Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies

Mutation Research, 149 (1985) 221-230

221

Elsevier MTR 04006

Discrimination between the effects of X-ray irradiation of the mouse oocyte and uterus on the induction of dominant lethals and congenital anomalies I. Embryo-transfer experiments John D. West *, K. Margaret Kirk, Yvonne Goyder and Mary F. Lyon M R C Radiobiology Unit, Harwell, Didcot, Oxon, 0 X l l ORD (Great Britain)

(Received 18 May 1984) (Revision received24 October 1984) (Accepted 31 October 1984)

Summary In order to test whether irradiation of the postimplantation maternal environment had any effect on the apparent induction of dominant lethals or congenital anomalies by radiation, preimplantation embryos were surgically transferred between females which had been irradiated before conception or left untreated. A high proportion of preimplantation embryos, collected from females that had been irradiated 15-21 days prior to conception with 3.6 G y X-rays, were either arrested or developmentally retarded compared with those collected from untreated females. The transfer experiments indicate that irradiation of the uterus has no significant effect on the frequency of subsequent postimplantation mortality or on mean fetal weight. However, it remains unclear whether irradiation of the uterus contributes to the induction of congenital anomalies.

The induction of embryonic loss or abnormalities in offspring of laboratory mammals provides a number of simple and useful tests for mutagenicity. The most widely used is the dominant lethal test (e.g. Bateman and Epstein, 1971) which provides estimates of both preimplantation embryonic loss and postimplantation ~eath. More detailed information on preimplantation loss and abnormalities has been obtained by examining histological sections of embryos within the oviducts (Russell and Russell, 1954; Generoso, * Present address: Department of Obstetrics and Gynaecology, Centre for ReproductiveBiology,37 Chalmers Street, Edinburgh, EH3 9EW (Scotland)

1968) and by culturing embryos in vitro (Goldstein and Spindle, 1976; Burki and Sheridan, 1978; Goldstein et al., 1978; Pedersen and Goldstein, 1979). Additional information on postimplantation abnormalities can be provided by screening for congenital anomalies such as malformed, dwarf and retarded fetuses. Knudsen et al. (1977) suggested combining such a screen with the estabfished dominant lethal test and work by Nomura (1979, 1982) and Kirk and Lyon (1982, 1984) has confirmed the feasibility of such an approach. Difficulties arise when the embryos under study are produced by mutagen-treated females because the mutagenic agent may adversely affect the

0027-5107/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)

222

maternal reproductive physiology. Thus embryonic losses and abnormalities may arise both as a direct consequence of mutations induced in oocytes and also as a result of damage to the maternal environment. Damage to the uterine epithelium, for example, may impair its ability to sustain normal fetal development. We now described the results of embryo-transfer experiments designed to discriminate between the effects of X-ray irradiation of the oocyte and the uterus (plus most of the rest of the postimplantation maternal environment) on subsequent embryonic development. Preimplantation embryos were surgically transferred between the uteri of irradi~tted and untreated female mice so that the maternal environment was altered just before the embryo implanted into the uterus. We then examined the effect of this procedure on the incidence of embryonic mortality and congenital anomalies, and on fetal weight. These experiments also yielded information on the effect of X-irradiation of oocytes on subsequent preimplantation development. Materials and methods

Irradiation Virgin female ( C 3 H / H e H ~ × 1 0 1 / H 3 )F 1 mice (henceforth abbreviated to 3H1) were exposed to acute part-body X-ray treatment when approximately 9 weeks old. An absorbed dose of 3.6 Gy (360 rad), at a rate of 0.72 G y / m i n (250 kV, half-value thickness 1.1 mm Cu) was given to the abdomen, including the ovaries, with the head and thorax shielded. 3H1 males and 3H~ females for other groups were left untreated. The mice were mated 15-21 days after exposure to X-rays.

