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X-ray-induced multiple aberrations among oocytes of Drosophila melanogaster
I22
Mutation Research Elsevier Publishing Company, Amsterdan Printed in The Netherlands
X-Ray-induced multiple aberrations among oocytes of Drosophila rnela, nogaster When Drosophila melanogaster females with attached-X chromosonles arc irradiated and mated to appropriate males, it is possible to detect phenotypicall 3 chromatid interchanges that had been induced in germ cells. Both the relative frequency of interchange and the chromosomes involved can be determined2,7, '~. Light- and electron-microscope analyses of D. rnelanogaster ovaries have beer conducted by KING at al. 4 and KOCH AND KING5, who divided oogenesis into severa stages. The two ovaries in each fly were found to contain an average of 24 egg-producing tubes called ovarioles, 12 in each ovary. Each ovariole is differentiated into at: anterior germanium and a series of egg chambers. At tile anterior end of the germarium is a region of about 50 mitotically active cells. A germarial cell that is to become an oocyte first undergoes four synchronous divisions to form a cyst of I6 daughtet cells which are referred to as cystocytes. One cystocyte will become the oocyte while the remaining 15 will become nurse cells. When tile cyst is completely surrounded by a layer of follicle cells, it is termed an egg chamber. KING el al.* have divided oogenesis into 14 stages with the germarial cyst and first egg chamber as stages I and 2 respectively. Newly enclosed females have 6 7 developing eggs per ovariole, eystocytes, oogonia and finally stem cells. KOCH AND KING~ suggest that a newly eclosed female may have to lay as many as 5oo eggs before mature ooeytes would be deposited which were stem cells when oviposition started. The time necessary for development of these stages can be inferred from the work of GRELL AND CHANDLEY3 who utilized !3H]thymidine incorporation studies of oogenesis and concluded that a minimum of 6 days was needed for cystocytes that have undergone the last DNA replication to become mature eggs. In a study in which newly eclosed attached-X females were irradiated with 3ooo R, individually nlated, and a sample of 48 or less eggs per female was collected, it was observed that some females had more aberrations among their offspring than would have been expected if the events were randomly distributed among the flies 9. Eggs sampled from follicles already established and containing 15 nurse cells and one oocyte each at the time of irradiation and yielding nonrandomly recovered interchange products require some basis other than proliferation of stem-cell-induced events to account for the evident nonrandomness. Hence, it seemed necessary to make additional observations bearing on the question of the origin of such nmltiple aberrations in the progenies of single treated females. Newly eclosed (0-6 h) females with the genotypes y v / B ~ Y . y + were irradiated with 3ooo R (6oo R / m i n , 25o kV, 15 mA, I mm A1 and 4 mm Lucite filtration) and were mated individually with three 3-4-day-old males that had a y sc ~1 B In 49 c/ sc 8 X- and a normal Y-chromosome (see L I N D S L E Y AND G R E L L 6 for an explanation of the symbols used). Each vial was numbered and this number and the associated males and females were transferred to new food every two days for io broods. This technique does not insure that each brood will represent specific oogenic stages, however, they should represent progressively earlier stages at the time of irradiation. Control tests differed only in that the females were not irradiated. In Expts. I and 2, which were replicates, 148 of 15o and 19o of 200 females in the X-ray experiments IVIutation Res., 7 (1969) 122-125
SHORT
I23
COMMUNICATIONS
and 41 of 5o and 95 of IOO females in the control experiments individually produced more than 50 offspring. The 26 females that produced fewer than 50 offspring were not included in the analysis. Since the majority of them produced no offspring, excluding these flies did not alter significantly the analyses made. These females were eliminated since it was necessary for all flies to have approximately the same chance for aberrations to occur if meaningful comparisons were to be made. The vials containing the eggs from each brood were stored at 25 ~: I°C for 16-18 days before scoring. Since all detachment classes were phenotypically different, both the total frequencv of detachment and the intercharge partners associated with the aberrations could be determined. In addition, aberrations infolving the Y-, but not the X-chromosomes could be detected. It is difficult to show the actual brood pattern for the recovery of aberrations among the offspring of each female, therefore, a graphical presentation has been sought. A theoretical expectation for the aberration frequencies was calculated on the basis of a Poisson distribution in which the aberrations were assumed to have been recovered as independent events. From Table I it can be seen that more females had either no or large multiple aberrations than was expected based on this assumption. TABLE
I
T H E N U M B E R OF F E M A L E S ~ r l T H O, I , OR M U L T I P L E X - R A Y - I N D U C E D OFFSPRING
A B E R R A T I O N S AMONG T H E I R
Number ofaberrationsfromsinglefemales o
I
2
3
4
5
6
7
8
9
Io
II
I2
13
Experiment i 2
Total observed Total expected a
36 64
43 61
21 32
26 17
8 8
5 4
2 I
3 I
i I
i o
I o
0
0
I
I
O
I
ioo
lO4
53
43
16
9
3
4
2
i
I
I
0
2
65
lO 7
88
49
20
7
2
a T h i s e x p e c t e d P o i s s o n d i s t r i b u t i o n of a b e r r a t i o n s is b a s e d o n a r e c o v e r y of 5 5 8 a b e r r a t i o n s a m o n g 3 3 8 f e m a l e s for a n a v e r a g e of 1.65 a b e r r a t i o n s p e r f e m a l e .
