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The induction of translocations in mouse spermatozoa I. Kinetics of dose response with acute X-irradiation
Mutation Research, 22 (1974) I57-I74
:~',Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
157
T H E I N D U C T I O N O F T R A N S L O C A T I O N S IN MOUSE S P E R M A T O Z O A I. K I N E T I C S O F DOSE R E S P O N S E W I T H A C U T E X - I R R A D I A T I O N
A. G. SEARLE*, C. E. FORD**, E. P. EVANS**, C. V. BEECHEY*, M. D. BURTENSHA\V** AND HILARY M. CLEGG** with Appendix by D. G. PAPVVORTH* * Medical Research Council Radiobiology Unit, Harwell, Didcot, Berhs., and ** Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford (Great Britain)
(Received July 3rd, 1973) (Revision received October i2th, 1973)
SUMMARY A d u l t male mice were given gonadal doses of o-I2OO rad acute X - i r r a d i a t i o n a n d m a t e d the same day. 531 sons, conceived within a week of the t r e a t m e n t , were tested for fertility a n d their testes e x a m i n e d cytologically for chromosome a b e r r a t i o n s in spermatocytes. 55/57 of those diagnosed as semi-sterile a n d 35/4 ° of those diagnosed as sterile were judged to be heterozygous for one or more reciprocal translocations. N u m b e r s of o, i, 2... translocations per mouse showed a good fit to a Poisson distribution, in contrast to previous findings with spermatogonial irradiation. Although the dose response fitted a linear relationship, the power law e q u a t i o n of best fit had a dosee x p o n e n t of 1.41. F u r t h e r analysis along similar lines to those used previously in Drosophila b y CATCHESIDE, LEA AND HALDANE, which assumed r a n d o m rejoining of breaks a n d direct p r o p o r t i o n a l i t y between dosage a n d n u m b e r of breaks, gave a close fit between the actual results a n d those expected if ~q = 2.8. IO 3/rad, where ~ is the m e a n n u m b e r of breaks per nucleus a n d q is the proportion which rejoin or restitute. B y c o m b i n i n g these d a t a with those for litter-size reduction in F1 (taken as a measure of i n d u c e d d o m i n a n t lethality) c~ was e s t i m a t e d to be 3.4 × lO-3 per rad. W h e n compared with the value of 0.8 × lO -3 per rad o b t a i n e d in Drosophila b y HALDANE a n d LEA, this suggested t h a t mouse haploid nuclei are more radiosensitive to chromosome breakage t h a n Drosophila haploid nuclei b y a factor of a b o u t 4. The m e a n n u m b e r of i m p l a n t s per p r e g n a n t female m a t e d to cytologically a b n o r m a l males was a b o u t 15% lower t h a n with n o r m a l males. This p r e - i m p l a n t a t i o n loss was t h o u g h t to be m a i n l y the result of a reduction in the rate of fertilization in this group r a t h e r t h a n to early death of u n b a l a n c e d zygotes. There was no evidence for the i n d u c t i o n of a n y u n d e t e c t e d types of chromosomal a b e r r a t i o n or gene m u t a t i o n which could cause i n t r a u t e r i n e death in the progeny of F1 males.
Dedicated to Professor Dr. Dr. h. c. Paula Hertwig, in recognition of her outstanding pioneer work in this field.
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INTRODUCTION
In recent years, the induction of reciprocal translocations by irradiation of spermatogonia in the mouse has been extensively studied by a number of workers (for a review of this work, see LI~ONARD19),mainly by the cytological examination of descendant spermatocytes at diakinesis-metaphase I in the irradiated males themselves. Results of these studies have posed a number of problems. For instance, doseresponse curves for acute X-irradiation have not shown the expected quadratic component, since translocation frequencies have been proportional to dose up to about 700 R (refs. 8, 20, 22, 30) and have then declined sharply~2,2",32; only a slight dose-rate effect has been found with X-irradiation but a marked one with )J-irradiationS4, 35; the distribution of o, I, 2... translocations per cell has not fitted a Poisson relationship35; the observed frequency of transmission of translocations to progeny of the exposed males has been only about one half of that expected u. These divergences from expectation have been thought to result mainly from (i) differential radiosensitivity of different stages of the A type spermatogonia being sampled, (ii) events during the many germ-cell divisions intervening between irradiation and observation, (iii) the induction of asymmetrical exchanges and other types of lethal aberration in the same cells as the symmetrical exchanges which give rise to reciprocal transloeations, and (iv) the existence of a group of balanced reciprocal translocations which were germcell letbals at various stages of gametogenesisS, ~6. In order to obtain a wider view-point on translocation induction in mammals it seemed desirable to study its kinetics in a non-dividing germ-cell stage which was, moreover, highly radioresistant to killing, i.e. the spermatozoon. No previous cytogenetic attempt has been made to do this in the laboratory mouse. However, HERTWIG (refs. 14, 15) showed that the incidence of partially and completely sterile sons of irradiated male mice, mated in the pre-sterile period, rose from 4.4% at o R to 57.1% at 15oo-16oo R. Most of the sons' infertility presumably stemmed from the presence of translocations. L~ONARD AND DEKNUDT21 found that 5.1% of sons of males given 300 R acute X-irradiation carried reciprocal translocations when spermatozoa inferred to have been irradiated in the epididymis and vas deferens were sampled. The incidence was higher when less mature spermatozoa and spermatids were treated. MATERIALS AND METHODS
Fz (C3H/HeH × I o I / H ) male mice, aged between 8 and 18 weeks, were given gonadal doses of o, 50, IOO, 2o0, 4oo, 600, 800, IOOO, or 12oo rad acute X-irradiation (250 kVp, HVT 1.2 mm Cu, 217 rad/min). On the same day they were mated to pairs of hybrid females of the same stock, from which they were separated one week later. Numbers and sexes of offspring were recorded at birth and weaning. For the purpose of analysis, mixed litters (derived from parturition of both females in a cage on the same day) were regarded as of equal size (when even-numbered) or differing by one (when odd-numbered). At the age of 8 weeks, sons of the irradiated males were mated to pairs of outbred " R " females, from the Fz generation of a specific locus experiment. Vaginal plugs were recorded and pregnant females were killed 14 days later, when numbers of implantations of live and dead embryos were counted. The fertility status was then
I N D U C T I O N OF T R A N S L O C A T I O N IN MOUSE SPERMATOZOA. I .
159
determined by the method of CARTERet al. ~ in which pregnant females were diagnosed as fertile (F), semi-sterile (S) or giving an inconclusive result (I) according to numbers of live and dead implantations. The tested male was then regarded as (I) Fertile if 2F, or I F + I I with respect to the two mates; (2) Semi-sterile if 2S, or I S + I I ; (3) Test inconclusive if 2I, or I F + I S . If neither mate appeared pregnant after a period of at least 4 weeks the male was considered sterile. However, confirmation of this findings was frequently sought by replacing the females with a further pair. This was also done when one of the two original females failed to become pregnant in 4 weeks. Males were killed as soon as possible after their fertility status had been established. Air-dried preparations were made from the testes by the method of EVANS et al. 7, and primary spermatocytes at diakinesis-metaphase I were examined and the nature of the chromosome associations recorded. As a routine, 25 spermatocytes were examined from each male diagnosed as fertile, while ioo were examined from those diagnosed as sterile, semi-sterile or with an inconclusive result. However, the number required from the "test inconclusive" group was reduced to 50 later, since this seemed sufficient for the purpose. Whenever there was cytological evidence for presence of a reciprocal translocation at least IOO spermatocytes were scored, while even more were studied when the frequency of abnormal configurations was low. Scoring was mainly shared between M.D.B., H.C., E.P.E. and C.E.F., but C.V.B. scored 15 of the sterile males. Additional information obtained from sterile males included sperm-counts and testis weights. In addition, mitotic chromosome preparations were obtained from sterile males, either from the cornea by a modification of FREDGA'Smethod 1~ or from the bone-marrow by the air-drying method of FORD1°. Daughters of males given doses of 600 rad or more were also tested for the presence of reciprocal translocations. Results of these studies will be given in a separate paper in this series. The normal criterion for diagnosing translocation heterozygosity in F1 males was the presence of characteristic multivalent configurations in spermatocytes at diakinesis-metaphase I. Those giving hexavalent configurations were regarded as having two reciprocal translocations involving three pairs of chromosomes, rather than as the result of cyclic exchanges. Ten sterile males in which no metaphase spermatocytes or later spermatogenic stages could be found were considered heterozygous for one reciprocal translocation. This was because LYoN AND ~]IEREDITH~ have demonstrated that heterozygosity for particular autosomal reciprocal translocations can cause failure of spermatogenesis, with very few cells reaching meiotic metaphase I in some cases. CATTANACHe~ al. 5 failed to find any metaphase spermatocytes in one transloeation of this type. Confirmatory evidence was obtained from our own sterile males lacking spermatocytes in metaphase I by the observation of one or two mitotic marker chromosomes, suggestive of reciprocal translocation, in somatic tissue of five of the ten mice concerned. RESULTS
R e c i p r o c a l translocations
Table I shows that there was close agreement between diagnoses of fertility or
16o
a.G.
SEARLE
631
al.
