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Relation between the X-ray dose and the rate of dominant lethals induced by irradiation of mouse spermatozoa
MUTATION RESEARCH
73
R E L A T I O N B E T W E E N T H E X-RAY DOSE AND T H E RATE OF DOMINANT L E T H A L S INDUCED BY I R R A D I A T I O N OF MOUSE SPERMATOZOA
A. Lt~ONARD Laboratoire de Gdndtique, D@artement de Radiobiologie, Centre d't2tude de l'~nergie Nucldaire, Mol (Belgique)
(Received September 9th, 1965)
SUMMARY
Male mice received testicular irradiation with increasing doses from IOO to 6000 R of X-rays. After irradiation, each male was mated with 3 virgin females. Dissection of the females showed that the percentage of pregnant females decreases with the dose of X-irradiation received by the male. Our results show that the relationship between dominant lethal mutation rate and X-ray dose is linear. The ratio deaths before implantation/deaths after inlplantation increases only for high doses.
INTRODUCTION The studies which have followed tile discovery of dominant lethals15 induced by irradiation of the male reproductive cells have shown that the rate of lethality induced in the offspring is related to the X-ray dose, provided that the matings occur during the initial period of fertility of the irradiated male mice. Though very complete studies were performed on Drosophila",1% on Habrobracon 11,22 and oi_, the bee 14, the data concerning small mammals are rathe~ scanty. This problem has now been studied systematically from th e point of view of the relation between X-ray dose and rate of lethality as well as the influence of the dose on the time of embryonic death. MATERIALAND METHODS Male mice from the BALB/C strain, eleven to thirteen weeks old, each weighing about 25 g, received local irradiation to the testis with doses of IOO, 200, 300, 400, 500, 600, 700, 800, 900, IOOO, ILOO, 12oo, 15oo, 3000 and 6000 R of X-rays. For Xirradiation, male mice were taped individually oil a Plexiglass plate and covered by a lead shield with a hole (2..5 c m × 3 cm) located over the testis. The conditions of irradiation were 300 kV, 20 mA, filtration 2 mm Cu, dose rate IOO R/min. Immediately after irradiation, each male was mated, for a period of 7 days, Mutation Res., 3 (1966) 73-78
:',. LEONARD
74
w i t h 3 v i r g i n f e m a l e s . O n t h e s e v e n t e e n t h d a y a f t e r t h e b e g i n n i n g of t h e m a t i n g , all t h e f e m a l e s w e r e d i s s e c t e d a n d t h e n u m b e r of c o r p o r a l u t e a a n d of l i v i n g a n d d e a d embryos recorded. RESULTS A s s h o w n in T a b l e 1 , 1 4 4 f e m a l e s w e r e o p e n e d a t e a c h d o s e l e v e l a n d t h e m m : b e r of p r e g n a n t f e m a l e s d e c r e a s e s r e g u l a r l y , f r o m 1 lO for t h e c o n t r o l s t o 7 for t h e f e m a l e s TABLE I GENERAL
RESULTS
OF THE
DISSECTIONS
X-ray dose (R)
Total females
Pregnant ~males
Corpora lutea
lmplanlations
Live embryos
Dead embryos
o loo 200 300 4 °0 5 °0 600 700 8oo 900 iooo Itoo I2oo 15°° 3000 6ooo
144 I44 i44 I44 144 144 i44 I44 14.t i44 144 144 144 ~44 144 144
ilo 1o8 94 lo5 91 9:3 99 lO3 95 79 82 59 64 5° 12 7
1244 1193 Io45 12o 9 too 5 IO52 lO86 116l lO94 893 9o9 650 716 56t 133 80
lO29 974 798 88I 722 734 756 74 ° 660 522 5Ol 354 347 234 42 32
932 841 649 634 517 429 407 369 327 225 217 135
97 133 I49 247 205 305 .349 37 ~ 1333 297 284 219
136
211
82 23 20
152 ~9 12
m a t e d w i t h m a l e s i r r a d i a t e d w i t h 6000 R (76% a n d 5 % ) . T h u s , b e c a u s e t h e p e r c e n t a g e of p r e g n a n t f e m a l e s is t o o l o w a f t e r i r r a d i a t i o n of t h e m a l e s w i t h 3 o o o or 6 o o o R , w e will c o n s i d e r m a i n l y t h e r e s u l t s u p t o 15oo R . ;
90-
80 "
a / ~
7 5
/
m 7o o ~
o/f
60
o
o
o''-~
/
50
A
45
~
e / o ~
/
0//
35
2O ~
,
!