Embryo collection and transfers Preimplantation embryos at 3.5 days post coitum (p.c.) (time of finding vaginal plug is day 0.5) were flushed from the excised uteri using the Hepes-buffered medium M2 described by Quinn et al. (1982). All embryos from a single 3H1 female were classified according to developmental stage and transferred to the uterus of a pseudopregnant 3H1 female that had been mated to a genetically sterile male, heterozygous for the male-sterile translocations T145H or Is40H. Recipients were

normally 2.5 days pseudopregnant but some were 3.5 days pseudopregnant at the time of transfer. The transfer technique was similar to that described by McLaren and Michie (1956). The recipient was anaesthetised with Avertin and the upper reproductive tract pulled through incisions in the dorsal body wall and skin. The top of the uterus was viewed with a dissecting microscope and held by forceps while it was punctured with a mounted needle, coated with a little aqueous dye (Giemsa) at its tip. Embryos were then tranferred, in M2 medium, to the uterus using a mouth controlled, finely drawn-out pasteur pipette and the skin closed with one or two metal wound clips. The dye was used to help relocate the hole (as recommended by Dr. W.K. A1-Murrani and Dr. A. McLaren). Several air bubbles were drawn into the pipette before picking up the embryos in order to improve the pipette control (as recommended by P.H. Glenister). The number of embryos transferred to each horn was variable. Initially all the embryos from a single donor female were transferred to the right uterine horn. In later experiments clutches of 8 or more were divided so that 6 were transferred to the right and the remainder to the left. Sham transfers were used in one control experiment. These were performed in exactly the same way, by transferring M2 medium into the right uterine horn. All instruments were flame-sterilised.

Analysis of uterine contents Most of the recipients were killed 15 days after the transfer date so that the fetuses were 18.5 days p.c. but a few were examined at 17.5 days p.c. No allowance was made for delay caused by asynchronous transfers into recipients at 2.5 days p.c rather than 3.5 days p.c. Although it is normal to time pregnancy from the date of the recipient plug rather than the donor plug we found that the fetuses were not retarded by a full day and the recipients frequently gave birth before the examination date if this was delayed by a further day. Uterine contents were examined and classified as described by Kirk and Lyon (1982) except that only dwarf or visibly abnormal fetuses and control littermates were fixed in Bouin's fluid for later dissection. A fetus was considered to be dwarf if it

223 weighed less t h a n 75% of the m e a n of the others in the litter. The index of i n d u c e d p o s t i m p l a n t a t i o n dominant lethality was c a l c u l a t e d using the formula: % Induced p o s t i m p l a n t a t i o n d o m i n a n t lethality = [

( Livefetuses, treated / L i v e fetuses, control )] 1- Total implants, treated / Total implants, control x 100

This index differs f r o m that used b y K i r k a n d Lyon (1982) in t h a t a b n o r m a l fetuses that were alive at the time of dissection were i n c l u d e d a m o n g the live fetuses even t h o u g h s o m e w o u l d subsequently have died. C o r p o r a lutea were c o u n t e d for the untransferred c o n t r o l g r o u p s A a n d B ( T a b l e 1) and used to e s t i m a t e p r e i m p l a n t a t i o n loss using the formula given in T a b l e 2. Statistical tests were performed using a H e w l e t t P a c k a r d 97 p r o g r a m mable calculator, p r o g r a m m e d b y Mr. D . G . Papworth to c o m p u t e X 2, F i s h e r ' s exact test, binomial s t a n d a r d errors a n d analysis of variance. Results

The various e x p e r i m e n t a l a n d c o n t r o l g r o u p s of females were as shown in T a b l e 1. T h e i r r a d i a t i o n positive c o n t r o l g r o u p A a n d the u n t r e a t e d negative control g r o u p B were r e p e a t s of two of the groups considered b y K i r k a n d L y o n (1982). T h e incidence of p o s t i m p l a n t a t i o n d o m i n a n t l e t h a l i t y in group A ( T a b l e 2) was 37.8 + 4.0% w h i c h is

TABLE 2 INDUCTION OF CONGENITAL ANOMALIES AND DOMINANT LETHALS BY X-RAYS PRIOR TO CONCEPTION Group

A Irradiated

B Untreated

Number of 2 ¢ Corpora lutea

36 551

37 452

242 109 5 0

349 27 10 15

Implantations Total Small moles Large moles Dead fetuses Abnormal fetuses (dwarf and other) Normal fetuses