TABLE I RELATIONSHIP BET'WEEN DIAGNOSIS OF FERTILITY STATUS AND NUMBERS L O C A T I O N S C A R R I E D B Y S O N S O F I R R A D I A T E D A N D C O N T R O L IvIALES
Fertility diagnosis
Translocations per mouse 0 I 2
Fertile Test inconclusive Semi-sterile Sterile
.385 37 2a 5
.3 S 50 25
Total
429
8()
OF RECIPROCAL
3
Total of mic~ examined
o
.388
o r 5 7
o
46
o 3
57 4°
I3
3
53 I
TRANS-
a I with insertion. s e m i - s t e r i l i t y a n d a b s e n c e or p r e s e n c e of r e c i p r o c a l t r a n s l o c a t i o n s , r e s p e c t i v e l y . All t h r e e of t h o s e d i a g n o s e d as fertile y e t f o u n d to c a r r y t r a n s l o c a t i o n s g a v e s o m e d e a d i m p l a n t a t i o n s (2/12, 4/15 a n d I/IO). Of tile t w o s e m i - s t e r i l e m a l e s w h i c h failed to g i v e c y t o l o g i c a l e v i d e n c e for r e c i p r o c a l t r a n s l o e a t i o n s , one p r o v e d to c a r r y an i n s e r t i o n (which will be d e s c r i b e d elsewhere). T h e other, w h i c h was in tile c o n t r o l series, g a v e 7 d e a d a m o n g 14 i n p l a n t a t i o n s . F o u r of t h e s e (all f r o m one female) h a d d i e d as foetuses at a b o u t 13 d a y s p o s t - c o i t u s ; t h e y l o o k e d f l a t t e n e d a n d d i s t o r t e d b u t h a d no specific a b n o r m a l i t y . T h e o t h e r t h r e e h a d d i e d earlier a n d w e r e r e c o g n i s e d as d e c i d u o m a t a ("small moles"). A b o u t 2 o % of t h o s e m a l e s in w h i c h t h e f e r t i l i t y t e s t was i n c o n c l u s i v e p r o v e d to c a r r y one or m o r e r e c i p r o c a l t r a n s l o c a t i o n s , as did n e a r l y 9 o % of sterile males. A full a c c o u n t of t h e s e will be g i v e n in a s e p a r a t e p a p e r . I n all, 113 s i m p l e r e c i p r o c a l t r a n s l o c a t i o n s a n d 4 a n i m a l s w i t h h e x a v a l e n t s w e r e d i a g n o s e d , as well as tile i n s e r t i o n a n d an X Y Y a n i m a l , in 531 sons tested. F r e q u e n c i e s of serui-sterile a n d sterile sons rose s t e a d i l y w i t h i n c r e a s i n g p a t e r n a l dose (Table II) a n d s h o w e d g o o d a g r e e m e n t w i t h HERTWIG'S earlier results~4, ~a. In c o n t r a s t to findings a f t e r s p e r m a t o g o n i a l i r r a d i a t i o n , t h e t r a n s l o c a t i o n f r e q u e n c y a f t e r s p e r m a t o z o a l i r r a d i a t i o n also rose s t e a d i l y w i t h dose (except at I o o rad) a n d s h o w e d no decline at h i g h e r levels (Table I I I ) . D o s e - r e s p o n s e c u r v e s w e r e f i t t e d b y t h e m e t h o d of m a x i m u m likelihood, a s s u n f i n g a Poisson d i s t r i b u t i o n of o, i , 2, 3 .... t r a n s l o c a t i o n s p e r m o u s e . T h e o b s e r v e d d i s t r i b u t i o n did, in fact, s h o w a g o o d fit to a Poisson (P -o . 9 I ). T h e s t r a i g h t line of best fit to t h e d a t a (Fig. I) h a d t h e f o r n m l a Y : 4.78 -! 0.43 TABLE II FERTILITY
Dose (rad)
o 5° I00
DIAGNOSES
AT DIFFERENT
Fertile
57 53 54
DOSES IN SONS OF IRRADIATED
N u m b e r s diagnosed as [nconSemiclusive sterih
7 2 2
Sterile
I i o
o l 1
MALES
Percentage Semi-sterile or sterile
1.5 3.5 1.8
200
42
4
2
0
4.2
4 °0
47
6
4
5
I4.5
600 800 1ooo I200 Total
49 4° 28 I8 388
5 7 7 6 46
1o t3 ~4 ~2 57
5 7 to II 4°
2 i. 7 29.q 40.7 48.9
I N D U C T I O N OF T R A N S L O C A T I O N IN M O U S E S P E R M A T O Z O A . TABLE
I.
16I
lli
DISTRIBUTION AND FREQUENCY OF RECIPROCAL TRANSLOCATIONS IN SONS OF MALE MICE GIVEN ACUTE X-IRRADIATION (SPERMATOZOAL SAMPLING)
Dose (rad)
Number of mice
Translocations per mouse o • 2 3
o 50 IOO 200 400 600 800 IOOO 12oo Total
65 57 57 t8 62 69 67 59 47 531
65 56 57 46 53 52 44 33 a 23 429
o I o , 8 i4 21 22 i8 86
o o o o I 3 2 2 5 13
o o o o o o o 2 I 3
Total Frequency translocations per mouse ( % 0
i o 2 IO 20 25 32 31
0,0
1.8 o.o 4.2 16.1 28. 5 37-3 54.2 66.0
a i with insertion
PERCENT TRANSLOCATIONS70
I
6050 40 30 20 10
--t"'"""" t'"'"'" t'"'"'-" }""
--"'"'"'"
200
400
600
800
1000 1200
OOSE (.Ao) l q g . i. F r e q u e n c i e s ( w i t h s t a n d a r d e r r o r s ) of r e c i p r o c a l t r a n s l o c a t i o n s in s o n s of m a l e m i c e g i v e n v a r i o u s d o s e s of a c u t e X - i r r a d i a t i o n t o s p e r m a t o z o a . T h e i n t e r r u p t e d line is t h e s t r a i g h t line of b e s t fit.
• xo 4 D, where Y is the translocation yield a n d D the dose. Although the fit was good (P = o.57) it was n o t e d t h a t deviations of observed values from expectations were all negative at low doses (5o-4oo rad) b u t positive at higher doses of xooo a n d I2oo rad. This suggested t h a t the true relationship was curvilinear, as would be expected on theoretical grounds. The e q u a t i o n of best fit to a quadratic was Y - - 2.25 _+_ I.O3 • IO 4 D + 3 . o 9 ~_ 1.26.IO -7 D 2 (P = 0.84), while t h a t of a power law was Y = 3.o3 ! 3.98"Io 5 D1.41 (p = o.87). The s t a n d a r d error of the dose e x p o n e n t is o.2o; thus it differs significantly from a value of I (u = 2.i2, P = o.o34).
Lethality in F1 and F2 generations I n this experiment, the a m o u n t of d o m i n a n t lethality resulting from the spermatozoal irradiation can be estimated only from the rate of decrease of m e a n litter-size with increasing dose, shown in Table IV. There is a roughly e x p o n e n t i a l relationship between m e a n litter-size a n d dose (see Fig. 3 of Appendix), as would be expected if the d i s t r i b u t i o n of o, I, 2... d o m i n a n t lethal m u t a t i o n s is Poisson a n d if the rate of induction of p r e n a t a l d o m i n a n t lethals is directly p r o p o r t i o n a l to r a d i a t i o n dose. The equation 3'~ -- yoe-~o was fitted to the litter-size observations at different doses (y D)by the m e t h o d of least-squares, each observation being weighted inversely as its variance,
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A. G. SEARLE el al.
T A B L E IV LITTER-SIZES
AT B I R T H
Dose (tad) o
5o IOO 200 4oo 6oo 80o IOOO 1200
AND WEANING
IN OFFSPRING
OF IRRADIATED
MALES
Total born
No. of litters
Mean litter-size
Total weaned
Per cent weaned
128 149 117 lO4 127 16.5 14° 124 9.5
13 19 15 14 21 39 34 48 48
9.85 7.84 7.80 7.43 6.05 4,23 4 .12 2.52 1.98
126 139 116 lO3 123 156 134 119 76
98. 4 93.3 99.2 99.0 96.9 94.5 95.7 96.o 80.0
± ~ ± ~ i i -c ± ~
0.34 o.49 o.46 0.45 0.30 0.25 0.30 o.14 o.15
which was calculated from the observed distribution of litter-sizes. The derived value ofyo was 9.44 ~ o.35 and of k was 1.28 _~ o.o7-1o -3. Therefore the estimated rate of induction of dominant lethals is 1.28.Io-a/gamete/rad. This is about three times the rate per rad for translocations, assuming a linear relationship. It should be realised, however, that this estimate of dominant lethality can only be approximate, based as it is on newborn litters, some of them mixed. Survival of F1 offspring to weaning age remained high at all dose levels (Table IV) although lowest at 12oo rad. This drop was probably a consequence of the very low litter-size at this level, since it is known that under these circumstances the stimulus for lactation may be insufficient. Thus, out of 19 litters of one in the 12oo rad series, 9 died before weaning age. All F1 males were mated to two or more females and the amount of embryonic lethality at or near the I4th day of gestation was determined. Overall results for the different cytological categories are summarized in Table V. The mean numbers of live embryos per female in the cytologically abnormal categories are less than half that in the normal category. The mean number of implants is also markedly reduced by an amount which is clearly significant. This suggests some pre-implantation loss which may partly result from reduced fertilization of eggs, as discussed later. The embryonic survival (in terms of live embryos per female) in those with I reciprocal translocation was 42.4% of the survival in those without detectable abnormality. Since a value of 5o% is expected with normal disjunction, the observed figure suggests an average level of adjacent-2 disjunction of around 15% (ref. 33). However, this TABLE
V
EMBRYONIC
LETHALITY
IN PROGENY
OF MALES V¢ITH OR WITHOUT
DETECTABLE
TYPES
OF CHROMO-
SOME ABERRATION
(RT, r e c i p r o c a l t r a n s l o c a t i o n ) Category of male
Number of pregnant 9Q
Total implants
Live embryos
Dead implants
Per cent dead
Implants per 9
Live embryos per 9
With i RT With 2 RT With insertion Normal Total
12o 8 2 849 979
8o6 58 14 6743 7621
369 18 7 6161 6555
437 4° 7 582 lO66
54 .2 69.0 5 °.0 8.63 13.99
6.72 ~ o . 2 o 7.25 7 .00 7.94 7.78
3.o8 _~o.I 5 2.25 3.50 7 .26
I N D U C T I O N OF T R A N S L O C A T I O N I N M O U S E S P E R M A T O Z O A . TABLE
I.
