malformation of the extremity in the mouse foetus caused by x

J. Embryol. exp. Morph., Vol. II, Part 3, pp. 549-569, September 1963Printed in Great Britain

Malformation of the Extremity in the Mouse FoetusCaused by X-radiation of the Mother During

Pregnancy

by UJIHIRO MURAKAMI, YOSHIRO KAMEYAMA and

HIROSHI NOGAMI1

From the Research Institute of Environmental Medicine, Nagoya University

WITH ONE PLATE

INTRODUCTION

W H I L E producing malformations in the mouse foetus with X-rays, the authorsrecognized an abnormal shortening of the extremities in a group treated on the11th day of pregnancy. Such shortenings were caused by the absence, shorten-ing, curvature, flexure or synostosis of the long bones of the extremities.Furthermore, in foetuses treated on the 12th day of pregnancy, numerous casesof ectrodactylism were found.

Recently it has been noted that thalidomide taken during the early stages ofpregnancy is liable to produce congenital malformations of the extremities suchas phocomelia, defects of radii and thumbs, ectrodactylism, etc. in humanbeings. Lenz & Knapp (1962) pointed out that there is a critical stage whensuch serious malformations of the extremities caused by the use of the drugbecome manifest. The critical stage for the onset of serious malformation ofextremities in the mouse foetus resulting from X-radiation corresponded wellwith the critical stage of the reduction malformations of the extremity causedby thalidomide. This paper describes an experimental attempt, using labora-tory animals, to determine the critical stage for certain other malformationsknown in human beings. It also describes a mechanism responsible forectrodactylism.

MATERIALS AND METHODS

Animals used were the ddN strain of mice, originally supplied by theZikkendobutsu Chuo Kenkyujo, 90 days or older and weighing from 22 to 24 g.In each case one oestrous female was kept overnight in a cage with one male.

1 Authors' address: Research Institute of Environmental Medicine, Nagoya University,Furo-Cho, Chikusa-Ku, Nagoya, Japan.

550 UJIHIRO MURAKAMI et al

The next morning, all the females showing a vaginal plug were considered tobe in the 1st day of pregnancy. The pregnant mice were subjected to a singledose of whole body X-radiation of 300 r., 200 r. or 150 r. between the 8thand the 13th day of pregnancy (Tables 1-9). The mice were divided into six

100

90

80-

70

60-

50

40-

30-

20-

10-

8 10 11 12 13th day

TEXT-FIG. 1. Incidence of malformations of the extremitiescaused by maternal X-radiation during pregnancy.

groups, i.e., group A corresponding to the 8th, group B to the 9th, group Cto the 10th, group D to the 11th, group E to the 12th, and group F to the 13thday of pregnancy.

Each group treated with a 300 r. X-radiation, contained ten to twelve micewhereas in each group subjected to 200 r. or 150 r. there were twenty mice.For each experimental series, corresponding to different doses of X-radiation,an equal number of untreated mice were examined as controls (Tables 1-9).

TA

BL

E

1

Pre

gnan

cy s

tatu

s of

mic

e tr

eate

d w

ith

300

r. X

-rad

iati

on a

t di

ffer

ent

stag

e of

pre

gnan

cy

Gro

ups

ofan

imal

scl

assi

fied

by th

e da

y of

trea

tmen

t

A B C D E F

CO

NT

RO

L

Day

of

preg

nanc

yw

hen

trea

ted

8th

9th

10th

11th

12th

13th

No.

of

mic

etr

eate

d

12 10 10 12 12 12 20

Tot

alnu

mbe

r of

foet

uses

89 81 81 76 116

85 181

* F

ive

dead

Mea

nli

tter

size

7-42

8-10

81

0

6-07

9-67

9-08

9-05

Foe

tal

deat

hsin

clud

ing

plac

enta

lre

mna

nts

80 89-8

9%

60 74-0

7%

47 58-0

2%

45 59-2

1%

33 28-4

5%

33 38-8

2%

19 10-5

0%

ones

are

inc

lude

d.