/
- /
o, ........
I0
o
6o
~;o
3;o
4;0
60
68o
760
A
Preimplanlation
O
Poslimplanta
~8o
sGo
loss lion
loss
,o'oo .8o
Eoo
,3;0 ~4'oo ,s;o
X - r a y dose
Fig. I. Relation between total, pro- and post-implantation loss and the X-ray (loses.
2YIulation ICes., 3 (1966) 73 78
X-RAY DOSE AND RATE OF DOMINANT LETHALS
75
Tile total loss given by the ratio corpora lutea--live embryos/corpora lutea increases from about 25% for the controls to about 85% after irradiation of the males with 15oo R (Fig. I) and the mean number of live embryos at the day of dissection decreases from 8.5 to 1.6. In the same way the distribution of females according to the number of live embryos shifts to the left (Fig. 2) when the X-ray dose increases. The pre-implantation loss, corpora lutea--implantations/corpora lutea, seems to increase linearly (Fig. I) with the X-ray dose, while implantations/9 decrease from 9-4 for tile controls to 4-7 after 15oo R (Table II).
TABLE ANALYSIS
lI OF TABLE
I DATA
X-ray dose ( R)
o,, / o of pregnant females
Implantations/~,
Live embryos~9
Dead embryos/~_
Pre-implanrations deaths post-implan lations deaths
Live embryos corpora lutea
o lOO 200 3o0 4°0 5°0 600 700 800 9°0 iooo ilOO 12oo 15oo 3°00 6000
76.4 75.o 65. 3 72.9 63.2 64.6 68. 7 71.5 66.0 54-9 56.9 41.o 44.4 34.7 8.3 4.9
9.4 9.0 8. 5 8. 4 7.9 7.9 7.6 7.2 6.9 6.6 6.1 6.0 5-4 4-7 3.5 4 .6
8.5 7.8 6. 9 6.0 5.7 4 .6 4.1 3.6 3.4 2.8 2.6 2.3 2.1 1.6 1.9 2.9
o.9 1.2 i .6 2. 3 2.2 3.3 3.5 3.6 3-5 3.8 3.5 3.7 3-3 3.0 1.6 1.7
2.2 1. 7 i-7 1. 3 1. 4 i .o 0.9 i.i i .3 1.2 1.4 1-4 1.8 2.2 4.8 4.0
o.7491 o.7o38 0.621 i 0.5244 o.5144 0.4078 0.3748 o.3t78 0.2989 0.2520 0.2387 0.2077 o.1913 o. 1461 o. t 7 2 9 0-2500
The post-implantation loss, implantations--live embryos/corpora lutea, contains implanted embryos dying a short time after implantation (deciduomata) and the late deaths. As the frequency of late deaths was almost the same in control and irradiated matings, the post-implantation loss was not divided into two classes. Fig. I shows that the post-implantation loss increases from 8% for the controls to 27°/"o after irradiation of the males with 9oo R, while the dead embryos/9 increases from o. 9 to 3.8 (Table II). The time of embryonic death was studied from the ratio deaths before implantation/deaths after implantation. To 6oo R (Table II) we have a decrease in the ratio (from 2.2 for the controls to o. 9 after 6oo R) because most of the induced dominant lethality occurs after implantation. From 7oo R, the death before implantation and the ratio increases to reach a value of about 4.8 for the females mated with males irradiated with 3ooo R. The rate of induction of dominant lethal mutations was calculated according to the method used by EDWARDSAND SEARLEv. If we consider the ratio live embryos/ corpora lutea as a measure of overall survival, then the incidence of induced dominant lethality (corrected for embryonic loss in the controls) is given by P -- I -- (survival in irradiated series/survival in controls). The rate of induction of dominant lethality Mutation Res., 3 (1966) 7 3 - 7 8
76
a. LI~.ONARD
600 R
©
1100 R
I
1001 vs 200
700 R
R
11200 R
./ /
1.6 ¸ /
800 R
]1500
R
P
Y = 0.001142
1.4
X
~) 1.2 ¸
0
4J D 1.0
. . . . . . .
E
I ,oo. 2S
/\ ,/'-'#"
",
~
0.8
--
0.6.