TABLE
II
T H E B R O O D S IN W H I C H O N E OR M U L T I P L E A B E R R A T I O N S VCERE R E C O V E R E D F R O M S I N G L E F E M A L E S
Number ofaberrationsfromsinglefemales ±
2
3
4
5
6
7
8
9
Io
I~
12
z3
A B C D
61 28 6 2
56 31 8 2
68 34 12 7
29 18 5 4
14 12 6 2
2 2 I I
3 2 4 7
o I 2 2
o 2 I 2
I o I 2
I
o
I
E F G H I
I I o 3 o
I 4 I 2 I
2 3 3
I 3 2 2
3 4 I 2 I
3 6 I I o
4 5 I 2
3 I 4 2 o
4
I I 2 I o
I I 2 3 I 2
o o o o o o o
4 5 5 6 2 2 I
J
2
Two day broods
I
I
I
2~Iutation R e s . , 7 ( 1 9 6 9 )
122-125
124 TABLE
SHORT COMMUNICATION~ III
T H E A V E R A G E N U M B E R O F O F F S P R I N G P R O D U C E D BY" F E M A L E S W I T H O, I , O R M U L T I P L E A B E R R A T I O N ~ AMONG THEIR OFFSPRING
N u m b e r o f a b e r r a t i o n s f r o m a single f e m a l e
Experiment i 2
o
•
2
3
4
5
6
7
8
9
zo
122 lO6
162 113
154 154
I66 131
16o 119
178 204
273 127
184 67
258
113
25o
11
t2
13
174
--
147 95
Although not as great a difference was noted, fewer females were recovered than wa~ expected t h a t had one, two, three, or four aberrations. It can be argued t h a t the us~ of Poisson distribution is not warranted since the mean expectation of 1.65 aberrations per culture would hold only if all cultures were of the same size. It can be stated t h a t those females which produced more aberrations than were expected on the basis ot randonmess alone did not do so as a result of producing more offspring and therefore having a greater chance for the events to occur. This is demonstrated in Table I I I where it can be seen t h a t no meaningful differences exist in the average number o! -offspring produced by females associated with no, one, or multiple aberrations. It fi, felt t h a t the lack of complete homogeneity of culture sizes from groups of females with different numbers of aberrations among their progenies alters the expected frequencies only slightly, and t h a t the general observation of nonrandomness is valid. The d a t a in Table II show the broods in which the aberrations from individual females were recovered. It can be seen t h a t most aberrations occurred among the earliest broods, which is in agreement with the findings of PARKER7 and ABRAHAM"SONI. Furthermore, when individual females had multiple aberrations among their offspring, they occurred as early as brood A before cells t h a t had been oogonia at the time of irradiation should have been recovered. Therefore, it seems unreasonable t h a t these events could be explained on the basis of stem-cell clusters. The number of ovarioles in the detachment stock used has been 22-24 with very little deviation for the several years t h a t it has been tested. It is assumed t h a t oogenesis in these flies is similar to t h a t described b y KING et al. ~. Other evidence can be presented t h a t these multiple aberrations do not represent proliferations of stem-cell events based on the interchange partners involved in the aberrations. Interchanges involving X - Y B s, X - Y y ÷. X-autosomes, or Y-autosomes could be observed in these experiments and the events could be classified as the same, different, or reciprocal. This will give only a minimal estimate for differences since similar or reciprocal appearing events, b y these phenotypical classifications, m a y