7 (2 + 5) a 2 (2 + 0) 121 295

Corpora lutea/females Implants/females

15.3 6.7

12.2 9.4

% Induced preimplantation loss b % Postimplantation mortality

43.1 + 3.1 47.1 + 3.2

14.9 + 1.9

% Induced postimplantation dominant lethality % Abnormal fetuses c

37.8 + 4.0 5.5 + 2.0

0.7 + 0.5

a Abnormal fetuses comprise 2 dwarfs, 1 exencephalic, 1 umbilical hernia, 1 bilateral open eyelids, 1 dwarf with excencephaly and left eyelids open and 1 dwarf with exencephaly and cleft palate. b % Induced preimplantation loss = 1- (

Implants, treated Z Implants, control Corpora lutea, treated / C ~ r o l

)]

x100 c % Abnormal fetuses =

TABLE 1 EXPERIMENTAL AND CONTROL GROUPS OF FEMALESUSED Group

Embryo donor

Embryo recipient

Untransferredcontrols a

A 13

Irradiated Untreated

-

Experimental

C D E

Irradiated Untreated Untreated

Untreated Irradiated Untreated

* In addition to the untransferred control groups A and B shown here, 31 untreated pseudopregnant females were examined to test for artefactual induction of small moles. Sham-embryo transfers were performed on 16 of these females.

Abnormal fetuses Xl(~ (Abnormal + normal fetuses)

c o n s i s t e n t with 29.0 + 4.5%, c a l c u l a t e d from the results of K i r k a n d L y o n (1982) using the s a m e formula. T h e f r e q u e n c y of a b n o r m a l fetuses in this g r o u p (5.5 +_ 2.0%) was also r e a s o n a b l y close to the p r e v i o u s finding of 8.4 _+ 5.5%.

Preimplantation development O n e effect of 3.6 G y X - r a y t r e a t m e n t to female mice 1 5 - 2 1 d a y s p r i o r to c o n c e p t i o n is to increase the p r o p o r t i o n of e m b r y o s lost before i m p l a n t a tion. This is shown for the positive c o n t r o l g r o u p

224 TABLE 3 P R E I M P L A N T A T I O N E M B R Y O S C O L L E C T E D F R O M D O N O R F E M A L E S A T 3.5 DAYS p.c. Treatment of donor

Embryos collected Fragmented, lysed or abnormal (1-8 cell) 1 cell 2 cell 3 - 4 cell 5 - 8 cell Abnormal morulae Abnormal blastocysts Normal morulae b Normal blastocysts b Total

Irradiated

Untreated

C

D a

Ea

6

1

61 55 31 38 20 28 2 369 (48.6%) 156 (20.5%) 760

Pregnant females E m b r y o s / p r e g n a n t females

94 8.1

% preimplantation abnormalities % induced preimplantation abnormalities c

30.9 + 1.7 28.3 + 1.8

6

1

0 0 0 4 0 71 (26.6%) 180 (67.4%) 267

0 1 0 3 0 100 (30.1%) 226 (68.1%) 332

33 8.1

42 7.9

6.0 + 1.5 -

1.8 + 0.7 -

a Embryos from series D donors were transferred to irradiated recipients and those from series E were transferred to untreated recipients (see Tables 1 and 5). b Includes those without a zona pellucida but otherwise normal. c % induced preimplantation abnormalities =

[1 (ormalmolaeanblastocyststreatedormalmorulaeandblast / ystsntrol t] Total embryos, treated

/

(A) in Table 2 as the discrepancy between the number of implants and corpora lutea. The classification of preimplantation embryos collected at 3.5 days p.c. (Table 3) indicates that irradiation leads to a significant increase in retarded or arrested development. Both the proportion of abnormal or retarded embryos (pre-morula stage) and the ratio of normal morulae to normal blastocysts at 3.5 days p.c. is very significantly higher in the irradiated group than in the untreated group (Table 4). The embryos classified as 1-8 cell would probably have died before implantation but it is not clear from this single observation whether the high morula:blastocyst ratio reflects a group of embryos that arrest at or shortly after the morula stage or whether they are simply developing more slowly. The elevated frequency of corpora lutea/female in group A (Table 2) is in keeping with earlier

Total embryos, control

× 100

observations that irradiation of female mice can cause superovulation (Russell and Russell, 1954, 1956). However, the superovulation effect reported earlier was limited to-those mice that were irradiated during the two weeks prior to fertilization. No TABLE 4 D I F F E R E N C E S IN P R E I M P L A N T A T I O N DEVELOPM E N T IN I R R A D I A T E D A N D U N T R E A T E D FEMALES

Irradiated donors Untreated donors X z (1 d.f.) Significance

Normal morulae and blastocysts/ total

Normal blastocysts/ normal morulae and blastocysts

525/760 (69.1%)

156/525 (29.7%)

577/599 (96.3%) 160 P < 0.001

406/577 (70.4%) 180 P < 0.001

225

significant effect was found for those that mated 2-3 weeks after irradiation, which was the interval used in the present study.