....
.XC_,....
7,] ,oo
./
1000 R
i", 1 6000 R
.~ 0.4-
E 0
C~ 0 . 2 -
2
,
6 ~ ,0 *~ I~
2 4
W I ,* ,2 I~
Living embryos
o
/
/
./
./
'26o'46o'66o's6o
ldoo" ffoo
X-ray
15bo
dose
Fig. 2. D i s t r i b u t i o n of t h e f e m a l e s a c c o r d i n g t o t h e n u m b e r of live e m b r v o s a n d t h e X - r a y d o s e . .... , females mated with control males; , f e m a l e s m a t e d witil i r r a d i a t e d males. F i g . 3. D o m i n a n t l e t h a l m u t a t i o n rate.
(corrected for embryonic loss in the controls) is given by --loge (I--P). As shown in Fig. 3, this relationship between dominant lethal mutation rate and dose, is linear up to 15oo R with a regression coefficient b = 0.o01142 ~_ o.oooo164 (P < o.ooi). DISCUSSION
The decrease in the percentage of pregnant females may be due to various factors including a sharp decrease in the number of male reproductive cells or even their complete disappearance, a loss of the physiological properties of the spermatozoa or even a diminution of the mating instinct in the irradiated males. Various studies on spermatozoa of mice that had been subjected to high doses of irradiationS, 2° did not reveal any morphological changes induced by irradiation. FOGG AND COWING8--10, using doses from 300 to 5050 R, did not observe any change in the number of spermatozoa during the first days after treatment. For X-ray induced changes in the properties of spermatozoa, most authors:~,4, ~ 10 agree that these changes are minimal. In 1963, VALENTA, KOLOUSEK AND FULKA2] studies the action of various kinds (X, fl, 7) and doses (from IO to 15oooo R) of ionizing radiation on male germ cells. They concluded that the vitality and mobility of spermatozoa are affected only by doses of the order of IOOO and ~oooo R. Some changes in the fertilizing capacity of spermatozoa and in the content of nucleic acids are discernible from IOO R doses. Thus, the ~ I u t a l i o n Res., 3 ( [ 9 6 6 ) 7 3 - 7 8
77
X-RAY DOSE AND RATE OF DOMINANT LETHALS
d i m i n u t i o n in t h e n u m b e r of p r e g n a n t females can p r o b a b l y be ascribed to a loss in the fertilizing c a p a c i t y of t h e s p e r m a t o z o a c o m b i n e d with a physiological effect on the male. Previous d a t a h a v e shown t h a t m a t i n g of males during the first weeks after X - i r r a d i a t i o n gave litters of r e d u c e d size. SNELL 18 a n d HERTWIG 12, after performing i r r a d i a t i o n of male mice with increasing X - r a y doses from 2oo to 14oo R, observed a decrease in m e a n litter size in relation with the X - r a y dose given to the male. F r o m the results of SNELL 18, BRENNEKE ~ or BATEMAN 1,2 (Table I I I ) it was possible to conclude t h a t this decrease in l i t t e r size was due to an increase in pre- and p o s t - i m p l a n TABLE PREVIOUS
Rejere~zce No.
IIl DATA
ON TIME
X-ray d o s e
18
o 800
4 17 1
OF DEATH
OF
EMBRYOS
lmpla~tations/?