have been found to have been different upon further testing. However, of the 23 females that had 5 or more aberrations among their offspring, none had all phenotypically identical aberrations, 7 could be explained by postulating reciprocal events, and '9 and 7 cases could be explained only if 2 or 3 separate events were postulated, respectively. The aberrations recovered in these experiments must have been associated with the irradiation since only 9 aberrations were found among 41o32 offspring in the controls. They were distributed throughout the different broods, and in no case was more than one aberration associated with a single female. No satisfactory explanation can be offered at this time for the apparent differ2VIutation R e s . , 7 (1969) 122-125
SHORT COMMUNICATIONS
125
ential radiosensitivity of the ooeytes in the different females. The multiple aberrations do occur among early broods, which should not contain products of cells that had been stem cells at the time of irradiation, and m a n y events are independent based on phenotypic observations. Further studies to explain these observations are in progress. This work was supported by U.S.A.E.C. contracts AT (o4-3)-59 ° to R.R.R. and AT (04-3)-529 to F.J.R. I t also received support from the Association of E u r a t o m and the University of Leiden, Contract No. o52-64-1 BIAN, the International Atomic Energy Agency and The Health Research Organization T.N.O. It is a pleasure to thank Dr. F. H. SOBELS for facilities utilized by ROBERT R. RINEHART during a part of the work and Dr. K. SANKARANARAYANANfor comments on the manuscript. We also acknowledge the very efficient technical assistance of J E A N PRENDERGAST and GLORIA WESTIN.
Biology Department, San Diego, State College, San Diego, Calif. (U.S.A.)
ROBERT R. R I N E H A R T * F. J. RATTY
i ABRAHAMSON, SEYMOUR, C h r o m o s o m e r e a r r a n g e m e n t s i n d u c e d b y X - r a y s in i m m a t u r e g e r m cells of Drosophila, Nature, 191 (1961) 523-524 . 2 ABRAHAMSON, SEYMOUR, IRWIN H. HERSKOWlTZ AND H. J. MULLER, I d e n t i f i c a t i o n of halft r a n s l o c a t i o n s p r o d u c e d b y X - r a y s in d e t a c h i n g a t t a c h e d - X c h r o m o s o m e s of D. melanogaster, Genetics, 41 (1956) 41o-419. 3 GRELL, RHODA F., AND ANN C. CHANDLEY, E v i d e n c e b e a r i n g on t h e coincidence of e x c h a n g e a n d D N A replication in t h e oocyte of Drosophila melanogaster, Proc. Natl. Acad. Sci. (U.S.), 53 (1956 ) 134o-1346. 4 KING, R. C., ANx C. RUmNSON AND R. F. SMITH, Oogenesis in a d u l t Drosophila melanogaster, Growth, 20 (1956) 121-157. 5 KOCH, ELIZABETH A., AND ROBERT C. KING, T h e origin a n d early differentiation of t h e egg c h a m b e r of Drosophila rnelanogaster, J. Morphol., 119 (1966) 283 304 . 6 LINDSLEY, DAN" L., AND E. H. GRELL, Genetic v a r i a t i o n s of Drosophila melanogaster, Carnegie [nst. Wash. Publ., 627 (1968). 7 PARKER, D. R., R a d i a t i o n i n d u c e d e x c h a n g e s in Drosophila females, Proc. Natl. Acad. Sci. (U.S.), 4 ° (1954) 795-800. 8 PARKER, D. R., AND J. McCRONE, A genetic a n a l y s i s of s o m e r e a r r a n g e m e n t s i n d u c e d in oocytes of Drosophila, Genetics, 43 (1958 ) 172 186. 9 RINEHART, R. R., U n p u b l i s h e d data.