The transfer of embryos from untreated to irradiated females (group D) induced negligible postimplantation dominant lethality. Correspondingly, the postimplantation mortality was not significantly higher than for embryos transferred between two untreated females (group E), and was very significantly lower than for embryos transferred from irradiated to untreated females (C). These observations demonstrate that irradiation of the postimplantation maternal environment, 15-21 days prior to conception, makes no significant contribution to the frequency of induced postimplantation dominant lethals. On 3 occasions the total number of implants was one greater than the number of embryos transferred. In each case the discrepancy was accounted for by an extra small mole and there were never any extra fetuses. There was no evidence to suggest that the sterile males were incompletely sterile. Instead it seems likely that the

Postimplantation mortality The frequencies of postimplantation mortality and induced postimplantation dominant lethality are shown in Tables 2 and 5. The embryos transferred from irradiated to untreated females (group C) produced a high yield of induced dominant lethality, even higher than in the positive control group A. In each case the frequency of postimplantation mortality was very significantly greater (Table 6) than in the corresponding negative control group (groups B and E respectively). Thus, the frequency of postimplantation mortality produced when embryos were transferred from irradiated donors to untreated recipient females (group C) is sufficient to account for all of those produced in the positive control group (A).

TABLE 5 TRANSFER OF PREIMPLANTATION EMBRYOS Group Recipients Number of recipient ¢ ~

Irradiated ~ Untreated C

Untreated ~ Irradiated D

Untreated --o Untreated E

All

All

All

Pregnant

Pregnant

Pregnant

94

52

33

29

42

37

Embryos transferred Total Normal morulae and blastocysts

759 a 525

546 392

264 a 251

238 227

332 326

301 298

Implantations Total Small moles Large moles Dead fetuses Abnormal fetuses (dwarf+ other) Normal fetuses

340 186 6 0 7 (7 + 0) 141

Implants/pregnant females Implants/embryos transferi'ed c Implants/normal morulae and blastocysts c Postimplantation mortality Induced postimplantation dominant lethality Abnormal fetuses

6.5 0.62 0.87 56.5 + 2.7 48.4 + 3.5 4.7 + 1.7

195 22 9 6 5 (2 + 3) b 153 6.7 0.82 0.86 19.0 + 2.8 4.0 _+4.2 3.2 + 1.4

244 27 4 7 2 (2 + 0) 204 6.6 0.81 0.82 15.6 + 2.3 0.97 ___0.68

Total is lower than that shown in Table 3 because a few embryos were lost. b Abnormal fetuses comprised 2 dwarfs, 2 exencephalic and 1 exencephalic with exophthalamos (bulging eyes). c Considering only pregnant recipient females.

226 TABLE 6 DIFFERENCES IN FREQUENCIES OF POSTIMPLANTATION MORTALITY AND ABNORMAL FETUSES Groups compared

Postimplantation mortality

Abnormal fetuses

Frequency

Significance

Frequency

Significance

Irradiated positive control Untreated negative control

(A) (B)

114/242 52/349

X2 = 71.81 ***

7/128 2/297

P = 0.0040 **

Irradiated ~ Untreated Untreated --*Untreated

(C) (E)

192/340 38/244

X2 = 97.82 ***

7/148 2/206

P = 0.038 *

Irradiated positive control Irradiated ~ Untreated

(A) (C)

114/242 192/340

X2 = 4.60 *

7/128 7/148

P = 0.791

Untreated ---,Irradiated Untreated ~ Untreated

(D) (E)

37/195 38/244

X2 = 0.66

5/158 2/206

P = 0.247

Irradiated positive control Untreated -o Irradiated

(A) (D)

114/242 37/195

X2 = 36.56 ***

7/128 5/158

P = 0.384

Irradiated ---*Untreated Untreated -o Irradiated

(C) (D)