Live embryos/~_
Dead embrvos/~
7.56 6.50
7.22 2.63
12oo
3.79
0-74
3.05
O 8OO
9.17 7.56
8.19 3.92
0.98 3.64
I2.OO
I 1.0 9
O. 9 I
t 1.92 ii.73 9.38
~o.58 8.62 5.o8
J.34 3.i~ 4.3 o
O
125 200 5oo
0.33 3.87
t a t i o n loss. However, if the previous d a t a were not extensive enough to conclude d e f i n i t i v e l y on the relation between d o m i n a n t lethal m u t a t i o n rate a n d X - r a y dose, t h e results of BATEMAN 1,2 with 125, 2oo, 5oo, IOOO a n d 2ooo R acute X - i r r a d i a t i o n of mouse s p e r m a t o z o a seem to show t h a t the m u t a t i o n rate e s t i m a t e d b y in ( I - - d e c i d u o m a t e / i m p l a n t a t i o n s ) has a t e n d e n c y to give a concave d o s e - d e p e n d e n t curve. Using --loge ( l - - P ) as an e s t i m a t i o n of the rate induction of d o m i n a n t lethal m u t a tions, we can conclude from our results t h a t this relation is linear for increasing doses from IOO to 15oo R. RUSSELL 16, a n a l y z i n g the results of SNELL IS a n d HERTWIG 12, p o i n t e d out t h a t an increase in the dose of i r r a d i a t i o n from 800 to 12oo R seems to produce an increase in the ratio d e a t h s before i m p l a n t a t i o n / d e a t h s after i m p l a n t a t i o n , because, as LEA 1:~ has s t a t e d , an increase in n u m b e r of s p e r m a t o z o a c a r r y i n g more t h a n one lethal will t e n d to increase the frequency of p r e - i m p l a n t a t i o n e m b r y o n i c death. F r o m our results a n d BATEMAN'S data1, 2 it m a y be concluded t h a t the increase of X - r a y dose from IOO to 600 R increases the d e a t h s before i m p l a n t a t i o n , b u t chiefly the d e a t h s after i m p l a n t a t i o n , while from 700 R we observe an increase in the n u m b e r of s p e r m a t o z o a c a r r y i n g m o r e t h a n one lethal t o g e t h e r with a d r o p in the n u m b e r of s p e r m a t o z o a c a r r y i n g only one lethal (Fig. I). ACKNOWLEDGEMENT
I a m d e e p l y grateful to Dr. A. G. SEARLE a n d Professor K. (/. L{;NIN(; for their helpful criticisms. 2Vh#alion Res., 3 (1966) 73-78
78
A. LI~ONARD
This work was done thanks to the contract Euratom-C.E.N. No. o53-64-3-BIOB and with the aid of grants from the "Fonds de la Recherche Scientifique Fondamentale Collective". I~EFERENCES
I BATEMAN, A. J., Mutagenic sensitivity of n l a t u r i n g germ cells in the inale mouse. Heredity, 12 (1958) 213-232. 2 BATEMAN, A. J., The p a r t i t i o n of d o m i n a n t lethals in the nlouse between u n i m p l a n t e d eggs and deciduomata. Heredity, 12 (1958) 467-475 . 3 BERCONIE, J. AND g. TR1BONDEAU, Actions des r a y o n s X sur le testicule de r a t blanc. Compt. Rend. Soe. Biol., 57 (19o4) 592-5974 ]3RENNEKE, H., Strahlensch/idigung y o n M/iuse- u n d R a t t e n s p e r m a , b e o b a c h t e t an der Friihe n t w i c k l u n g der Eier. Strahlentherapie, 60 (1937) 214-238. 5 CHANG, hi. C. AND E. ]3. HARVtgY, 1Lffects of ionizing radiation of g a m e t e s and zygotes on the e m b r y o n i c d e v e l o p m e n t of r a b b i t s and h a m s t e r s , i n \¥. D. CARLSON AND F. X. (;ASSI~ER, Effect oJ lonizing Radiation on the Reproductive System, New York, 1964, p. 73-89. 6 DEMEREC, M. AND U. FANG, F r e q u e n c y of d o m i n a n t lethals induced by radiation in s p e r m s of Drosophila melanogaster. Genetics, 29 (1944) 348-36o. 7 EDWARDS, R. G. AND A. G. SEARLE, Genetic radiosensitivity of specific post-dictyate stages in m o u s e o6cytes. Genet. Res., 4 (1963) 389-398. 