLLECTED POSTNATALLY AT AGE I O - - I 2 WEEKS COMPARED WITH NON-IRRADIATED CONTROLS
Irradiated Non-irradiated
TABLE
Number Total oocytes of mice collected
Number of oocytes /mouse
Matured in vitro Number %
Analyzable metaphases
21 IO
67 75
777 388
5 lo 237
i4o5 75o
55.3 51.7
II
FREQUENCIES OF STRUCTURAL CHROMOSOME ABERRATIONS IN OOCYTES AND SPERMATOCYTES OF MICE EXPOSED TO 1 5 0 t { ~-RADIATION ON THE I 2 t h DAY" OF GESTATION AND U N I R R A D I A T E D CONTROLS
Irradiated: Controls :
Females Males Females Males
Cells analyzed
$ bnormal cells Number Percent
Number of aberrations Chromatid Fragments Multivalents breaks
51o 728 237 819
14 18 3 23
7 io i 9
2.8 2. 5 i. 2 2.8
7 6 i 13
i 2 i i
CHROMOSOME ABERRATIONS IN PRENATALLY IRRADIATED MICE
279
i n d i c a t i n g t h a t e x p o s u r e to 15o R on t h e I 2 t h d a y of g e s t a t i o n affected n e i t h e r t h e v i a b i l i t y of t h e oocytes nor t h e a b i l i t y of d i c t y a t e oocytes to resume meiosis in vitro. Of t h e oocytes collected from t h e females i r r a d i a t e d in utero, 2.8% h a d s t r u c t u r a l a b e r r a t i o n s c o m p a r e d w i t h 1.8% in the controls (Table II). One m u l t i v a l e n t (chainof-four) was o b s e r v e d in b o t h i r r a d i a t e d a n d control cells. The rest were single c h r o m a t i d or i s o c h r o m a t i d breaks. A n a l y s e s of t h e s p e r m a t o c y t e s p r o d u c e d similar results. There were no significant differences between a n y of the frequencies. P r e n a t a l e x p o s u r e to r a d i a t i o n , therefore, d i d n o t increase the frequency of c h r o m o s o m a l a b e r r a t i o n s in the meiotic cells recovered from males a n d females p o s t - n a t a l l y nor was t h e r e a n y s e x - r e l a t e d difference in r a d i o s e n s i t i v i t y . DISCUSSION
To s t u d y t h e effect of r a d i a t i o n on oocytes the only m e t h o d a v a i l a b l e u n t i l r e c e n t l y has been t h e e x a m i n a t i o n of p r o g e n y with t h e use of genetic m a r k e r s or k a r y o t y p e s of fibroblasts. RUSSELL TM f o u n d t h a t sex h y p o p l o i d y can be increased b y i r r a d i a t i n g oocytes in p r e - l e p t o t e n e t h r o u g h diplotene stages b u t the increase is g r e a t e r when the d i c t y o t e n e oocytes of m a t u r e mice are i r r a d i a t e d . The m a j o r i t y of s t r u c t u r a l a b e r r a t i o n s c a n n o t be identified with the p r o g e n y m e t h o d , b u t if it can be assumed t h a t c h r o m o s o m e loss results m a i n l y from c h r o m o s o m e breakage, then our results can be c o m p a r e d . I t a p p e a r s t h e n t h a t p r i m o r d i a l g e r m cells are even more r e s i s t a n t to r a d i a t i o n t h a n p r e - d i c t y a t e oocytes. Similar results h a v e been o b s e r v e d in males. SEARLE a n d his colleagues TM,18 f o u n d t h a t p r i m o r d i a l g e r m cells are more r a d i o r e s i s t a n t t h a n s p e r m a t o g o n i a . Their o b s e r v a t i o n s are given f u r t h e r s u p p o r t b y our results a n d those of