192/340 37/195

X2 = 69.64 ***

7/148 5/158

P = 0.563

P values for two-sided X2 tests are as follows: * P < 0.05; ** P < 0.01; *** P < 0.001. P values for abnormal fetuses were calculated using a two-sided Fisher's exact test. Asterisks are used for easy comparison with the X2 results for postimplantation mortality.

moles were the result of a d e c i d u a l r e s p o n s e elicited either b y the e m b r y o - t r a n s f e r t e c h n i q u e or p e r h a p s even b y the presence of the recipient f e m a l e ' s own unfertilized eggs. Such technical artefacts d o n o t affect the conclusions b e c a u s e the i n d u c e d p o s t i m p l a n t a t i o n d o m i n a n t lethality shown in T a b l e 5 was c a l c u l a t e d with reference to an e m b r y o transfer negative c o n t r o l g r o u p (group E). I n a n y case the f r e q u e n c y of small moles in this g r o u p ( 2 7 / 2 4 4 = 11%) is n o t significantly higher t h a n for the u n t r e a t e d c o n t r o l g r o u p ( G r o u p B in T a b l e 1: 2 7 / 3 4 9 = 7.7%; X 2 = 1.54). Also, n o i m p l a n t s were f o u n d a m o n g a s e p a r a t e c o n t r o l g r o u p of 31 u n i r r a d i a t e d p s e u d o p r e g n a n t females even .though unilateral s h a m - e m b r y o transfers h a d b e e n perf o r m e d on 16 of these controls. T h e s e o b s e r v a t i o n s i n d i c a t e that the a r t e f a c t u a l i n d u c t i o n of small m o l e s is likely to l~e s p o r a d i c a n d in n o w a y c o m p r o m i s e s the conclusions d r a w n . D e a d fetuses were f o u n d in g r o u p s B, D a n d E b u t n o t in g r o u p s A a n d C w h i c h suffered h e a v y e m b r y o n i c loss at earlier stages (Tables 2 a n d 5). A similar o b s e r v a t i o n was m a d e b y K i r k a n d L y o n (1984) w h o a t t r i b u t e d m u c h of the fetal loss to u t e r i n e crowding.

Congenital anomalies T a b l e s 2, 5 a n d 6 show that the frequency of fetal a n o m a l i e s i n d u c e d when e m b r y o s were transferred f r o m i r r a d i a t e d d o n o r s to u n t r e a t e d recipients (group C) was n o t significantly lower t h a n in the positive c o n t r o l g r o u p (A). M o r e o v e r the f r e q u e n c y in g r o u p C was significantly higher t h a n w h e n neither d o n o r n o r recipient were i r r a d i a t e d ( g r o u p E). These two o b s e r v a t i o n s i m p l y that i r r a d i a t i o n o f the d o n o r a l o n e m a y b e sufficient to account for all the c o n g e n i t a l a n o m a l i e s i n d u c e d when the entire r e p r o d u c t i v e tract is i r r a d i a t e d . However, transfer of e m b r y o s f r o m u n t r e a t e d d o n o r s to i r r a d i a t e d recipients (D) p r o d u c e d an i n t e r m e d i a t e f r e q u e n c y of congenital a n o m a l i e s that was n o t significantly different f r o m either the positive control g r o u p (A) o r the negative e m b r y o - t r a n s f e r c o n t r o l g r o u p (E). It therefore r e m a i n s unclear w h e t h e r i r r a d i a t i o n of the uterus c o n t r i b u t e s to the i n d u c t i o n of congenital anomalies. The most commonly recorded congenital a n o m a l y was d w a r f i s m which a c c o u n t e d for 1 5 / 2 3 of the a b n o r m a l fetuses. 2 of the 8 fetuses with o t h e r a b n o r m a l i t i e s were also d w a r f as docum e n t e d in the f o o t n o t e s to T a b l e s 2 a n d 5.