Foe

tuse

sex

amin

ed

9 21 34 36*

83 52

162

Foe

tuse

sw

it/t

out g

ross

mal

form

atio

ns

2 22-2

2%

2 9-52

%

9 26-4

7%

18 50-0

0%

0 23 44-2

3%

162

100-

00%

Foe

tuse

sw

ith

gros

sm

alfo

rmat

ions

7 77-7

8%

19 90-4

8%

25 73-5

3%

18 50-0

0%

83 100-

00%

29 55-7

7%

0

MALFOR1 H O H-1

* THE MOl i_ m tn o m H XA


TABLE 2Groups of

animalsclassified

by the day oftreatment

A

B

C

D

E

F

CONTROL

Day ofpregnancy

whentreated

8th

9th

10th

11th

12th

13th

Brainhernia

222-22%

14-76%

0

0

0

0

0

Hydro-cephalus

0

838-10%

1852-94%

925-00%

4351-81%

0

0

Micro-cephalia

0

0

12-94%

616-67%

56-62%

0

0

Micro-ophthalmos

11111%

419-05%

411-76%

25-56%

7590-36%

0

0

Anoph-thalmos

1H-11%

1152-38%

411-76%

513-89%

1113-25%

0

0

After sacrificing the mothers on the 19th day of pregnancy, the foetuseswere removed and studied under a low-power microscope. All foetuses wereexamined once more under the same microscope after being cleared accordingto the Dowson method, i.e., clearing tissues with 1 per cent potassium hydroxidesolution and staining the bone with alizarin red S solution.

In another series of experiments, meant to examine the process leading toectrodactylism, the mice were exposed to 200 r. on the 12th day of pregnancy,and the embryos were taken out after 2, 6, 12, 18 or 24 hours. Thereafter, thefoetuses were taken out at 1-day intervals until the 19th foetal day. Theseembryos and foetuses were fixed with Bouin's solution, and their paraffinsections were stained with haematoxylin and eosin for histological examination.

For the X-radiation a therapeutic X-ray machine was used. The beam wasproduced by the factors; 170 KVP, 10 mA., 40 cm., and the filter was 0-5 mm.Al + 0-5 mm. Cu., 16 r./min.

RESULTS

As shown in the tables, malformations of the extremities were concentratedin two groups, i.e., mice treated on the 11th (group D) and those treated on the12th day of pregnancy (group E). Further, such malformations appeared mainlyin the two experimental series treated with 300 r. and 200 r. of X-radiation(Tables 3, 6 & 9; Text-fig. 1).

MALFORMATION IN THE MOUSE FOETUS 553

Classification of malformations (300 r.)

Taperedsnout

0

1047-62%

0

0

0

0

0

Abnormalmaxilla andmandible

0

11*52-38%

0

822-22%

0

0

0

Harelip

11M1%

0

0

0

0

0

0

Cleftpalate

111-11%

0

25-88%

1541-17%

7590-36%

47-69%

0

* Fused

Abdominalhernia

444-44%

0

0

0

0

0

0

premaxillae.

Tailabnormality

1H-11%

14-76%

2058-82%

1438-89%

8197-59%

3057-69%

0

Extremitymalformation

222-22%

0

12-94%

1747-22%

8197-59%

713-46%

0

Vertebralmalformatic

9100-00%

1571-43%

2470-59%

1952-78%

83100-00%

1325-00%

95-56%

Groups treated with 300 r. of X-radiation

In group D, scapular malformations appeared in nineteen cases (52 • 78 percent). Humero-radio-ulnar synostosis was observed in sixteen cases (44-44 percent), while in the hind limb, defects of the long bone formed in thirteen cases(33 • 33 per cent.). Cases with malposition of the foot, or clubfoot, were observedin fifteen cases (41-69 per cent.). Cases in which the bones constituting thepelvic elements were incompletely formed or defective numbered twenty-two(61-11 per cent.).

In group E, the major malformations were reductions in number and size ofthe digits. In the fore-limbs, ectrodactylism occurred in eighty-seven cases (97 • 59per cent.), while in the hind-limbs, there were seventy-three cases (87-95 percent.). Bone defects were rare in the fore-limb, while these were twenty-sevencases (32-53 per cent.) in the hind-limbs.

In other groups, malformations of the extremities were markedly reduced innumber. In group B none were found (Table 3).

Other malformations were also detected. There were nine cases of hydro-cephalus (25-07 per cent.) in group D, while in group E, there were forty-threecases (51-81 per cent.). There were only two cases (5-56 per cent.) of micro-phthalmia in group D, while in group E, there were seventy-five cases (90 • 36per cent.). There were fifteen cases of cleft palate (41-17 per cent.) in group D,while seventy-five cases (90 • 36 per cent.) were detected in group E. Almost all

TA

BL

E

3

Cla

ssifi

catio

n of

ext

rem

ity

mal

form

atio

ns

(300

r.)