IVANOV et al. 5. N e i t h e r males nor females showed a n y d e t e c t a b l e increase in c h r o m o s o m e d a m a g e from e x p o s u r e of p r i m o r d i a l g e r m cells to 15o R ~-radiation. I t appears, therefore, t h a t these cells are r e l a t i v e l y insensitive to this dose of r a d i a t i o n or an efficient selection or r e p a i r m e c h a n i s m has m a s k e d the chromosome b r e a k s induced. These results are in m a r k e d c o n t r a s t to the increased f r e q u e n c y of chromosome a b e r r a tions o b s e r v e d in t h e oocytes of i r r a d i a t e d m a t u r e females15,1~. ACKNOWLEDGEMENTS
W e are grateful to Dr. C. C. LIN for m a n y helpful discussions a n d to ELIZABETH BYRNES a n d VIOLA LEE for their technical assistance. REFERENCES I BORUM, K., Oogenesis in the mouse: A study of the meiotic prophase, Exptl. Cell Res., 24
(1961) 495-507 . 2 EVANS, E. P., Personal communication. 3 EVANS, E. P., G. BRECKON AND C. E. FORD, An air drying method for meiotic preparations from m a m m a l i a n testes, Cytogenetics, 3 (1964) 289-294. 4 HENDERSON, S. A., AND R. G. EDWARDS, Chiasma frequency and m a t e r n a l age in m a m m a l s ,
Nature, 218 (1968) 22-28. 5 IVANOV, B., A. L1~ONARD AND GH. DEKNUDT, Chromosome rearrangements induced in the mouse by embryonic X-irradiation, II. Irradiation of male fetuses at the ID.5th day of gestation, Mutation Res., 18 (1973) 89-92. 6 LI~ONARD, A., AND GH. OEKNUDT, Persistence of chromosome rearrangements induced in male mice by X-irradiation of premeiotic germ cells, Mutation Res., 9 (197o) 127-133.
280
W . S . TSUCHIDA, I. A. UCHIDA
7 L~ONARD, A., AND GH. DEKNUDT, C h r o m o s o m e r e a r r a n g e m e n t s induced in the mouse by embryonic X-irradiation, I. Pronuclear stage, Mutation Res., 4 (1967) 689-697. 8 LIMA-DE FARIA, A., AND K. BORUM, The period of D N A synthesis prior to meiosis in the mouse, J. Cell Biol., 14 (1962) 381-388. 9 RUSSELL. L. 13, in A. G. STEINBERG AND A. G. BEARN (Eds.), Progress in Medical Genetics, Grune & Stratton, New York, 1962, pp. 230 294. IO RUSSELL, L. B., in Effects of Radiation of Meiotic Systems, Intern. Atomic E n e r g y _Agency, Vienna, 1968, pp. 27-41. i i RUSSELL, W. L., in A. I-IOLLAENDER (Ed.), Genetic Effects of Radiation in Mammals, McGrawHill, New York, 1954, pp. 825-859 • 12 SEARLE, ,A_. G., E. 1°. EVANS AND B. J. WEST, Studies on the induction of translocations in mouse spermatogonia, II. Effects of fast n e u t r o n irradiation, Mutation Res., 7 (1969) 235 240. 13 SEARLE, A. G., AND R. J. S. PHILLIPS, The mutagenic effectiveness of fast n e u t r o n s in male and female mice, Mutation Res., I i (1971) 97-1o5. 14 TARKOWSKI, A. K., An air-drying m e t h o d for c h r o m o s o m e p r e p a r a t i o n s from mouse eggs, Cytogenetics, 5 (1966) 394-400. 15 GILLIAVOD, N., AND A. L~ONARD, Sensitivity of the mouse oocytes to the induction of translocations b y ionizing radiations, Can. J. Genet. Cytol., 15 (1973) 363-366. 16 TSUCHIDA, W. S., AND I. A. UCHIDA, Radiation-induced c h r o m o s o m e aberrations in mouse s p e r m a t o c y t e s and oocytes, submitted.