227

Fetal weights Distributions of fetal weights are shown in Fig. 1 and summarized in Table 7. Fetuses that were examined before 18.5 days p.c. and those that resulted from synchronous embryo transfers were excluded from this analysis. The distribution of fetal weights (Fig. 1) shows that although the dwarf embryos were clustered at the light end of each distribution they did not form a distinct population. This presumably reflects variation between litters and emphasises the need for within-litter comparisons. In Table 7 the mean fetal weights are compared to determine whether X-irradiation of the postimplantation maternal environment reduces fetal weight in subsequent pregnancies. Fetuses from the irradiated positive control group (A) were actually heavier than those from untreated negative control group (B). This is probably another effect of uterine crowding; group B has nearly twice the number of live fetuses per uterine horn and over twice as many per litter. The mean fetal weights of the embryo-transfer groups (C-E) cannot be compared with unop-

A

Irrodiated Controls

C

Irrod~ated~Unlreated

B

Untreated Conlrots

O

Untreated~Irrodlaled

~ 20

-03-02-01

0

O1 02

-03-02-01

0

01 02 03

E Untreoted~Untreoted

-02-03

0

O1

02 03

LOglO Feto[ Weight (groins) Fig. 1. Distribution of fetal weights (shown as lOglo fetal weight) in the 5 groups. Fetuses classified as dwarfs are shown as shaded areas.

erated controls (A and B) because development has been interrupted in the former groups. However, comparison of groups D and E provides a means of evaluating the effects of X-irradiation of the uterus on fetal weights since uterine crowding is similar. In this case, fetuses implanted into an irradiated uterus (group D) are slightly lighter than those implanted into an untreated uterus.

TABLE 7 FETAL WEIGHTS Group

A Irradiated positive control

B Untreated negative control

C Irradiated -*Untreated

D Untreated --*Irradiated

E Untreated ---,Untreated

1.20+0.016 (57) 1.22+0.025 (56) 1.21 -+0.015 (113)

1.11+0.009(128) 1.15_+0.011(106) 1.13-+0.007 (234)

1.05+0.018 (66) 1.11+0.014 (56) 1.08_+0.012 (122)

0.99+0.011 (79) 1.03+0.012 (57) 1.01 _+0.008 (136)

1.03+0.011 (97) 1.03-+0.010 (92) 1.03 _+0.007 (189)

0.626;0.81d 1.00d;1.025

0.71~;0.802

0.59~;0.72~ 0.79~;0.827 0.82~;0.84~ 0.865;

0.762;0.812

0.575;0.77d

3.56 1-9 3 2.24 1-6 1

8.10 1-12 7 and 10 4.12 1-8 4

2.35 1-6 2 2.35 1-6 2

5.31 1-8 6 4.31 1-7 6

5.76 1-10 5 3.96 1-7 5

Meanfetal weight (g) a ~ ~ Total

Dwarfweights

Uterinecrowding Live fetuses/litter

Live fetuses/ uterine horn b

mean range mode mean range mode

' Mean+ SE (N). b Uterine horns with no live fetuses were not included.

228 However, an analysis of variance of fetal weights in groups D and E showed that there was significant variation in fetus weight between litters (variance ratio, F(57,266) = 5.817; P = 6.4 x 10 -23) but no significant difference between groups D and E (variance ratio, F(1,57)= 1.010; P = 0.32). It, therefore, seems unlikely that irradiation of the postimplantation maternal environment 15-21 days prior to conception adversely affects fetal growth. Certainly if any such effect exists it is smaller than the effect of uterine crowding. The means and standard errors shown in Table 7 are based on individual fetal weights. These were recalculated as 1.007 ___0.016 (group D) and 1.028 + 0.014 (group E) to account for the variation between litters. (These larger standard errors were calculated using the estimated variance between litter means and the estimated variance within litters derived from the variance component analysis described by Kempthorne, 1957.) Discussion

The experiments reported here were designed to distinguish between the direct mutagenic effect of X-rays on the oocyte and effects on the postimplantation maternal environment (notably the uterus). Any effect on the preimplantation maternal environment would be indistinguishable from the mutagenic effect on the oocytes because embryos were transferred at the end of the preimplantation period. Examination of preimplantation embryos collected from females that had been exposed to 3.6 Gy X-rays 15-21 days prior to conception revealed a high proportion that were either arrested or retarded in development. These observations, although limited to a single time point, do parallel others. Generoso (1968) examined histological sections of preimplantat.ion mouse embryos within the oviducts of females that had been treated with ethyl methanesulphonate (EMS) 0.5-4.5 days prior to conception. At 2 days p.c. the majority of embryos in this treatment group were at the 2-cell stage whereas most of the untreated controls had advanced to 4-8 cells. Also, embryos sired by males, that had been irradiated (Goldstein and Spindle, 1976) or treated with triethylenemelamine, TEM (Bi~rki and Sheridan, 1978), and cul-