163
VI
EMBRYONIC LETHALITY IN THE PROGENY OF CYTOLOGICALLY NORMAL F 1 MALES AFTER VARIOUS DOSES OF PATERNAL X-IRRADIATION 1replants
Dead Total % dead
Dose ( r a d s ) o
5°
IOO
200
400
600
8oo
3000
12oo
81 994 8.2
73 886 8. 3
71 897 7.9
62 735 8.4
87 876 9.9
77 845 9.I
59 7°8 8.3
35 467 7.5
37 335 ii.o
Z ~ = 6.53, P = o.59.
must be regarded as an upper limit, for the comparative embryonic survival in terms of live embryos/total implants was 5o.1 ± 1.9°/o. Table VI shows that within the normal category (free of detectable aberrations) there is no significant heterogeneity in the proportion of dead implants with respect to X-ray dose. If some of this embryonic lethality was the result of the induction of undetected types of chromosomal aberration, or of sub-lethal gene mutation, then its frequency would have tended to increase with increasing dose; instead it remains remarkably constant. DISCUSSION
Comparison with Drosophila We have shown that the frequency of reciprocal translocations in sons of male mice given spermatozoal X-irradiation over a range of o to 12oo rad is roughly proportional to dose, although the curve of best fit has a dose exponent of 1. 4. This agrees fairly well with results of similar treatment on Drosophila spermatozoa, reviewed by MULLER 29, in which a dose exponent of around 1.5 was usually obtained, although this sometimes approached 2 at lower doses. CATCHESIDEa was the first to try and calculate expected frequencies of induced structural changes if these resulted from the fusion of broken ends of chromosomes and if there was direct proportionality between dosage and number of breaks. He also assumed (i) that all breakage ends rejoin, (ii) that they rejoin at random and (iii) that breaks occur one in each chromosome, so that only interchanges are involved. He found a reasonable fit between his observations and expectation over the limited range of doses used (100o-40oo R). MULLER28 also found a good fit to expectation over a wider range of doses. Further contributions to the mathematical analysis of structural changes came from FANO9 and LEA AND CATCHESIDE is, who considered inter alia the contribution of dyscentric interchanges and sister-strand unions to dominant lethals, and from HALDANE AND LEA is, who dealt with the occurrence of more than one break per chromosome arm and with expectations for different numbers of arms. The subject is also reviewed by LEA in his book 17. LEA AND CATCHESIDEis found that the theoretical curve (assuming many arms) best fitted experimental data on the proportion of viable sperm with chromosome aberrations in Drosophila after various doses if aq = 0.57 per IOOO R, where ~ is a constant, relating the mean number of breaks to the dose, and q is the probability that a given break shall either restitute or take part in interchange. HALDANE AND LEAla found that an scq value of 0.52 gave the best fit with their improved 5-arm formula. By considering also the results for induction of dominant lethals in Drosophila sperm,
16 4
A.G. SEARLE et al.
the a u t h o r s a r r i v e d at values for ~. of o.75 ( m a n y arms) or o.78 (5 arms) per IOOO R a n d for q of o.76 ( m a n y arms) or o.67 (5 arnls). The same calculations can be m a d e for the mouse, since d a t a are now available for the i n d u c t i o n of b o t h d o m i n a n t lethals a n d translocations in mouse s p e r m a t o z o a . The calculations are given in the A p p e n d i x b y D. G. PAPWORTH, in which a d j u s t m e n t s to HALDANE AND LEA'S t r e a t m e n t are m a d e to allow for the fact t h a t in the mouse the identification of reciprocal t r a n s l o c a t i o n s was n o r m a l l y based on the a p p e a r a n c e of n m l t i v a l e n t configurations at meiosis. This m e a n t t h a t cyclic exchanges involving 3, 4, 5... chromosomes would n o r m a l l y be classified as 2, 3, 4... reciprocal translocations in which 2 breaks had occurred in I, 2, 3... chromosomes. On this basis, exp e c t e d n u m b e r s of reciprocal t r a n s l o c a t i o n s per " v i a b l e " s p e r m are c a l c u l a t e d (where v i a b i l i t y m e a n s the a b i l i t y to give rise to viable offspring). This is a more precise measure t h a n the p r o p o r t i o n of viable sperm with chromosome aherrations, used b y previous authors. The fact t h a t n u m b e r s of offspring with o, I, 2... t r a n s l o c a t i o n s after a p a t e r n a l dose D showed a good fit to a Poisson d i s t r i b u t i o n p r o v i d e d some justification for the a s s u m p t i o n t h a t the n u m b e r of breaks p r o d u c e d in a nucleus after the same dose would also fit a Poisson. In contrast, we found a m a r k e d l y non-Poissonian d i s t r i b u t i o n of t r a n s l o c a t i o n s in mouse s p e r m a t o c y t e s at d i a k i n e s i s - m e t a p h a s e I after s p e r m a t o g o n i a l i r r a d i a t i o n 3s. The curve showing the relationship between the function of dose, a.qD a n d the m e a n n u m b e r of t r a n s l o c a t i o n s per " v i a b l e " s p e r m a t o z o o n (Fig, I of A p p e n d i x ) shows a good fit to the d o s e - r e s p o n s e relationship over the range of observations, n a m e l y from o-o. 7 t r a n s l o c a t i o n s per viable s p e r m a t o z o o n . The d e r i v e d value of ~q is 2.84. IO -s, a b o u t five times t h a t c a l c u l a t e d b y HALDANE AND LI~A for D r o s o p h i l a s p e r m a t o z o a . As the A p p e n d i x shows, s e p a r a t e values of c~ a n d q can be calculated from the d a t a on litter-sizes at different doses, if it is a s s u m e d t h a t the litter-size changes were e n t i r e l y due to the i n d u c t i o n of d o m i n a n t lethal m u t a t i o n s a n d t h a t there was a I : I relationship between the f r e q u e n c y of d o m i n a n t lethal m u t a t i o n s and of d y s c e n t r i c e x c h a n g e s + b r e a k s which n e i t h e r rejoin nor restitute. U n d o u b t e d l y , n e i t h e r of these a s s u m p t i o n s is s t r i c t l y true. I t is well k n o w n t h a t d o m i n a n t l e t h a l i t y is b e t t e r expressed in t e r m s of p r e - n a t a l d e a t h t h a n of litter-size at birth, especially when pairs of females are k e p t in the s a m e cage, so t h a t some m i x e d litters are observed. I t is not surprising therefore t h a t the fit of the t h e o r e t i c a l curve to t h a t o b s e r v e d was poor. Nevertheless, it is i n t e r e s t i n g to note t h a t the e s t i m a t e of q (0.823 -!: 0.052) is not g r e a t l y different from t h a t c a l c u l a t e d b y HALDANE AND LEA TM for Drosophila, n a m e l y 0.76 on the m a n y - a r m a n d 0.67 on the 5-arm formula, nor from the value of 0.74 c a l c u l a t e d b y CATCHESIDE AND LEA 4 from an analysis of sex-ratio distortion. The e s t i n l a t e d value of a, the m e a n n u m b e r of b r e a k s per mouse nucleus (3.445"IO-a p e r rad) is larger t h a n HALDANE aXI) LEA'S value for D r o s o p h i l a of o.78-IO -a per r a d (5-arm formula) b y a factor of 4.4. Similar e s t i m a t e s of the comp a r a t i v e r a d i o s e n s i t i v i t y of Drosophila a n d mouse were o b t a i n e d b y L#NING 2a and b y L Y o N et al. 27 (factors of 2-4 a n d 4-5, respectively), b u t these were based on the rates of i n d u c t i o n per genome of recessive lethal m u t a t i o n s in s p e r m a t o g o n i a . CARTER ~ o b t a i n e d a value of 23 for recessive lethals i n d u c e d in s p e r m a t o z o a , b u t this was with chronic 7-irradiation. A more meaningful comparison m a y be with the ratio of genome sizes expressed in m a p units. As Lt'TNING24 has p o i n t e d out to us, the best e s t i m a t e of this on present i n f o r m a t i o n is 4.5, v e r y close to our factor of 4.4.