8 FOGG, L. C. AND R. F. COWING, The changes in cell m o r p h o l o g y and h i s t o c h e m i s t r y of the testis following irradiation and their relation to o t h e r induced testicular changes. I. Q u a n t i t a t i v e r a n d o m s a m p l i n g of germinal cells at intervals following direct irradiation. Cancer Res., 1~ (I951) 23-28. 9 FOG(;, I.. C. AND t{. F. COWING, The changes in cell m o r p h o l o g y and histochenlistry of the testis following irradiation and their relation to other induced testicular changes. 1 I. Comparison of effects of doses of 144o and 5o5 ° r w i t h 3oo r. Cancer Res., II (I951) 8I 86. lo I;OC,(;, L. C. AND R. F. COWING, P o s t - i r r a d i a t i o n studies on m a n l m a l i a n testes. Effect at hourly intervals for first 24 hours. Proc. Soe. Exptl. Biol. Med., 79 (1952) 88--92" 1 I HE1DI~;NTHAL, G., The occurrence of X - r a y induced d o m i n a n t lethal m u t a t i o n s in H a b r o b r a c o n . Genetics, 3 ° (1945) 197 205 . 12 HERTWlG, P., Unterschiede in der Entwicklungsf/ihigkeit von F 1 M/iusen nach R6ntgenb e s t r a h l u n g yon Spermatogonien, fertigen und unfertigen Spermatozoen. Biol. Zentr., 58 (1938) 273-3Ol. 13 LEA, D. E., Effects ol radiation on germ-cells: D o m i n a n t lethals and hereditary partial sterility. Brit. J. Radiol. Suppl., I (1947) 12o 141. 14 LEE, W. B., The dosage response curve for radiation induced d o l n i n a n t lethal m u t a t i o n s in the honeybee. Genetics, 43 (1958) 48o-49215 REGAUD, C. AND G. DUBREUIL, P e r t u r b a t i o n s d a n s le d d v e l o p p e m e n t des (t~ufs f~condds p a r des spermatozoYdes RoentgGnis~s chez le lapin. Compl. Rend. Soc. Biol., 64 (19o8) l o l 4 lOI6. 16 RUSSELL, xcV. L., Genetic effects of radiation in nlammals. I n A. HOLLAENDER, Radiation Biology, Vol. 1, McGraw-Hill, New York, 1954, p. 825-859. 17 RUSSELL, W, L., J. C. ](ILE AND L. }3. RUSSELL, Failure of h y p o x i a to p r o t e c t against the radiation induction of d o m i n a n t lethals in mice. Genetics, 36 (1951) 574-578. 18 SNELL, G. D., X - r a y sterility in the house mouse. J. Expll. Zool., 65 (1933) 421 441. 19 SONNENBLICK, B. P., Cytology and d e v e l o p m e n t of e m b r y o s of X - r a y e d adult Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S., 26 (194 o) 373 381. 20 SPALDING, J. F., J. M. WELLNITZ AND V~r. H. SCHVCEITZER, Effects of rapid lnassive (loses of g a m m a r a y s on the testes and germ cells of the rat. Radiation Res., 7 (1957) 65 7 TM 21 VALEN'rA, M., J. KOULOUSEK AND J. FULKA, The influence of ionizing radiation on nucleic acids, vitality and fecundating ability of male sexual cells. Intern. J. Radiation Biol., 6 (1963) 81-9i. 22 WHITING, A. R., Genetics of H a b r o b r a c o n . Advan. Genet., IO (1961) 295-348.
Mutation Res., 3 (~966) 73-78
73
R E L A T I O N B E T W E E N T H E X-RAY DOSE AND T H E RATE OF DOMINANT L E T H A L S INDUCED BY I R R A D I A T I O N OF MOUSE SPERMATOZOA
A. Lt~ONARD Laboratoire de Gdndtique, D@artement de Radiobiologie, Centre d't2tude de l'~nergie Nucldaire, Mol (Belgique)
(Received September 9th, 1965)
SUMMARY
Male mice received testicular irradiation with increasing doses from IOO to 6000 R of X-rays. After irradiation, each male was mated with 3 virgin females. Dissection of the females showed that the percentage of pregnant females decreases with the dose of X-irradiation received by the male. Our results show that the relationship between dominant lethal mutation rate and X-ray dose is linear. The ratio deaths before implantation/deaths after inlplantation increases only for high doses.