tured in vitro from the 2-cell stage had a higher incidence of developmental failures in early cleavage, at the late morula stage and at the late blastocyst stage than control embryos. In vitro culture techniques could also be applied to preimplantation embryos collected from treated females to distinguish between mutagenic effects of the treatment on the oocyte and effects on the preimplantation environment. This would also clarify whether the high morula:blastocyst ratio among embryos collected from irradiated females in group C (Tables 3 and 4) reflects a general slowing of development, or represents a group of morulae that were unable to develop into blastocysts. The embryo-transfer experiments described here demonstrate that postimplantation mortality is not significantly increased as a result of irradiation of the postimplantation maternal reproductive environment, following exposure of female mice to 3.6 Gy X-rays, 15-21 days prior to conception. Similarly there is no significant reduction of fetal weight. These observation indicate that the irradiated uterus is just as capable of sustaining normal survival and growth as the untreated uterus. However, Chang and Hunt (1960) reached the opposite conclusion from their embryo-transfer experiments on rabbits exposed to 400 r of y-rays. In this study rabbits or preimplantation embryos in vitro were irradiated on days 2-6 after ovulation so that there was little time for any irradiation damage to repair before the embryos implanted. When 119 non-irradiated embryos were transferred to irradiated recipients, 74 implanted and 48 produced normal fetuses. There was no concurrent control group of unirradiated embryos transferred to unirradiated hosts, the only negative controls being a group of normal rabbits, pregnant after artificial insemination. The proportion of implants that survived to form fetuses in this group was 74/76 (97.4%), compared with 48/74 (64.9%) for the embryo-transfer experimental group using irradiated recipients. However, an embryo-transfer, negative control group is crucial for interpreting the results. In an historical embryotransfer, negative control group (Chang and Hunt, 1960, p. 513) 'about 42%' of embryos transferred at 4-6 days survived to form normal fetuses. This shows little difference from the value of 33/84

229

(39.3%) normal fetuses from embryos transferred at 4-6 days into irradiated females by Chang and Hunt. There is, therefore, insufficient evidence to support Chang and Hunt's claim that, for the rabbit, irradiation of the maternal environment has an adverse effect on embryonic development. Nevertheless, irradiation close to the time of implantation could have such an effect and our own results are only relevant for irradiation 15-21 days prior to conception in the mouse. The results for congenital anomalies were equivocal in our work and it remains uncertain whether irradiation of the maternal environment plays a role in inducing congenital anomalies. This ambiguity is partly because the frequency of induced congenital anomalies is low and so larger groups of fetuses are needed. An additional problem is that the endpoint is not entirely objective. For example, we excluded a number of very minor anomalies, such as minor bruising and small kinks of the tail tip. Also, the classification of a fetus as dwarf is dependent on the weights of its littermates. This is essential because there is considerable variation between litters but problems arise when litter sizes are very small as commonly occurs in the irradiated groups. Despite this classification problem, irradiation of females consistently produces significantly more congenital abnormalities than are found in untreated females (Nomura, 1979, 1982; Kirk and Lyon, 1982; present results, Tables 2 and 6) and this effect is dose-dependent. The issue of whether irradiation of the maternal environment contributes to this elevated frequency of congenital anomalies has been re-examined in the companion paper (West et al., 1985) using a technique for selective irradiation of the ovary or uterus. This approach is experimentally more efficient than the embryo-transfer technique reported here and this makes it easier to collect data for larger groups of fetuses. In conclusion, the eml~ryo-transfer experiments show that irradiation Of the uterus (and most of the postimplantation maternal environment) 15-21 days prior to conception has no significant effect on the subsequent viability or growth of fetuses. However, it remains unclear whether this treatment contributes to the induction of congenital anomalies.

Acknowledgements We are grateful to Mr. M. Corp and Mr. P. Adams for irradiating the mice, to Mr. D.G. Papworth for help with statistical tests and to Mr. G. Fisher for preparing the illustrations. We also thank Mr. P.H. Glenister for advice on embryotransfer technique. K.M.K. was supported by a grant from the National Radiological Protection Board.

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