Gro

ups

of

,an

imal

s D

ay o

fcl

assi

fied

preg

nanc

y Sc

apul

aby

the

day

of

whe

n m

al-

Syn-

trea

tmen

t tr

eate

d fo

rmat

ion

dact

yliaFor

e-lim

bsH

ind-

limbs

B C D E

8th

9th

10th

11th

12th

13th

0 0 0 19 1 1-21

%

0

Ect

ro-

dact

ylia

0 0 0

Bon

eab

norm

ali-

0 0 17*

Pel

vic

gird

lem

al-

Vol

arfle

xion

of

Pol

y-

0 0 0 22

2 22-2

2%

1 2-94

%

1552

-78%

2

-78

%

19-4

4%

47-2

2%

61-1

1%

41-6

9%

5-56

%

81

If

3 4

29

7-5

9%

1-

21%

3

-61

%

4-8

2%

2

-41

%

Syn-

for

mat

ion

the

foot

da

ctyl

ia

dact

ylia

Bon

eE

ctro

- ab

norm

ali-

dact

ylia

tie

s

0

0

0

0

0

0 13t

33-3

3%

73

27§

87-9

5%

32-5

3%

1-92

%

3-85

%3-

85%

1 6

1-92

%

11-5

4%

c! <—i

i—i o

,,. fS

ynos

tosi

s hu

mer

o-ra

dio-

ulna

ris

16 (

44-4

4%)

[Rad

ius

defe

ct

1 (

2-78

%)

t Sy

nost

osis

hu

mer

o-ra

dio-

ulna

ris.

. rF

ibul

a de

fect

s 13

(36

• 11

%)

* 1

Fem

ur a

bnor

mal

itie

s w

ith f

ibul

a de

fect

s 12

(33

-33%

)8

/Abn

orm

al o

r de

fect

ive

fibu

lae

# 27

\Abn

orm

al o

r de

fect

ive

tibia

e w

ith t

hat

of

fibu

lae

24


tail abnormalities were shortenings with curvatures or flexures, only fourteencases (38-89 per cent.) of them in group D, and eighty-one cases (97-59 percent.) in group E. The above malformations were usually associated withmalformations of the extremities (Table 2).

TEXT-FIG. 3. A line drawing of theoutline of the skeleton of a 19-daymouse foetus whose mother wastreated with 200 r. X-radiation onthe 11 th day of pregnancy. Scapulaand other long bones of the fore-limb are involved in malformationbut no synostosis is seen. In thehind-limb, the femur and tibia areabnormal and the fibula is absent.Hands and feet show malposition.

Abbreviations as in Text-fig. 2.

TEXT-FIG. 2. A line drawing ofthe outline of the skeleton ofa 19-day mouse foetus whosemother was treated with 200 r.X-radiation on the 1 lth day ofpregnancy. Hypoplasia of thescapula, humero-radio-ulnarsynostosis, hypoplastic tibia andabsence of the fibula are shown.The ilium also is malformed.Hands and feet are also inmalposition, s, the scapula; h,the humerus; r, the radius; u,the ulna; i, the ilium; f, thefemur; t, the tibia; fi.the fibula.


In group D, there were forty-eight cases (37 • 8 per cent.) of hypoplasia ofthe scapula (Text-figs. 2 and 3). Malformation of the fore-limbs appeared inthirteen cases (11-02 per cent.). Shortening, curvature, flexure, etc. of the boneexisted in nine cases (7-09 per cent.). In four cases (3-15 per cent.) the shorten-ing of the fore-limb was associated with volar flexure of the hand or clubhand.

In the hind-limbs, in addition to twenty-three cases (18-11 per cent.) ofmalformation of the primordial ilium (Text-fig. 2), there were eight instances(6 • 3 per cent.) of skeletal malformation of the hind-limb. Among of them,

36


malformations due to the absence, flexures or curvatures of the tibia or fibulawere detected in three cases (2 • 36 per cent.) (Text-fig. 2). Further, there wereeleven cases with volar flexure of the foot with or without extension of the hipjoint, many of them along with the above bone malformations.

In the group treated on the 11th day of pregnancy, there were some caseswith digital malformations. Only one case of polydactylia and two cases ofpolysyndactylia could be found in the fore-limbs. In the hind-limbs, a tendencyto volar flexure of the foot or clubfoot was present in 8 • 66 per cent. Further,one case of polydactylia and two cases of polysyndactylia in the hind-limb weredetected.