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Contrast with sperrnatogonia The close fit to expectation of the dose-response curves for translocation induction in both Drosophila and mouse, despite the simplifying initial assumptions, suggests that the influence of secondary events between induction of the interchanges and their detection is small. The period over which irradiated males were mated in the present experiment ensured that only products of mature spermatozoa, irradiated in epididymis or vas deferens, contributed to the results. This cell-stage homogeneity, coupled with the very low radiosensitivity of spermatozoa to killing6,1~ seem to be the essential differences between this experiment and earlier ones on the induction of translocations in spermatogonia, in which the spermatocytes examined would have come from A type spermatogonia irradiated as a mixture of cell-stages, heterogeneous in their radiosensitivity to killing and to aberration induction. The presumed induction of dyscentric as well as eucentric interchanges in the present experiment did not lead to a humped dose-response curve, nor was this expected from the theoretical calculations. Therefore it is hardly likely that the humped dose-response curve found after spermatogonial irradiation was due to this phenomenon by itself. It seems much more probable that the cell-stage heterogeneity was the essential factor, since OFTEDAL31 has shown that this leads to a humped response if there is a correlation between radiosensitivity to killing and to mutation induction, as would be expected here. The calculations have only been concerned with reciprocal translocations and cyclic exchanges and have assumed the induction of not more than I break per chromosome arm. However, some reciprocal transloeations can result from the interaction of 3 breaks, 2 in one chromosome and I in another. These cannot be distinguished from the usual 2-break transloeations. Two examples of another type of 3-break aberration, the insertion, were identified cytologically. Others may have escaped detection because of the shortness of the inserted segment. However, these were probably few in number, since there was no evidence for additional embryonic lethality in the progeny of the F1 generation which could be accounted for by the presence of undetected chromosome aberrations giving rise to unbalanced genomes. Therefore it seems likely that the frequency of insertions is genuinely low; the same is probably true for the frequency of 3-break aberrations masquerading as reciprocal translocations. The similarity between Drosophila and mouse spermatozoa in the form of their response to the induction of reciprocal translocations by X-irradiation raises the possibility that in the mouse, as in Drosophila, chromosome breaks induced in spermatozoa may stay open until after fertilization, thus allowing more opportunity for chromosome movement and a closer approach to random interaction. Pre-implantation loss The mean number of implants in females mated to translocation carriers was markedly lower than in those mated to normal males (Table V). This extra preimplantation loss may have reflected extra pre-implantation lethality due to early death of some very unbalanced zygotes, or a lowered rate of egg-fertilization, so that fewer zygotes were formed. CARTERet al. 2 found that most of the translocations they studied showed no sign of extra pre-implantation loss. However T6Ca showed it in progeny of heterozygous males while T8Ca showed it in progeny of both heterozygous males and females. Since a number of T6Ca heterozygous males are infertile or corn-
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p l e t e l y s t e r i l e , a n d s i n c e it is k n o w n t h a t m a n y o t h e r a u t o s o m a l t r a n s l o c a t i o n s c a n h a v e t h e s a m e e f f e c t in t h e m a l e 25 it s e e m s p r o b a b l e t h a t t h e e x t r a p r e - i m p l a n t a t i o n loss in T 6 C a s t e m s f r o m a l o w e r e d f e r t i l i z a t i o n r a t e , b e c a u s e of a low s p e r m - c o u n t . I n T8Ca, h o w e v e r , p r o g e n y of b o t h s e x e s s e e m e d t o b e a f f e c t e d , a l t h o u g h t h e effect w a s less m a r k e d in t h e f e w f e m a l e s e x a m i n e d a n d m i g h t well b e d u e t o c h a n c e f a c t o r s in t h i s sex. T h e r e f o r e it s e e m s l i k e l y t h a t t h e e x t r a p r e - i m p l a n t a t i o n loss in t h e p r e s e n t e x p e r i m e n t w a s m a i n l y d u e t o a t e n d e n c y for d e c r e a s e d s p e r m p r o d u c t i o n in I"l t r a n s l o c a t i o n c a r r i e r s , l e a d i n g t o a l o w e r e d r a t e of f e r t i l i z a t i o n . W e h a v e r e c e n t l y f o u n d a" t h a t a fall in t h e e p i d i d y m a l s p e r m - c o u n t t o a b o u t i o % of n o r m a l in t h e m o u s e l e a d s t o a m a r k e d d r o p in t h e p r o p o r t i o n of eggs f e r t i l i s e d , f r o m t h e n o r m a l level of n e a r l y lOO%. ACKNOWLEDGEMENTS
W e are g r e a t l y i n d e b t e d t o Mr. M. J. CORP for a r r a n g i n g t h e i r r a d i a t i o n s , a n d t o Mr. D. G. PAPWORTH for his s t a t i s t i c a l a n a l y s i s of t h e d a t a a n d for c o n t r i b u t i n g the Appendix. REFERENCF.S I CARTER, T. C., Recessive lethal mutation induced in the mouse by chronic v-irradiation,
Prec. Roy. Soc. B., 147 (19571 4o2-4 II. 2 CARTER, T. C., }f. F. LYON AND R. J. s. PHILLIPS, Gene-tagged chromosome translocations in
eleven stocks of mice, J. Gcnet., 53 (1955) 154-166. 3 CATCHESIDE, D. G., The effect of X-ray dosage upon the frequency of induced structural
changes in the chromosomes of Drosophila melanogaster, J. Genet., 36 (19381 307-320. 4 CATCHESIDE, D. G., AND D. E. LEA, Dominant lethals and chromosome breaks in ring-X
chromosomes of Drosophila melanogaster, J. Genet., 47 (19451 25-4 o. 5 CATTANACH, B. M., C. E. POLLARD AND J. H. ISAACSON, E t h y l m e t h a n e s u l f o n a t e - i n d u c e d
chromosonle breakage in the mouse, Mutation Res., 6 (1968) 297 307 . 6 EDVCARDS, R. G., The experimental induction of gynogenesis in the mouse, I. h'radiation of
the sperm by X-rays, Prec. Roy. Soc. B., 146 (1957) 469 487 • 7 EVANS, E. P., G. BRECKON AND C. E. FORD, An air-drying method for meiotic preparations from manlmalian testes, Cytogenetics, 3 (19641 289-292. b; EVANS, E. P., C. E. FORD, A. G. SEARLE AND B. J. WEST, Studies on the induction of trans locations in mouse spermatogonia, Ilf. Effects of X-irradiation, Mutation Res., 9 (197 o) 5 ol 506. 9 FANO, U., Mechanisms of induction of gross chromosomal rearrangements in Drosophila sperms, Prec. Natl. Aead. Sei., Wash., 29 (19431 12 18. lO FORD, C. E., The use of chromosome markers, Appendix I of H. S. MICKLEMAND J. F. LOUTIT, Tissue Grafting and Radiation, Academic Press, New York, 1966. 1 1 FORD, C. E., A. G. SEARLE, E. P. EVANS AND B. J. WEST, Differential transmission of translocations induced in spermatogonia of mice by irradiation. Cytogenetics, 8 (19691 447 47 °. 12 FREDGA, K., A simple technique for demonstration of the chromosomes and mitotic stages in a mammal. Chromosomes from cornea, Hereditas, 51 (1964) 268-27.3. 13 HALDANE, J. B. S., AND D. E. LEA, A mathematical theory of chromosomal rearrangenlents, .[. Genet., 48 (1947) I IO. 14 MERTWIG, P., Unterschiede in der Entwicklungsf~higkeit von F 1 MXusen nach RGntgenbestrahlung von Spermatogonien, fertigen und unfertigen Spermatozoen, Biol. Zentr., 58 (1938 ) 273-3Ol. 15 I-[ERTWlG, P., Vererbbare Semisterilit/~t bei M/~usen nach RGntgenbestrahlung verursacht durch reziproke Chromosomentranslokationen, Z. Indukt. Abstamm. Vererbungslehre, 79 (194 o) 1-27. 16 HERTWlG, P., AND H. BRENNEKE, Die Ursachen der herabgesetzen "WurfgrGsse bei MAusen nach RGntgenhestrahlung des Spermas, Z. Indukt. Abstamm. Vererbungslehre, 72 (19371 483 487 . 17 LEA, D. E., Actions of Radiations on Living Cells, 2nd ed., Cambridge University Press, Canlbridge, 1955 .