INTRODUCTION The studies which have followed tile discovery of dominant lethals15 induced by irradiation of the male reproductive cells have shown that the rate of lethality induced in the offspring is related to the X-ray dose, provided that the matings occur during the initial period of fertility of the irradiated male mice. Though very complete studies were performed on Drosophila",1% on Habrobracon 11,22 and oi_, the bee 14, the data concerning small mammals are rathe~ scanty. This problem has now been studied systematically from th e point of view of the relation between X-ray dose and rate of lethality as well as the influence of the dose on the time of embryonic death. MATERIALAND METHODS Male mice from the BALB/C strain, eleven to thirteen weeks old, each weighing about 25 g, received local irradiation to the testis with doses of IOO, 200, 300, 400, 500, 600, 700, 800, 900, IOOO, ILOO, 12oo, 15oo, 3000 and 6000 R of X-rays. For Xirradiation, male mice were taped individually oil a Plexiglass plate and covered by a lead shield with a hole (2..5 c m × 3 cm) located over the testis. The conditions of irradiation were 300 kV, 20 mA, filtration 2 mm Cu, dose rate IOO R/min. Immediately after irradiation, each male was mated, for a period of 7 days, Mutation Res., 3 (1966) 73-78
:',. LEONARD
74
w i t h 3 v i r g i n f e m a l e s . O n t h e s e v e n t e e n t h d a y a f t e r t h e b e g i n n i n g of t h e m a t i n g , all t h e f e m a l e s w e r e d i s s e c t e d a n d t h e n u m b e r of c o r p o r a l u t e a a n d of l i v i n g a n d d e a d embryos recorded. RESULTS A s s h o w n in T a b l e 1 , 1 4 4 f e m a l e s w e r e o p e n e d a t e a c h d o s e l e v e l a n d t h e m m : b e r of p r e g n a n t f e m a l e s d e c r e a s e s r e g u l a r l y , f r o m 1 lO for t h e c o n t r o l s t o 7 for t h e f e m a l e s TABLE I GENERAL
RESULTS
OF THE
DISSECTIONS
X-ray dose (R)
Total females
Pregnant ~males
Corpora lutea
lmplanlations
Live embryos
Dead embryos
o loo 200 300 4 °0 5 °0 600 700 8oo 900 iooo Itoo I2oo 15°° 3000 6ooo
144 I44 i44 I44 144 144 i44 I44 14.t i44 144 144 144 ~44 144 144
ilo 1o8 94 lo5 91 9:3 99 lO3 95 79 82 59 64 5° 12 7
1244 1193 Io45 12o 9 too 5 IO52 lO86 116l lO94 893 9o9 650 716 56t 133 80
lO29 974 798 88I 722 734 756 74 ° 660 522 5Ol 354 347 234 42 32
932 841 649 634 517 429 407 369 327 225 217 135
97 133 I49 247 205 305 .349 37 ~ 1333 297 284 219
136
211
82 23 20
152 ~9 12
m a t e d w i t h m a l e s i r r a d i a t e d w i t h 6000 R (76% a n d 5 % ) . T h u s , b e c a u s e t h e p e r c e n t a g e of p r e g n a n t f e m a l e s is t o o l o w a f t e r i r r a d i a t i o n of t h e m a l e s w i t h 3 o o o or 6 o o o R , w e will c o n s i d e r m a i n l y t h e r e s u l t s u p t o 15oo R . ;
90-
80 "
a / ~
7 5
/
m 7o o ~
o/f
60
o
o
o''-~
/
50
A
45
~
e / o ~
/
0//
35
2O ~
,
!
/
- /
o, ........
I0
o
6o
~;o
3;o
4;0
60
68o
760
A
Preimplanlation
O
Poslimplanta
~8o
sGo
loss lion
loss
,o'oo .8o
Eoo
,3;0 ~4'oo ,s;o
X - r a y dose
Fig. I. Relation between total, pro- and post-implantation loss and the X-ray (loses.
2YIulation ICes., 3 (1966) 73 78
X-RAY DOSE AND RATE OF DOMINANT LETHALS
75
Tile total loss given by the ratio corpora lutea--live embryos/corpora lutea increases from about 25% for the controls to about 85% after irradiation of the males with 15oo R (Fig. I) and the mean number of live embryos at the day of dissection decreases from 8.5 to 1.6. In the same way the distribution of females according to the number of live embryos shifts to the left (Fig. 2) when the X-ray dose increases. The pre-implantation loss, corpora lutea--implantations/corpora lutea, seems to increase linearly (Fig. I) with the X-ray dose, while implantations/9 decrease from 9-4 for tile controls to 4-7 after 15oo R (Table II).