TEXT-FIG. 4. A line drawing of the outline of theskeleton of a 19-day mouse whose long bones of theextremity are normal. Hands and feet are in normal

position. Abbreviations as in Text-fig. 2.

In the group treated on the 12th day of pregnancy ectrodactylism was themost frequent and typical malformation, and the incidence in the fore-limbwas very high (101 cases or 63 -33 per cent.), whereas in the hind-limb the samemalformation occurred only in thirty-three cases (22 per cent.). In the fore-limbs, there were eight cases (5 • 33 per cent.) of syndactylia and one case ofpolysyndactylism (Table 6). Reductions in the number or size of the digits andthe mechanism will be discussed later.

In group D there were twenty cases of microcephalia (15-75 per cent.) andfourteen cases (11 -02 per cent.) of hydrocephalic conditions, while in group Ethe cases of microcephalia were very few, the incidence of hydrocephalus,however, was increased (forty-nine cases or 32-67 per cent.). In group D, fewcases of micro- or anophthalmoses were detected, in group E, however, cases

TA

BL

E

4

Pre

gnan

cy s

tatu

s of

mic

e tr

eate

d w

ith

200

r. X

-rad

iatio

n at

dif

fere

nt s

tage

of p

regn

ancy

Gro

ups

ofan

imal

scl

assi

fied

by t

he d

ay o

ftr

eatm

ent

A B C D E F

CO

NT

RO

L

Day

of

preg

nanc

yw

hen

trea

ted

8th

9th

10th

11th

12th

13th

No.

of

mic

etr

eate

d

20 20 20 20 20 20 20

Tota

lnu

mbe

r of

foet

uses

169

178

149

160

175

170

181

Mea

nlit

ter

size

8-45

8-90

7-45

80

0

8-75

8-50

9 05

Foe

tal

deat

hsin

clud

ing

plac

enta

lre

mna

nts

26 15-3

8%

42 23

-6%

45 30-2

0%

39 24-3

8%

26 14-8

6%

169-

41%

19 10-5

0%

Foe

tuse

sex

amin

ed

143

137*

109*

127*

150*

157*

162

Foe

tuse

sw

ithou

tgr

oss

mal

-fo

rmat

ions

114 79

-72%

61 44-5

3%

45 41

-21

%

64 50-3

9%

19 12-6

7

124

78-9

8%

162

100-

00%

Foe

tuse

sw

ithgr

oss

mal

-fo

rmat

ions

29 20-2

8%

76 55-4

7%

64 58-7

2%

63 49-6

1%

131 87

-33%

33 21-0

2%

0

o H o •25

»—i

% H X w o (-1

*—1 00 W O W H C CO

* S

om

e de

ad o

nes

are

incl

uded

.

558 UJIHIRO MURAKAMI et a!

TABLE 5

Groups ofanimalsclassified


A

B

C

D

E

F

CONTROL

Day ofpregnancy

whentreated

8th

9th

10th

11th

12th

13th

Ex-encephaliaAcephalia

21-4%

107-3%

0

0

0

0

0

Brainhernia

96-29%

107-3%

10-92%

43-15%

10-67%

0

0

Hydro-cephalus

32-1%

96-57%

1715-6%

1411-02%

4932-67%

0

0

Micro-cephalia

0

21-46%

10-92%

2015-75%

21-33%

0

0

Micro-ophthalmos

10-7%

0

54-59%

43-15%

4932-67%

10-64%

0

An-ophthalmos

139-09%

3525-55%

98-26%

97-09%

2919-33%

0

0

of microphthalmoses were numerous (forty-nine cases or 32-67 per cent.),while there were twenty-nine cases (19-33 per cent.) of anophthalmoses(Table 5).

Further, in both groups approximately 30 per cent, of cleft palates weredetected. Tail abnormalities, such as curvature or flexures with or withoutshortening, existed in twenty cases (15-75 per cent.) in group D, as comparedto 106 cases (70-67 per cent.) in group E. Malformations of the extremitieswere sometimes associated with micro- or hydrocephalic conditions or cleftpalates. In addition to the peculiar appearance of the head, due to a micro-or hydrocephalic condition, the shape of the snout was also abnormal, theneck was short and thick, and the whole external appearance of thesefoetuses was peculiar. The above malformations were often associated withcleft palates and reductions of the eye and tail, abnormalities due to X-radiationat the same critical stage (Table 5).