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18 LEA, D. E., AND D. G. CATCHESIDE, The relation between recessive lethals, d o m i n a n t lethals and c h r o m o s o m e a b e r r a t i o n s in Drosophila, J. Genet., 47 (1945) lO-24. 19 LI~ONARD, A., Radiation-induced translocations in s p e r m a t o g o n i a of mice, Mutation Res., I I (1971) 71-88. 20 L]~ONARD, A., AND GH. DEKNUDT, Relation between the X - r a y dose and the rate of chromosome r e a r r a n g e m e n t s in s p e r m a t o g o n i a of mice, Radiation Res., 32 (1967) 35-41. 2i LI~ONARD, A., AND G. I-~. DEKNUDT, The sensivity of various germ-cell stages of the male mouse to radiation induced translocations, Can. J. Genet. CytoI., IO (1968) 495-507. 22 LI~ONARD, A., AND GH. DEKNUDT, Dose response relationship for translocations induced by X-irradiation in spernlatogonia of mice, Radiation Res., 4 ° (1969) 276 284. 23 LI3NING, K. G., Studies of irradiated m o u s e populations, I I I . Accumulation of recessive lethals, Mutation Res., i (1964) 86-98. 24 LONING, K. G., Personal communication. 25 LYON, M. F., AND R. MEREDITH, A u t o s o m a l translocations causing male sterility and viable aneuploidy in the mouse, Cytogenetics, 5 (1966) 335-354. 26 LYon, M. F., AND T. MORRIS, Gene and c h r o m o s o m e m u t a t i o n after large fractionated or unfraetionated radiation doses to mouse spermatogonia, Mutation Res., 8 (1969) 191-198. 27 LYon, M. F., R. J. S. PHILLIPS AND A. G. SEARLE, The overall rates of d o m i n a n t and recessive lethal and visible m u t a t i o n induced b y s p e r m a t o g o n i a l X-irradiation of mice, Genet. Res., 5 (1964) 448-467 . 28 MULLER, H. J., An analysis of the process of s t r u c t u r a l change in c h r o m o s o m e s of Drosophila, J. Genet., 4 ° (I94 o) I 66. 29 MULLER, H. J., The m a n n e r of production of m u t a t i o n s by radiation, in A. HOLLAENDER (Ed.), Radiation Biology, vol. I, McGraw-Hill, New York, 1954, pp. 475-6263 ° MURAMATSU, D., W. NAKAMURAAND H. ITO, Radiation induced translocations in mouse spermatogonia, Jap. J. Genet., 46 (1971) 281-283. 31 OFTEDAL, P., A theoretical s t u d y of m u t a n t yield and cell killing after t r e a t m e n t of heterogeneous cell populations, Hereditas, 60 (1968) 177-21o. 32 SAVKOVIC, N. V., AND M. F. LYON, D o s e - r e s p o n s e curve for X - r a y induced translocations in mouse spermatogonia, Mutation Res., 9 (197 o) 407-409. 33 SEARLE, A. G., C. E. FORD AND C. V. ]~EECHEY, Meiotic disjunction in mouse translocations and the d e t e r m i n a t i o n of centromere position, Genet. Res., 18 (1971) 215-235. 34 SEARLE, A. G., C. V. BEECHEY, M. J. CORP AND D. G. I~:)APWORTH, A dose rate effect on translocation induction b y X-irradiation of mouse spermatogonia, Mutation Res., I5 (1972) 89-91. 35 SEARLE, A. G., E. P. EVANS, C. E. FOR[) AND B. J. WEST, Studies on the induction of translocations in mouse spermatogonia, I. The effect of dose-rate, Mutation Res., 6 (1968) 427-436. 36 SEARLE, A. G., AND C. V . BEECHEY, Sperm-count, egg-fertilization and d o m i n a n t lethality after X-irradiation of mice, Mutation Res., 22 (1974) 63-72.
:~',Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
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T H E I N D U C T I O N O F T R A N S L O C A T I O N S IN MOUSE S P E R M A T O Z O A I. K I N E T I C S O F DOSE R E S P O N S E W I T H A C U T E X - I R R A D I A T I O N
A. G. SEARLE*, C. E. FORD**, E. P. EVANS**, C. V. BEECHEY*, M. D. BURTENSHA\V** AND HILARY M. CLEGG** with Appendix by D. G. PAPVVORTH* * Medical Research Council Radiobiology Unit, Harwell, Didcot, Berhs., and ** Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford (Great Britain)
(Received July 3rd, 1973) (Revision received October i2th, 1973)
SUMMARY A d u l t male mice were given gonadal doses of o-I2OO rad acute X - i r r a d i a t i o n a n d m a t e d the same day. 531 sons, conceived within a week of the t r e a t m e n t , were tested for fertility a n d their testes e x a m i n e d cytologically for chromosome a b e r r a t i o n s in spermatocytes. 55/57 of those diagnosed as semi-sterile a n d 35/4 ° of those diagnosed as sterile were judged to be heterozygous for one or more reciprocal translocations. N u m b e r s of o, i, 2... translocations per mouse showed a good fit to a Poisson distribution, in contrast to previous findings with spermatogonial irradiation. Although the dose response fitted a linear relationship, the power law e q u a t i o n of best fit had a dosee x p o n e n t of 1.41. F u r t h e r analysis along similar lines to those used previously in Drosophila b y CATCHESIDE, LEA AND HALDANE, which assumed r a n d o m rejoining of breaks a n d direct p r o p o r t i o n a l i t y between dosage a n d n u m b e r of breaks, gave a close fit between the actual results a n d those expected if ~q = 2.8. IO 3/rad, where ~ is the m e a n n u m b e r of breaks per nucleus a n d q is the proportion which rejoin or restitute. B y c o m b i n i n g these d a t a with those for litter-size reduction in F1 (taken as a measure of i n d u c e d d o m i n a n t lethality) c~ was e s t i m a t e d to be 3.4 × lO-3 per rad. W h e n compared with the value of 0.8 × lO -3 per rad o b t a i n e d in Drosophila b y HALDANE a n d LEA, this suggested t h a t mouse haploid nuclei are more radiosensitive to chromosome breakage t h a n Drosophila haploid nuclei b y a factor of a b o u t 4. The m e a n n u m b e r of i m p l a n t s per p r e g n a n t female m a t e d to cytologically a b n o r m a l males was a b o u t 15% lower t h a n with n o r m a l males. This p r e - i m p l a n t a t i o n loss was t h o u g h t to be m a i n l y the result of a reduction in the rate of fertilization in this group r a t h e r t h a n to early death of u n b a l a n c e d zygotes. There was no evidence for the i n d u c t i o n of a n y u n d e t e c t e d types of chromosomal a b e r r a t i o n or gene m u t a t i o n which could cause i n t r a u t e r i n e death in the progeny of F1 males.
Dedicated to Professor Dr. Dr. h. c. Paula Hertwig, in recognition of her outstanding pioneer work in this field.
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INTRODUCTION
In recent years, the induction of reciprocal translocations by irradiation of spermatogonia in the mouse has been extensively studied by a number of workers (for a review of this work, see LI~ONARD19),mainly by the cytological examination of descendant spermatocytes at diakinesis-metaphase I in the irradiated males themselves. Results of these studies have posed a number of problems. For instance, doseresponse curves for acute X-irradiation have not shown the expected quadratic component, since translocation frequencies have been proportional to dose up to about 700 R (refs. 8, 20, 22, 30) and have then declined sharply~2,2",32; only a slight dose-rate effect has been found with X-irradiation but a marked one with )J-irradiationS4, 35; the distribution of o, I, 2... translocations per cell has not fitted a Poisson relationship35; the observed frequency of transmission of translocations to progeny of the exposed males has been only about one half of that expected u. These divergences from expectation have been thought to result mainly from (i) differential radiosensitivity of different stages of the A type spermatogonia being sampled, (ii) events during the many germ-cell divisions intervening between irradiation and observation, (iii) the induction of asymmetrical exchanges and other types of lethal aberration in the same cells as the symmetrical exchanges which give rise to reciprocal transloeations, and (iv) the existence of a group of balanced reciprocal translocations which were germcell letbals at various stages of gametogenesisS, ~6. In order to obtain a wider view-point on translocation induction in mammals it seemed desirable to study its kinetics in a non-dividing germ-cell stage which was, moreover, highly radioresistant to killing, i.e. the spermatozoon. No previous cytogenetic attempt has been made to do this in the laboratory mouse. However, HERTWIG (refs. 14, 15) showed that the incidence of partially and completely sterile sons of irradiated male mice, mated in the pre-sterile period, rose from 4.4% at o R to 57.1% at 15oo-16oo R. Most of the sons' infertility presumably stemmed from the presence of translocations. L~ONARD AND DEKNUDT21 found that 5.1% of sons of males given 300 R acute X-irradiation carried reciprocal translocations when spermatozoa inferred to have been irradiated in the epididymis and vas deferens were sampled. The incidence was higher when less mature spermatozoa and spermatids were treated. MATERIALS AND METHODS
Fz (C3H/HeH × I o I / H ) male mice, aged between 8 and 18 weeks, were given gonadal doses of o, 50, IOO, 2o0, 4oo, 600, 800, IOOO, or 12oo rad acute X-irradiation (250 kVp, HVT 1.2 mm Cu, 217 rad/min). On the same day they were mated to pairs of hybrid females of the same stock, from which they were separated one week later. Numbers and sexes of offspring were recorded at birth and weaning. For the purpose of analysis, mixed litters (derived from parturition of both females in a cage on the same day) were regarded as of equal size (when even-numbered) or differing by one (when odd-numbered). At the age of 8 weeks, sons of the irradiated males were mated to pairs of outbred " R " females, from the Fz generation of a specific locus experiment. Vaginal plugs were recorded and pregnant females were killed 14 days later, when numbers of implantations of live and dead embryos were counted. The fertility status was then
I N D U C T I O N OF T R A N S L O C A T I O N IN MOUSE SPERMATOZOA. I .