TABLE ANALYSIS
lI OF TABLE
I DATA
X-ray dose ( R)
o,, / o of pregnant females
Implantations/~,
Live embryos~9
Dead embryos/~_
Pre-implanrations deaths post-implan lations deaths
Live embryos corpora lutea
o lOO 200 3o0 4°0 5°0 600 700 800 9°0 iooo ilOO 12oo 15oo 3°00 6000
76.4 75.o 65. 3 72.9 63.2 64.6 68. 7 71.5 66.0 54-9 56.9 41.o 44.4 34.7 8.3 4.9
9.4 9.0 8. 5 8. 4 7.9 7.9 7.6 7.2 6.9 6.6 6.1 6.0 5-4 4-7 3.5 4 .6
8.5 7.8 6. 9 6.0 5.7 4 .6 4.1 3.6 3.4 2.8 2.6 2.3 2.1 1.6 1.9 2.9
o.9 1.2 i .6 2. 3 2.2 3.3 3.5 3.6 3-5 3.8 3.5 3.7 3-3 3.0 1.6 1.7
2.2 1. 7 i-7 1. 3 1. 4 i .o 0.9 i.i i .3 1.2 1.4 1-4 1.8 2.2 4.8 4.0
o.7491 o.7o38 0.621 i 0.5244 o.5144 0.4078 0.3748 o.3t78 0.2989 0.2520 0.2387 0.2077 o.1913 o. 1461 o. t 7 2 9 0-2500
The post-implantation loss, implantations--live embryos/corpora lutea, contains implanted embryos dying a short time after implantation (deciduomata) and the late deaths. As the frequency of late deaths was almost the same in control and irradiated matings, the post-implantation loss was not divided into two classes. Fig. I shows that the post-implantation loss increases from 8% for the controls to 27°/"o after irradiation of the males with 9oo R, while the dead embryos/9 increases from o. 9 to 3.8 (Table II). The time of embryonic death was studied from the ratio deaths before implantation/deaths after implantation. To 6oo R (Table II) we have a decrease in the ratio (from 2.2 for the controls to o. 9 after 6oo R) because most of the induced dominant lethality occurs after implantation. From 7oo R, the death before implantation and the ratio increases to reach a value of about 4.8 for the females mated with males irradiated with 3ooo R. The rate of induction of dominant lethal mutations was calculated according to the method used by EDWARDSAND SEARLEv. If we consider the ratio live embryos/ corpora lutea as a measure of overall survival, then the incidence of induced dominant lethality (corrected for embryonic loss in the controls) is given by P -- I -- (survival in irradiated series/survival in controls). The rate of induction of dominant lethality Mutation Res., 3 (1966) 7 3 - 7 8
76
a. LI~.ONARD
600 R
©
1100 R
I
1001 vs 200
700 R
R
11200 R
./ /
1.6 ¸ /
800 R
]1500
R
P
Y = 0.001142
1.4
X
~) 1.2 ¸
0
4J D 1.0
. . . . . . .
E
I ,oo. 2S
/\ ,/'-'#"
",
~
0.8
--
0.6.
....
.XC_,....
7,] ,oo
./
1000 R
i", 1 6000 R
.~ 0.4-
E 0
C~ 0 . 2 -
2
,
6 ~ ,0 *~ I~
2 4
W I ,* ,2 I~
Living embryos
o
/
/
./
./
'26o'46o'66o's6o
ldoo" ffoo
X-ray
15bo
dose
Fig. 2. D i s t r i b u t i o n of t h e f e m a l e s a c c o r d i n g t o t h e n u m b e r of live e m b r v o s a n d t h e X - r a y d o s e . .... , females mated with control males; , f e m a l e s m a t e d witil i r r a d i a t e d males. F i g . 3. D o m i n a n t l e t h a l m u t a t i o n rate.