As shown in Table 9, there were only two cases (1 -44 per cent.) in group Din which the fibula was absent, while in group E there were several cases ofreduction in the number or size of the digits. However, the incidence waslower than in the 300 r. and 200 r. experimental series. All the other malforma-tions were also less frequent, as shown in Tables 8 and 9.



Taperedsnout

32-1%

34*24-82%

0

43-15%

3t2-0%

0

0

Abnormalmandible

21-4%

21-46%

0

64-72%

0

0

0

Harelip \

53-5%

85-84%

0

0

0

0

0

Cleftpalate

64-2%

139-49%

54-59%

3729-13%

4429-33%

85-10%

0

Abdominalhernia

85-59%

10-73%

0

0

0

0

0

Tailabnormal-

ity

32-1%

0

5348-62%

2015-75%

10670-67%

2415-29%

0

Extremitymal-

formation

64-2%

64-38%

1211-01%

2217-32%

11778-00%

138-28%

0

Vertebralmal-

formation

8156-64%

8461-31%

6458-72%

6047-24%

6543-33%

106-37%

95-56%

* Fused premaxillae 44 (32-12%). f Abnormal nasal bone.

Now, some interesting points on reduction in number and size of digitsand the mechanism by which X-radiation acts will be made. In the fore-limbs, ectrodactylism most frequently affected the second digit, many of whichlacked ossification in part of the metacarpal bone. The group treated on the13th day of pregnancy showed only syndactyly and none had osseoussyndactylies.

In both fore- and hind-limbs, ectrodactylias were the predominant digitalabnormalities, in the hind-limb, however, the predominant site was in the thirddigit. There were few cases of polydactylias, polysyndactylias and the so-called'annular grooves'. The fourth digit was the most resistant to malformationboth in fore- and hind-limbs.

As to the mechanism forming digital reductions, no digital radiations couldbe observed in the hand plate at the 12th-day stage. Twelve hours afterX-radiation, findings that suggested digital abnormalities were already present,mostly in the parts corresponding to the second digit and sometimes also inthe parts corresponding to the fifth digit. After 24 hr., a marked absence ofthe second or fifth digital radiation was noticed. In preparations taken at the15-day stage, i.e. 72 hr. after X-radiation, absence or reduction in size of thesecond digit or the fifth digit were evident (Plate 1, Fig. 5).

Plate 1, Figs. 1 and 2, show a section parallel to the palm of the left-handplate of a mouse embryo 2 hr. after X-radiation of 200 r. The arrow indicatesan evident focus of cellular necrosis localized in the radial side. In other


TABLE 6

Groups ofanimals Day of

Fore-limbs

classified pregnancy Scapula Volar Splitby the day of when mal- flexion of hand-like Syn- Ectro-of treatment treated formation the hand state ' dactylia dactylia

8th

9th

0 0 0 0

Boneabnormali-

ties

0

0

c

D

E

F

CONTROL

10th

11th

12th

13th

0

4837-80%

10-67%

0

0

10

43

0

0

0

•92%

•15%

0

0

0

7

0

0

0

85-33%

0

0

0

32-36%

10167-33%

0

0

0

97-09%

0

0

0

cases, foci of cellular necrosis are evident on both the radial and ulnar sidesof the hand and foot plate. In some cases, besides the above findings, there isalso a focus of cellular necrosis at the base of the hand and foot plate cor-responding to the third phalanx and foreshadowing its shortening. In a similarpreparation, taken 6 hr. after X-radiation of 200 r., such necrotic cells weredetected in profusion (Plate 1, Figs. 3 and 4). After 24 hr., the concentration ofpre-cartilage cells to form the digital radiation became evident everywhereexcept in those parts involved in necrosis. At this stage, nuclei involved inpyknosis or karyorrhexis were almost eliminated.

In other cases, various transitional forms of developing ectrodactyliaswere present as shown in the figures indicating the condition on the 17th and19th day. The distal part of the second digit, for example, moves towards theradial side, until it fuses with the first digit and, by resorption, is graduallyreduced to a rudimentary condition. The detail of the whole process will bereported by Nogami elsewhere.

COMMENT

In this paper some malformations of the extremities in foetuses of femalemice exposed to X-radiation, at certain developmental stages, are discussed.However, some hereditary reduction malformations of the extremities in themouse are also known. Rabaud (1914) described a stock of mice called 'luxate',and Carter (1951) gave a detailed description of the trait. Landauer (1953) re-ported a malformed hereditary condition in mice called' brachypodia'. Recently,Gleucksohn-Waelsch et al. (1956) described a hereditary strain in mice havingpeculiar shortening of the extremity called 'phocomelic'.