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determined by the method of CARTERet al. ~ in which pregnant females were diagnosed as fertile (F), semi-sterile (S) or giving an inconclusive result (I) according to numbers of live and dead implantations. The tested male was then regarded as (I) Fertile if 2F, or I F + I I with respect to the two mates; (2) Semi-sterile if 2S, or I S + I I ; (3) Test inconclusive if 2I, or I F + I S . If neither mate appeared pregnant after a period of at least 4 weeks the male was considered sterile. However, confirmation of this findings was frequently sought by replacing the females with a further pair. This was also done when one of the two original females failed to become pregnant in 4 weeks. Males were killed as soon as possible after their fertility status had been established. Air-dried preparations were made from the testes by the method of EVANS et al. 7, and primary spermatocytes at diakinesis-metaphase I were examined and the nature of the chromosome associations recorded. As a routine, 25 spermatocytes were examined from each male diagnosed as fertile, while ioo were examined from those diagnosed as sterile, semi-sterile or with an inconclusive result. However, the number required from the "test inconclusive" group was reduced to 50 later, since this seemed sufficient for the purpose. Whenever there was cytological evidence for presence of a reciprocal translocation at least IOO spermatocytes were scored, while even more were studied when the frequency of abnormal configurations was low. Scoring was mainly shared between M.D.B., H.C., E.P.E. and C.E.F., but C.V.B. scored 15 of the sterile males. Additional information obtained from sterile males included sperm-counts and testis weights. In addition, mitotic chromosome preparations were obtained from sterile males, either from the cornea by a modification of FREDGA'Smethod 1~ or from the bone-marrow by the air-drying method of FORD1°. Daughters of males given doses of 600 rad or more were also tested for the presence of reciprocal translocations. Results of these studies will be given in a separate paper in this series. The normal criterion for diagnosing translocation heterozygosity in F1 males was the presence of characteristic multivalent configurations in spermatocytes at diakinesis-metaphase I. Those giving hexavalent configurations were regarded as having two reciprocal translocations involving three pairs of chromosomes, rather than as the result of cyclic exchanges. Ten sterile males in which no metaphase spermatocytes or later spermatogenic stages could be found were considered heterozygous for one reciprocal translocation. This was because LYoN AND ~]IEREDITH~ have demonstrated that heterozygosity for particular autosomal reciprocal translocations can cause failure of spermatogenesis, with very few cells reaching meiotic metaphase I in some cases. CATTANACHe~ al. 5 failed to find any metaphase spermatocytes in one transloeation of this type. Confirmatory evidence was obtained from our own sterile males lacking spermatocytes in metaphase I by the observation of one or two mitotic marker chromosomes, suggestive of reciprocal translocation, in somatic tissue of five of the ten mice concerned. RESULTS
R e c i p r o c a l translocations
Table I shows that there was close agreement between diagnoses of fertility or
16o
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SEARLE
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al.
TABLE I RELATIONSHIP BET'WEEN DIAGNOSIS OF FERTILITY STATUS AND NUMBERS L O C A T I O N S C A R R I E D B Y S O N S O F I R R A D I A T E D A N D C O N T R O L IvIALES
Fertility diagnosis
Translocations per mouse 0 I 2
Fertile Test inconclusive Semi-sterile Sterile
.385 37 2a 5
.3 S 50 25
Total
429
8()
OF RECIPROCAL
3
Total of mic~ examined
o
.388
o r 5 7
o
46
o 3
57 4°
I3
3
53 I
TRANS-
a I with insertion. s e m i - s t e r i l i t y a n d a b s e n c e or p r e s e n c e of r e c i p r o c a l t r a n s l o c a t i o n s , r e s p e c t i v e l y . All t h r e e of t h o s e d i a g n o s e d as fertile y e t f o u n d to c a r r y t r a n s l o c a t i o n s g a v e s o m e d e a d i m p l a n t a t i o n s (2/12, 4/15 a n d I/IO). Of tile t w o s e m i - s t e r i l e m a l e s w h i c h failed to g i v e c y t o l o g i c a l e v i d e n c e for r e c i p r o c a l t r a n s l o e a t i o n s , one p r o v e d to c a r r y an i n s e r t i o n (which will be d e s c r i b e d elsewhere). T h e other, w h i c h was in tile c o n t r o l series, g a v e 7 d e a d a m o n g 14 i n p l a n t a t i o n s . F o u r of t h e s e (all f r o m one female) h a d d i e d as foetuses at a b o u t 13 d a y s p o s t - c o i t u s ; t h e y l o o k e d f l a t t e n e d a n d d i s t o r t e d b u t h a d no specific a b n o r m a l i t y . T h e o t h e r t h r e e h a d d i e d earlier a n d w e r e r e c o g n i s e d as d e c i d u o m a t a ("small moles"). A b o u t 2 o % of t h o s e m a l e s in w h i c h t h e f e r t i l i t y t e s t was i n c o n c l u s i v e p r o v e d to c a r r y one or m o r e r e c i p r o c a l t r a n s l o c a t i o n s , as did n e a r l y 9 o % of sterile males. A full a c c o u n t of t h e s e will be g i v e n in a s e p a r a t e p a p e r . I n all, 113 s i m p l e r e c i p r o c a l t r a n s l o c a t i o n s a n d 4 a n i m a l s w i t h h e x a v a l e n t s w e r e d i a g n o s e d , as well as tile i n s e r t i o n a n d an X Y Y a n i m a l , in 531 sons tested. F r e q u e n c i e s of serui-sterile a n d sterile sons rose s t e a d i l y w i t h i n c r e a s i n g p a t e r n a l dose (Table II) a n d s h o w e d g o o d a g r e e m e n t w i t h HERTWIG'S earlier results~4, ~a. In c o n t r a s t to findings a f t e r s p e r m a t o g o n i a l i r r a d i a t i o n , t h e t r a n s l o c a t i o n f r e q u e n c y a f t e r s p e r m a t o z o a l i r r a d i a t i o n also rose s t e a d i l y w i t h dose (except at I o o rad) a n d s h o w e d no decline at h i g h e r levels (Table I I I ) . D o s e - r e s p o n s e c u r v e s w e r e f i t t e d b y t h e m e t h o d of m a x i m u m likelihood, a s s u n f i n g a Poisson d i s t r i b u t i o n of o, i , 2, 3 .... t r a n s l o c a t i o n s p e r m o u s e . T h e o b s e r v e d d i s t r i b u t i o n did, in fact, s h o w a g o o d fit to a Poisson (P -o . 9 I ). T h e s t r a i g h t line of best fit to t h e d a t a (Fig. I) h a d t h e f o r n m l a Y : 4.78 -! 0.43 TABLE II FERTILITY
Dose (rad)
o 5° I00
DIAGNOSES
AT DIFFERENT
Fertile
57 53 54
DOSES IN SONS OF IRRADIATED
N u m b e r s diagnosed as [nconSemiclusive sterih
7 2 2
Sterile
I i o
o l 1
MALES
Percentage Semi-sterile or sterile
1.5 3.5 1.8
200
42
4
2
0
4.2
4 °0
47
6
4
5
I4.5
600 800 1ooo I200 Total
49 4° 28 I8 388
5 7 7 6 46
1o t3 ~4 ~2 57
5 7 to II 4°
2 i. 7 29.q 40.7 48.9
I N D U C T I O N OF T R A N S L O C A T I O N IN M O U S E S P E R M A T O Z O A . TABLE
I.
16I
lli
DISTRIBUTION AND FREQUENCY OF RECIPROCAL TRANSLOCATIONS IN SONS OF MALE MICE GIVEN ACUTE X-IRRADIATION (SPERMATOZOAL SAMPLING)
Dose (rad)
Number of mice
Translocations per mouse o • 2 3
o 50 IOO 200 400 600 800 IOOO 12oo Total
65 57 57 t8 62 69 67 59 47 531
65 56 57 46 53 52 44 33 a 23 429
o I o , 8 i4 21 22 i8 86
o o o o I 3 2 2 5 13
o o o o o o o 2 I 3
Total Frequency translocations per mouse ( % 0
i o 2 IO 20 25 32 31
0,0
1.8 o.o 4.2 16.1 28. 5 37-3 54.2 66.0
a i with insertion
PERCENT TRANSLOCATIONS70
I
6050 40 30 20 10
--t"'"""" t'"'"'" t'"'"'-" }""
--"'"'"'"
200
400
600
800
1000 1200
OOSE (.Ao) l q g . i. F r e q u e n c i e s ( w i t h s t a n d a r d e r r o r s ) of r e c i p r o c a l t r a n s l o c a t i o n s in s o n s of m a l e m i c e g i v e n v a r i o u s d o s e s of a c u t e X - i r r a d i a t i o n t o s p e r m a t o z o a . T h e i n t e r r u p t e d line is t h e s t r a i g h t line of b e s t fit.