Received October ioth, 1968 * P r e s e n t a d d r e s s : D e p a r t m e n t of R a d i a t i o n Genetics, U n i v e r s i t y of Leiden, a n d I n s t i t u t e for R a d i o p a t h o l o g y a n d R a d i o p r o t e c t i o n , L e i d e n (The N e t h e r l a n d s )
Mutation Res., 7 (1969) 122-125
Mutation Research Elsevier Publishing Company, Amsterdan Printed in The Netherlands
X-Ray-induced multiple aberrations among oocytes of Drosophila rnela, nogaster When Drosophila melanogaster females with attached-X chromosonles arc irradiated and mated to appropriate males, it is possible to detect phenotypicall 3 chromatid interchanges that had been induced in germ cells. Both the relative frequency of interchange and the chromosomes involved can be determined2,7, '~. Light- and electron-microscope analyses of D. rnelanogaster ovaries have beer conducted by KING at al. 4 and KOCH AND KING5, who divided oogenesis into severa stages. The two ovaries in each fly were found to contain an average of 24 egg-producing tubes called ovarioles, 12 in each ovary. Each ovariole is differentiated into at: anterior germanium and a series of egg chambers. At tile anterior end of the germarium is a region of about 50 mitotically active cells. A germarial cell that is to become an oocyte first undergoes four synchronous divisions to form a cyst of I6 daughtet cells which are referred to as cystocytes. One cystocyte will become the oocyte while the remaining 15 will become nurse cells. When tile cyst is completely surrounded by a layer of follicle cells, it is termed an egg chamber. KING el al.* have divided oogenesis into 14 stages with the germarial cyst and first egg chamber as stages I and 2 respectively. Newly enclosed females have 6 7 developing eggs per ovariole, eystocytes, oogonia and finally stem cells. KOCH AND KING~ suggest that a newly eclosed female may have to lay as many as 5oo eggs before mature ooeytes would be deposited which were stem cells when oviposition started. The time necessary for development of these stages can be inferred from the work of GRELL AND CHANDLEY3 who utilized !3H]thymidine incorporation studies of oogenesis and concluded that a minimum of 6 days was needed for cystocytes that have undergone the last DNA replication to become mature eggs. In a study in which newly eclosed attached-X females were irradiated with 3ooo R, individually nlated, and a sample of 48 or less eggs per female was collected, it was observed that some females had more aberrations among their offspring than would have been expected if the events were randomly distributed among the flies 9. Eggs sampled from follicles already established and containing 15 nurse cells and one oocyte each at the time of irradiation and yielding nonrandomly recovered interchange products require some basis other than proliferation of stem-cell-induced events to account for the evident nonrandomness. Hence, it seemed necessary to make additional observations bearing on the question of the origin of such nmltiple aberrations in the progenies of single treated females. Newly eclosed (0-6 h) females with the genotypes y v / B ~ Y . y + were irradiated with 3ooo R (6oo R / m i n , 25o kV, 15 mA, I mm A1 and 4 mm Lucite filtration) and were mated individually with three 3-4-day-old males that had a y sc ~1 B In 49 c/ sc 8 X- and a normal Y-chromosome (see L I N D S L E Y AND G R E L L 6 for an explanation of the symbols used). Each vial was numbered and this number and the associated males and females were transferred to new food every two days for io broods. This technique does not insure that each brood will represent specific oogenic stages, however, they should represent progressively earlier stages at the time of irradiation. Control tests differed only in that the females were not irradiated. In Expts. I and 2, which were replicates, 148 of 15o and 19o of 200 females in the X-ray experiments IVIutation Res., 7 (1969) 122-125
SHORT
I23
COMMUNICATIONS
and 41 of 5o and 95 of IOO females in the control experiments individually produced more than 50 offspring. The 26 females that produced fewer than 50 offspring were not included in the analysis. Since the majority of them produced no offspring, excluding these flies did not alter significantly the analyses made. These females were eliminated since it was necessary for all flies to have approximately the same chance for aberrations to occur if meaningful comparisons were to be made. The vials containing the eggs from each brood were stored at 25 ~: I°C for 16-18 days before scoring. Since all detachment classes were phenotypically different, both the total frequencv of detachment and the intercharge partners associated with the aberrations could be determined. In addition, aberrations infolving the Y-, but not the X-chromosomes could be detected. It is difficult to show the actual brood pattern for the recovery of aberrations among the offspring of each female, therefore, a graphical presentation has been sought. A theoretical expectation for the aberration frequencies was calculated on the basis of a Poisson distribution in which the aberrations were assumed to have been recovered as independent events. From Table I it can be seen that more females had either no or large multiple aberrations than was expected based on this assumption. TABLE
I
T H E N U M B E R OF F E M A L E S ~ r l T H O, I , OR M U L T I P L E X - R A Y - I N D U C E D OFFSPRING
A B E R R A T I O N S AMONG T H E I R
Number ofaberrationsfromsinglefemales o
I
2
3
4
5
6
7
8
9
Io
II
I2
13
Experiment i 2
Total observed Total expected a
36 64
43 61
21 32
26 17
8 8
5 4
2 I
3 I
i I
i o
I o
0
0
I
I
O
I
ioo
lO4
53
43
16
9
3
4
2
i
I
I
0
2
65
lO 7
88
49
20
7
2
a T h i s e x p e c t e d P o i s s o n d i s t r i b u t i o n of a b e r r a t i o n s is b a s e d o n a r e c o v e r y of 5 5 8 a b e r r a t i o n s a m o n g 3 3 8 f e m a l e s for a n a v e r a g e of 1.65 a b e r r a t i o n s p e r f e m a l e .