(corrected for embryonic loss in the controls) is given by --loge (I--P). As shown in Fig. 3, this relationship between dominant lethal mutation rate and dose, is linear up to 15oo R with a regression coefficient b = 0.o01142 ~_ o.oooo164 (P < o.ooi). DISCUSSION
The decrease in the percentage of pregnant females may be due to various factors including a sharp decrease in the number of male reproductive cells or even their complete disappearance, a loss of the physiological properties of the spermatozoa or even a diminution of the mating instinct in the irradiated males. Various studies on spermatozoa of mice that had been subjected to high doses of irradiationS, 2° did not reveal any morphological changes induced by irradiation. FOGG AND COWING8--10, using doses from 300 to 5050 R, did not observe any change in the number of spermatozoa during the first days after treatment. For X-ray induced changes in the properties of spermatozoa, most authors:~,4, ~ 10 agree that these changes are minimal. In 1963, VALENTA, KOLOUSEK AND FULKA2] studies the action of various kinds (X, fl, 7) and doses (from IO to 15oooo R) of ionizing radiation on male germ cells. They concluded that the vitality and mobility of spermatozoa are affected only by doses of the order of IOOO and ~oooo R. Some changes in the fertilizing capacity of spermatozoa and in the content of nucleic acids are discernible from IOO R doses. Thus, the ~ I u t a l i o n Res., 3 ( [ 9 6 6 ) 7 3 - 7 8
77
X-RAY DOSE AND RATE OF DOMINANT LETHALS
d i m i n u t i o n in t h e n u m b e r of p r e g n a n t females can p r o b a b l y be ascribed to a loss in the fertilizing c a p a c i t y of t h e s p e r m a t o z o a c o m b i n e d with a physiological effect on the male. Previous d a t a h a v e shown t h a t m a t i n g of males during the first weeks after X - i r r a d i a t i o n gave litters of r e d u c e d size. SNELL 18 a n d HERTWIG 12, after performing i r r a d i a t i o n of male mice with increasing X - r a y doses from 2oo to 14oo R, observed a decrease in m e a n litter size in relation with the X - r a y dose given to the male. F r o m the results of SNELL 18, BRENNEKE ~ or BATEMAN 1,2 (Table I I I ) it was possible to conclude t h a t this decrease in l i t t e r size was due to an increase in pre- and p o s t - i m p l a n TABLE PREVIOUS
Rejere~zce No.
IIl DATA
ON TIME
X-ray d o s e
18
o 800
4 17 1
OF DEATH
OF
EMBRYOS
lmpla~tations/?
Live embryos/~_
Dead embrvos/~
7.56 6.50
7.22 2.63
12oo
3.79
0-74
3.05
O 8OO
9.17 7.56
8.19 3.92
0.98 3.64
I2.OO
I 1.0 9
O. 9 I
t 1.92 ii.73 9.38
~o.58 8.62 5.o8
J.34 3.i~ 4.3 o
O
125 200 5oo
0.33 3.87
t a t i o n loss. However, if the previous d a t a were not extensive enough to conclude d e f i n i t i v e l y on the relation between d o m i n a n t lethal m u t a t i o n rate a n d X - r a y dose, t h e results of BATEMAN 1,2 with 125, 2oo, 5oo, IOOO a n d 2ooo R acute X - i r r a d i a t i o n of mouse s p e r m a t o z o a seem to show t h a t the m u t a t i o n rate e s t i m a t e d b y in ( I - - d e c i d u o m a t e / i m p l a n t a t i o n s ) has a t e n d e n c y to give a concave d o s e - d e p e n d e n t curve. Using --loge ( l - - P ) as an e s t i m a t i o n of the rate induction of d o m i n a n t lethal m u t a tions, we can conclude from our results t h a t this relation is linear for increasing doses from IOO to 15oo R. RUSSELL 16, a n a l y z i n g the results of SNELL IS a n d HERTWIG 12, p o i n t e d out t h a t an increase in the dose of i r r a d i a t i o n from 800 to 12oo R seems to produce an increase in the ratio d e a t h s before i m p l a n t a t i o n / d e a t h s after i m p l a n t a t i o n , because, as LEA 1:~ has s t a t e d , an increase in n u m b e r of s p e r m a t o z o a c a r r y i n g more t h a n one lethal will t e n d to increase the frequency of p r e - i m p l a n t a t i o n e m b r y o n i c death. F r o m our results a n d BATEMAN'S data1, 2 it m a y be concluded t h a t the increase of X - r a y dose from IOO to 600 R increases the d e a t h s before i m p l a n t a t i o n , b u t chiefly the d e a t h s after i m p l a n t a t i o n , while from 700 R we observe an increase in the n u m b e r of s p e r m a t o z o a c a r r y i n g m o r e t h a n one lethal t o g e t h e r with a d r o p in the n u m b e r of s p e r m a t o z o a c a r r y i n g only one lethal (Fig. I). ACKNOWLEDGEMENT
I a m d e e p l y grateful to Dr. A. G. SEARLE a n d Professor K. (/. L{;NIN(; for their helpful criticisms. 2Vh#alion Res., 3 (1966) 73-78
78
A. LI~ONARD
This work was done thanks to the contract Euratom-C.E.N. No. o53-64-3-BIOB and with the aid of grants from the "Fonds de la Recherche Scientifique Fondamentale Collective". I~EFERENCES
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Mutation Res., 3 (~966) 73-78