• xo 4 D, where Y is the translocation yield a n d D the dose. Although the fit was good (P = o.57) it was n o t e d t h a t deviations of observed values from expectations were all negative at low doses (5o-4oo rad) b u t positive at higher doses of xooo a n d I2oo rad. This suggested t h a t the true relationship was curvilinear, as would be expected on theoretical grounds. The e q u a t i o n of best fit to a quadratic was Y - - 2.25 _+_ I.O3 • IO 4 D + 3 . o 9 ~_ 1.26.IO -7 D 2 (P = 0.84), while t h a t of a power law was Y = 3.o3 ! 3.98"Io 5 D1.41 (p = o.87). The s t a n d a r d error of the dose e x p o n e n t is o.2o; thus it differs significantly from a value of I (u = 2.i2, P = o.o34).
Lethality in F1 and F2 generations I n this experiment, the a m o u n t of d o m i n a n t lethality resulting from the spermatozoal irradiation can be estimated only from the rate of decrease of m e a n litter-size with increasing dose, shown in Table IV. There is a roughly e x p o n e n t i a l relationship between m e a n litter-size a n d dose (see Fig. 3 of Appendix), as would be expected if the d i s t r i b u t i o n of o, I, 2... d o m i n a n t lethal m u t a t i o n s is Poisson a n d if the rate of induction of p r e n a t a l d o m i n a n t lethals is directly p r o p o r t i o n a l to r a d i a t i o n dose. The equation 3'~ -- yoe-~o was fitted to the litter-size observations at different doses (y D)by the m e t h o d of least-squares, each observation being weighted inversely as its variance,
162
A. G. SEARLE el al.
T A B L E IV LITTER-SIZES
AT B I R T H
Dose (tad) o
5o IOO 200 4oo 6oo 80o IOOO 1200
AND WEANING
IN OFFSPRING
OF IRRADIATED
MALES
Total born
No. of litters
Mean litter-size
Total weaned
Per cent weaned
128 149 117 lO4 127 16.5 14° 124 9.5
13 19 15 14 21 39 34 48 48
9.85 7.84 7.80 7.43 6.05 4,23 4 .12 2.52 1.98
126 139 116 lO3 123 156 134 119 76
98. 4 93.3 99.2 99.0 96.9 94.5 95.7 96.o 80.0
± ~ ± ~ i i -c ± ~
0.34 o.49 o.46 0.45 0.30 0.25 0.30 o.14 o.15
which was calculated from the observed distribution of litter-sizes. The derived value ofyo was 9.44 ~ o.35 and of k was 1.28 _~ o.o7-1o -3. Therefore the estimated rate of induction of dominant lethals is 1.28.Io-a/gamete/rad. This is about three times the rate per rad for translocations, assuming a linear relationship. It should be realised, however, that this estimate of dominant lethality can only be approximate, based as it is on newborn litters, some of them mixed. Survival of F1 offspring to weaning age remained high at all dose levels (Table IV) although lowest at 12oo rad. This drop was probably a consequence of the very low litter-size at this level, since it is known that under these circumstances the stimulus for lactation may be insufficient. Thus, out of 19 litters of one in the 12oo rad series, 9 died before weaning age. All F1 males were mated to two or more females and the amount of embryonic lethality at or near the I4th day of gestation was determined. Overall results for the different cytological categories are summarized in Table V. The mean numbers of live embryos per female in the cytologically abnormal categories are less than half that in the normal category. The mean number of implants is also markedly reduced by an amount which is clearly significant. This suggests some pre-implantation loss which may partly result from reduced fertilization of eggs, as discussed later. The embryonic survival (in terms of live embryos per female) in those with I reciprocal translocation was 42.4% of the survival in those without detectable abnormality. Since a value of 5o% is expected with normal disjunction, the observed figure suggests an average level of adjacent-2 disjunction of around 15% (ref. 33). However, this TABLE
V
EMBRYONIC
LETHALITY
IN PROGENY
OF MALES V¢ITH OR WITHOUT
DETECTABLE
TYPES
OF CHROMO-
SOME ABERRATION
(RT, r e c i p r o c a l t r a n s l o c a t i o n ) Category of male
Number of pregnant 9Q
Total implants
Live embryos
Dead implants
Per cent dead
Implants per 9
Live embryos per 9
With i RT With 2 RT With insertion Normal Total
12o 8 2 849 979
8o6 58 14 6743 7621
369 18 7 6161 6555
437 4° 7 582 lO66
54 .2 69.0 5 °.0 8.63 13.99
6.72 ~ o . 2 o 7.25 7 .00 7.94 7.78
3.o8 _~o.I 5 2.25 3.50 7 .26
I N D U C T I O N OF T R A N S L O C A T I O N I N M O U S E S P E R M A T O Z O A . TABLE
I.
163
VI
EMBRYONIC LETHALITY IN THE PROGENY OF CYTOLOGICALLY NORMAL F 1 MALES AFTER VARIOUS DOSES OF PATERNAL X-IRRADIATION 1replants
Dead Total % dead
Dose ( r a d s ) o
5°
IOO
200
400
600
8oo
3000
12oo
81 994 8.2
73 886 8. 3
71 897 7.9
62 735 8.4
87 876 9.9
77 845 9.I
59 7°8 8.3
35 467 7.5
37 335 ii.o
Z ~ = 6.53, P = o.59.
must be regarded as an upper limit, for the comparative embryonic survival in terms of live embryos/total implants was 5o.1 ± 1.9°/o. Table VI shows that within the normal category (free of detectable aberrations) there is no significant heterogeneity in the proportion of dead implants with respect to X-ray dose. If some of this embryonic lethality was the result of the induction of undetected types of chromosomal aberration, or of sub-lethal gene mutation, then its frequency would have tended to increase with increasing dose; instead it remains remarkably constant. DISCUSSION
Comparison with Drosophila We have shown that the frequency of reciprocal translocations in sons of male mice given spermatozoal X-irradiation over a range of o to 12oo rad is roughly proportional to dose, although the curve of best fit has a dose exponent of 1. 4. This agrees fairly well with results of similar treatment on Drosophila spermatozoa, reviewed by MULLER 29, in which a dose exponent of around 1.5 was usually obtained, although this sometimes approached 2 at lower doses. CATCHESIDEa was the first to try and calculate expected frequencies of induced structural changes if these resulted from the fusion of broken ends of chromosomes and if there was direct proportionality between dosage and number of breaks. He also assumed (i) that all breakage ends rejoin, (ii) that they rejoin at random and (iii) that breaks occur one in each chromosome, so that only interchanges are involved. He found a reasonable fit between his observations and expectation over the limited range of doses used (100o-40oo R). MULLER28 also found a good fit to expectation over a wider range of doses. Further contributions to the mathematical analysis of structural changes came from FANO9 and LEA AND CATCHESIDE is, who considered inter alia the contribution of dyscentric interchanges and sister-strand unions to dominant lethals, and from HALDANE AND LEA is, who dealt with the occurrence of more than one break per chromosome arm and with expectations for different numbers of arms. The subject is also reviewed by LEA in his book 17. LEA AND CATCHESIDEis found that the theoretical curve (assuming many arms) best fitted experimental data on the proportion of viable sperm with chromosome aberrations in Drosophila after various doses if aq = 0.57 per IOOO R, where ~ is a constant, relating the mean number of breaks to the dose, and q is the probability that a given break shall either restitute or take part in interchange. HALDANE AND LEAla found that an scq value of 0.52 gave the best fit with their improved 5-arm formula. By considering also the results for induction of dominant lethals in Drosophila sperm,
16 4
A.G. SEARLE et al.