TABLE
II
T H E B R O O D S IN W H I C H O N E OR M U L T I P L E A B E R R A T I O N S VCERE R E C O V E R E D F R O M S I N G L E F E M A L E S
Number ofaberrationsfromsinglefemales ±
2
3
4
5
6
7
8
9
Io
I~
12
z3
A B C D
61 28 6 2
56 31 8 2
68 34 12 7
29 18 5 4
14 12 6 2
2 2 I I
3 2 4 7
o I 2 2
o 2 I 2
I o I 2
I
o
I
E F G H I
I I o 3 o
I 4 I 2 I
2 3 3
I 3 2 2
3 4 I 2 I
3 6 I I o
4 5 I 2
3 I 4 2 o
4
I I 2 I o
I I 2 3 I 2
o o o o o o o
4 5 5 6 2 2 I
J
2
Two day broods
I
I
I
2~Iutation R e s . , 7 ( 1 9 6 9 )
122-125
124 TABLE
SHORT COMMUNICATION~ III
T H E A V E R A G E N U M B E R O F O F F S P R I N G P R O D U C E D BY" F E M A L E S W I T H O, I , O R M U L T I P L E A B E R R A T I O N ~ AMONG THEIR OFFSPRING
N u m b e r o f a b e r r a t i o n s f r o m a single f e m a l e
Experiment i 2
o
•
2
3
4
5
6
7
8
9
zo
122 lO6
162 113
154 154
I66 131
16o 119
178 204
273 127
184 67
258
113
25o
11
t2
13
174
--
147 95
Although not as great a difference was noted, fewer females were recovered than wa~ expected t h a t had one, two, three, or four aberrations. It can be argued t h a t the us~ of Poisson distribution is not warranted since the mean expectation of 1.65 aberrations per culture would hold only if all cultures were of the same size. It can be stated t h a t those females which produced more aberrations than were expected on the basis ot randonmess alone did not do so as a result of producing more offspring and therefore having a greater chance for the events to occur. This is demonstrated in Table I I I where it can be seen t h a t no meaningful differences exist in the average number o! -offspring produced by females associated with no, one, or multiple aberrations. It fi, felt t h a t the lack of complete homogeneity of culture sizes from groups of females with different numbers of aberrations among their progenies alters the expected frequencies only slightly, and t h a t the general observation of nonrandomness is valid. The d a t a in Table II show the broods in which the aberrations from individual females were recovered. It can be seen t h a t most aberrations occurred among the earliest broods, which is in agreement with the findings of PARKER7 and ABRAHAM"SONI. Furthermore, when individual females had multiple aberrations among their offspring, they occurred as early as brood A before cells t h a t had been oogonia at the time of irradiation should have been recovered. Therefore, it seems unreasonable t h a t these events could be explained on the basis of stem-cell clusters. The number of ovarioles in the detachment stock used has been 22-24 with very little deviation for the several years t h a t it has been tested. It is assumed t h a t oogenesis in these flies is similar to t h a t described b y KING et al. ~. Other evidence can be presented t h a t these multiple aberrations do not represent proliferations of stem-cell events based on the interchange partners involved in the aberrations. Interchanges involving X - Y B s, X - Y y ÷. X-autosomes, or Y-autosomes could be observed in these experiments and the events could be classified as the same, different, or reciprocal. This will give only a minimal estimate for differences since similar or reciprocal appearing events, b y these phenotypical classifications, m a y have been found to have been different upon further testing. However, of the 23 females that had 5 or more aberrations among their offspring, none had all