the a u t h o r s a r r i v e d at values for ~. of o.75 ( m a n y arms) or o.78 (5 arms) per IOOO R a n d for q of o.76 ( m a n y arms) or o.67 (5 arnls). The same calculations can be m a d e for the mouse, since d a t a are now available for the i n d u c t i o n of b o t h d o m i n a n t lethals a n d translocations in mouse s p e r m a t o z o a . The calculations are given in the A p p e n d i x b y D. G. PAPWORTH, in which a d j u s t m e n t s to HALDANE AND LEA'S t r e a t m e n t are m a d e to allow for the fact t h a t in the mouse the identification of reciprocal t r a n s l o c a t i o n s was n o r m a l l y based on the a p p e a r a n c e of n m l t i v a l e n t configurations at meiosis. This m e a n t t h a t cyclic exchanges involving 3, 4, 5... chromosomes would n o r m a l l y be classified as 2, 3, 4... reciprocal translocations in which 2 breaks had occurred in I, 2, 3... chromosomes. On this basis, exp e c t e d n u m b e r s of reciprocal t r a n s l o c a t i o n s per " v i a b l e " s p e r m are c a l c u l a t e d (where v i a b i l i t y m e a n s the a b i l i t y to give rise to viable offspring). This is a more precise measure t h a n the p r o p o r t i o n of viable sperm with chromosome aherrations, used b y previous authors. The fact t h a t n u m b e r s of offspring with o, I, 2... t r a n s l o c a t i o n s after a p a t e r n a l dose D showed a good fit to a Poisson d i s t r i b u t i o n p r o v i d e d some justification for the a s s u m p t i o n t h a t the n u m b e r of breaks p r o d u c e d in a nucleus after the same dose would also fit a Poisson. In contrast, we found a m a r k e d l y non-Poissonian d i s t r i b u t i o n of t r a n s l o c a t i o n s in mouse s p e r m a t o c y t e s at d i a k i n e s i s - m e t a p h a s e I after s p e r m a t o g o n i a l i r r a d i a t i o n 3s. The curve showing the relationship between the function of dose, a.qD a n d the m e a n n u m b e r of t r a n s l o c a t i o n s per " v i a b l e " s p e r m a t o z o o n (Fig, I of A p p e n d i x ) shows a good fit to the d o s e - r e s p o n s e relationship over the range of observations, n a m e l y from o-o. 7 t r a n s l o c a t i o n s per viable s p e r m a t o z o o n . The d e r i v e d value of ~q is 2.84. IO -s, a b o u t five times t h a t c a l c u l a t e d b y HALDANE AND LI~A for D r o s o p h i l a s p e r m a t o z o a . As the A p p e n d i x shows, s e p a r a t e values of c~ a n d q can be calculated from the d a t a on litter-sizes at different doses, if it is a s s u m e d t h a t the litter-size changes were e n t i r e l y due to the i n d u c t i o n of d o m i n a n t lethal m u t a t i o n s a n d t h a t there was a I : I relationship between the f r e q u e n c y of d o m i n a n t lethal m u t a t i o n s and of d y s c e n t r i c e x c h a n g e s + b r e a k s which n e i t h e r rejoin nor restitute. U n d o u b t e d l y , n e i t h e r of these a s s u m p t i o n s is s t r i c t l y true. I t is well k n o w n t h a t d o m i n a n t l e t h a l i t y is b e t t e r expressed in t e r m s of p r e - n a t a l d e a t h t h a n of litter-size at birth, especially when pairs of females are k e p t in the s a m e cage, so t h a t some m i x e d litters are observed. I t is not surprising therefore t h a t the fit of the t h e o r e t i c a l curve to t h a t o b s e r v e d was poor. Nevertheless, it is i n t e r e s t i n g to note t h a t the e s t i m a t e of q (0.823 -!: 0.052) is not g r e a t l y different from t h a t c a l c u l a t e d b y HALDANE AND LEA TM for Drosophila, n a m e l y 0.76 on the m a n y - a r m a n d 0.67 on the 5-arm formula, nor from the value of 0.74 c a l c u l a t e d b y CATCHESIDE AND LEA 4 from an analysis of sex-ratio distortion. The e s t i n l a t e d value of a, the m e a n n u m b e r of b r e a k s per mouse nucleus (3.445"IO-a p e r rad) is larger t h a n HALDANE aXI) LEA'S value for D r o s o p h i l a of o.78-IO -a per r a d (5-arm formula) b y a factor of 4.4. Similar e s t i m a t e s of the comp a r a t i v e r a d i o s e n s i t i v i t y of Drosophila a n d mouse were o b t a i n e d b y L#NING 2a and b y L Y o N et al. 27 (factors of 2-4 a n d 4-5, respectively), b u t these were based on the rates of i n d u c t i o n per genome of recessive lethal m u t a t i o n s in s p e r m a t o g o n i a . CARTER ~ o b t a i n e d a value of 23 for recessive lethals i n d u c e d in s p e r m a t o z o a , b u t this was with chronic 7-irradiation. A more meaningful comparison m a y be with the ratio of genome sizes expressed in m a p units. As Lt'TNING24 has p o i n t e d out to us, the best e s t i m a t e of this on present i n f o r m a t i o n is 4.5, v e r y close to our factor of 4.4.
I N D U C T I O N O F T R A N S L O C A T I O N IN M O U S E S P E R M A T O Z O A . I.
165
Contrast with sperrnatogonia The close fit to expectation of the dose-response curves for translocation induction in both Drosophila and mouse, despite the simplifying initial assumptions, suggests that the influence of secondary events between induction of the interchanges and their detection is small. The period over which irradiated males were mated in the present experiment ensured that only products of mature spermatozoa, irradiated in epididymis or vas deferens, contributed to the results. This cell-stage homogeneity, coupled with the very low radiosensitivity of spermatozoa to killing6,1~ seem to be the essential differences between this experiment and earlier ones on the induction of translocations in spermatogonia, in which the spermatocytes examined would have come from A type spermatogonia irradiated as a mixture of cell-stages, heterogeneous in their radiosensitivity to killing and to aberration induction. The presumed induction of dyscentric as well as eucentric interchanges in the present experiment did not lead to a humped dose-response curve, nor was this expected from the theoretical calculations. Therefore it is hardly likely that the humped dose-response curve found after spermatogonial irradiation was due to this phenomenon by itself. It seems much more probable that the cell-stage heterogeneity was the essential factor, since OFTEDAL31 has shown that this leads to a humped response if there is a correlation between radiosensitivity to killing and to mutation induction, as would be expected here. The calculations have only been concerned with reciprocal translocations and cyclic exchanges and have assumed the induction of not more than I break per chromosome arm. However, some reciprocal transloeations can result from the interaction of 3 breaks, 2 in one chromosome and I in another. These cannot be distinguished from the usual 2-break transloeations. Two examples of another type of 3-break aberration, the insertion, were identified cytologically. Others may have escaped detection because of the shortness of the inserted segment. However, these were probably few in number, since there was no evidence for additional embryonic lethality in the progeny of the F1 generation which could be accounted for by the presence of undetected chromosome aberrations giving rise to unbalanced genomes. Therefore it seems likely that the frequency of insertions is genuinely low; the same is probably true for the frequency of 3-break aberrations masquerading as reciprocal translocations. The similarity between Drosophila and mouse spermatozoa in the form of their response to the induction of reciprocal translocations by X-irradiation raises the possibility that in the mouse, as in Drosophila, chromosome breaks induced in spermatozoa may stay open until after fertilization, thus allowing more opportunity for chromosome movement and a closer approach to random interaction. Pre-implantation loss The mean number of implants in females mated to translocation carriers was markedly lower than in those mated to normal males (Table V). This extra preimplantation loss may have reflected extra pre-implantation lethality due to early death of some very unbalanced zygotes, or a lowered rate of egg-fertilization, so that fewer zygotes were formed. CARTERet al. 2 found that most of the translocations they studied showed no sign of extra pre-implantation loss. However T6Ca showed it in progeny of heterozygous males while T8Ca showed it in progeny of both heterozygous males and females. Since a number of T6Ca heterozygous males are infertile or corn-
166
A.O. SEARLE et al.
p l e t e l y s t e r i l e , a n d s i n c e it is k n o w n t h a t m a n y o t h e r a u t o s o m a l t r a n s l o c a t i o n s c a n h a v e t h e s a m e e f f e c t in t h e m a l e 25 it s e e m s p r o b a b l e t h a t t h e e x t r a p r e - i m p l a n t a t i o n loss in T 6 C a s t e m s f r o m a l o w e r e d f e r t i l i z a t i o n r a t e , b e c a u s e of a low s p e r m - c o u n t . I n T8Ca, h o w e v e r , p r o g e n y of b o t h s e x e s s e e m e d t o b e a f f e c t e d , a l t h o u g h t h e effect w a s less m a r k e d in t h e f e w f e m a l e s e x a m i n e d a n d m i g h t well b e d u e t o c h a n c e f a c t o r s in t h i s sex. T h e r e f o r e it s e e m s l i k e l y t h a t t h e e x t r a p r e - i m p l a n t a t i o n loss in t h e p r e s e n t e x p e r i m e n t w a s m a i n l y d u e t o a t e n d e n c y for d e c r e a s e d s p e r m p r o d u c t i o n in I"l t r a n s l o c a t i o n c a r r i e r s , l e a d i n g t o a l o w e r e d r a t e of f e r t i l i z a t i o n . W e h a v e r e c e n t l y f o u n d a" t h a t a fall in t h e e p i d i d y m a l s p e r m - c o u n t t o a b o u t i o % of n o r m a l in t h e m o u s e l e a d s t o a m a r k e d d r o p in t h e p r o p o r t i o n of eggs f e r t i l i s e d , f r o m t h e n o r m a l level of n e a r l y lOO%. ACKNOWLEDGEMENTS
W e are g r e a t l y i n d e b t e d t o Mr. M. J. CORP for a r r a n g i n g t h e i r r a d i a t i o n s , a n d t o Mr. D. G. PAPWORTH for his s t a t i s t i c a l a n a l y s i s of t h e d a t a a n d for c o n t r i b u t i n g the Appendix. REFERENCF.S I CARTER, T. C., Recessive lethal mutation induced in the mouse by chronic v-irradiation,
Prec. Roy. Soc. B., 147 (19571 4o2-4 II. 2 CARTER, T. C., }f. F. LYON AND R. J. s. PHILLIPS, Gene-tagged chromosome translocations in
eleven stocks of mice, J. Gcnet., 53 (1955) 154-166. 3 CATCHESIDE, D. G., The effect of X-ray dosage upon the frequency of induced structural
changes in the chromosomes of Drosophila melanogaster, J. Genet., 36 (19381 307-320. 4 CATCHESIDE, D. G., AND D. E. LEA, Dominant lethals and chromosome breaks in ring-X
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