phenotypically identical aberrations, 7 could be explained by postulating reciprocal events, and '9 and 7 cases could be explained only if 2 or 3 separate events were postulated, respectively. The aberrations recovered in these experiments must have been associated with the irradiation since only 9 aberrations were found among 41o32 offspring in the controls. They were distributed throughout the different broods, and in no case was more than one aberration associated with a single female. No satisfactory explanation can be offered at this time for the apparent differ2VIutation R e s . , 7 (1969) 122-125
SHORT COMMUNICATIONS
125
ential radiosensitivity of the ooeytes in the different females. The multiple aberrations do occur among early broods, which should not contain products of cells that had been stem cells at the time of irradiation, and m a n y events are independent based on phenotypic observations. Further studies to explain these observations are in progress. This work was supported by U.S.A.E.C. contracts AT (o4-3)-59 ° to R.R.R. and AT (04-3)-529 to F.J.R. I t also received support from the Association of E u r a t o m and the University of Leiden, Contract No. o52-64-1 BIAN, the International Atomic Energy Agency and The Health Research Organization T.N.O. It is a pleasure to thank Dr. F. H. SOBELS for facilities utilized by ROBERT R. RINEHART during a part of the work and Dr. K. SANKARANARAYANANfor comments on the manuscript. We also acknowledge the very efficient technical assistance of J E A N PRENDERGAST and GLORIA WESTIN.
Biology Department, San Diego, State College, San Diego, Calif. (U.S.A.)
ROBERT R. R I N E H A R T * F. J. RATTY
i ABRAHAMSON, SEYMOUR, C h r o m o s o m e r e a r r a n g e m e n t s i n d u c e d b y X - r a y s in i m m a t u r e g e r m cells of Drosophila, Nature, 191 (1961) 523-524 . 2 ABRAHAMSON, SEYMOUR, IRWIN H. HERSKOWlTZ AND H. J. MULLER, I d e n t i f i c a t i o n of halft r a n s l o c a t i o n s p r o d u c e d b y X - r a y s in d e t a c h i n g a t t a c h e d - X c h r o m o s o m e s of D. melanogaster, Genetics, 41 (1956) 41o-419. 3 GRELL, RHODA F., AND ANN C. CHANDLEY, E v i d e n c e b e a r i n g on t h e coincidence of e x c h a n g e a n d D N A replication in t h e oocyte of Drosophila melanogaster, Proc. Natl. Acad. Sci. (U.S.), 53 (1956 ) 134o-1346. 4 KING, R. C., ANx C. RUmNSON AND R. F. SMITH, Oogenesis in a d u l t Drosophila melanogaster, Growth, 20 (1956) 121-157. 5 KOCH, ELIZABETH A., AND ROBERT C. KING, T h e origin a n d early differentiation of t h e egg c h a m b e r of Drosophila rnelanogaster, J. Morphol., 119 (1966) 283 304 . 6 LINDSLEY, DAN" L., AND E. H. GRELL, Genetic v a r i a t i o n s of Drosophila melanogaster, Carnegie [nst. Wash. Publ., 627 (1968). 7 PARKER, D. R., R a d i a t i o n i n d u c e d e x c h a n g e s in Drosophila females, Proc. Natl. Acad. Sci. (U.S.), 4 ° (1954) 795-800. 8 PARKER, D. R., AND J. McCRONE, A genetic a n a l y s i s of s o m e r e a r r a n g e m e n t s i n d u c e d in oocytes of Drosophila, Genetics, 43 (1958 ) 172 186. 9 RINEHART, R. R., U n p u b l i s h e d data.
Received October ioth, 1968 * P r e s e n t a d d r e s s : D e p a r t m e n t of R a d i a t i o n Genetics, U n i v e r s i t y of Leiden, a n d I n s t i t u t e for R a d i o p a t h o l o g y a n d R a d i o p r o t e c t i o n , L e i d e n (The N e t h e r l a n d s )
Mutation Res., 7 (1969) 122-125