MALFORMATION IN THE MOUSE FOETUS

Classification of extremity malformations (200 r.)

561

Pelvicgirdlemal-

formation

0

0

0

231811%

63-97%0

0

Volarflexion of

the foot

32-10%

42-92%

87-34%

118-66%

74-67%

10-64%

0

Malformedfoot

0

0

10-92%

0

32-00%

10-64%

0

Hind-limbs

Splitfoot like

state

0

0

0

0

0

42-55%

Poly-dactylia

32-10%

10-73%

0

10-79%

32-00%

0

0 0* Polysyndactylia.

Syn-dactylia

10-7%

0

0

2*1-58%

1*0-67%

0

0

Ectro-dactylia

0

10-73%

0

0

3322-00%

0

0

Boneabnormali-

ties

0

0

0

83-6%

42-67%

0

0

In luxates, in addition to limb malformations, a urogenital syndrome is seenin homozygous individuals. In phocomelics, the head is small and the palateis usually cleft, in association with malformations of the nasal and pre-maxillary bones, in almost all individuals.

It is noteworthy that cases of X-ray malformations of the extremities areassociated with an abnormally shaped head, a microcephalic or hydrocephaliccondition, peculiarly shaped snout or cleft palate, etc. Such multiple malforma-tions produced by X-rays can be explained by the common onset of thesemalformations at the same critical stage.

Experimental production of extremity malformations, other than by X-radiation, was carried out in pregnant rats or mice by means of a rachitogenicdiet (Warkany & Schraffenberger, 1944) and by riboflavin deficiency inpregnant rats (Warkany, Nelson & Schraffenberger, 1943, Giroud, Lefebvres& Prost, 1953). Kalter & Warkany (1957) produced such malformationsin mouse foetuses from mothers fed with a galactoflavin-containing, riboflavindeficient, diet. Asling, Nelson, Wright & Evans (1955) also described a similarkind of malformation in rats induced by pteroylglutamic acid deficiency. Otherextrinsic factors also interfered with the differentiation of the extremities inembryos and caused malformations whose details were discussed by Millen(1962).

Recently, it has been reported that thalidomide taken during early stages ofpregnancy caused reduction malformations of the extremities in human beings.Lenz & Knapp (1962) noticed amelia, phocomelia, absence of the radius and


thumb, ectrodactylias etc. in the upper-limbs, and frequent amelia, poly-dactyalia etc. in the lower-limbs. However, they stated that it was not clearwhether there were also cases of clubfoot or congenital dislocation of the hip,as such malformations were not specifically listed. Since 1961 many otherinvestigators have reported such cases too.

Lenz & Knapp stated that in human embryos the critical stage for severemalformations of the extremities caused by thalidomide, such as micromeliaor phocomelia, occurred from the 27th to the 33rd day of pregnancy. Further,they indicated that when the drug had been taken between about the 27th and33rd day of pregnancy, malformations were localized in the upper limbs, whileif it had been taken at a later stage, the malformation occurred in the upper-limbs but to a minor degree, with a tendency to extend to the lower limbs.They concluded that the critical stage for thaUdomide injury extended from the27th to the 40th day after conception. In the authors' present experiments,X-radiation induced extremity malformations in mouse foetuses, such asshortenings, became manifest after treatment on the 11th day, while digitalmalformations, the reduction of number and size of the digits, became manifestafter treatment on the 12th day of pregnancy. According to Otis & Brent (1954),the above critical stage in the mouse pregnancy corresponds to the 27th to 33rdor 35th day in human embryos, a fact that is in accordance with the criticalstage for thalidomide extremity malformations indicated by Lenz & Knapp.

It was said that the pattern of malformations caused by X-radiation resemblesthe one produced by riboflavin deficiency but with some additional skeletalchanges, such as humero-radial synostosis which were not found in the ribo-flavin deficiency cases (Warkany, Nelson & Schraffenberger, 1943; Warkany& Schraffenberger, 1947). Another interesting fact is that thaUdomide alsointerferes with riboflavin metabolism and causes vitamin-B deficiency (Leek& Miller, 1962).

EXPLANATION OF PLATE 1

FIG. 1. A section of the left-hand plate of a mouse foetus 2 hr. after 200 r. X-radiationparallel to the palmar plane. At the base of the hand plate there are three foci of cellularnecrosis, one is on the radial, the other on the ulnar side and the third one is in the middle part.

FIG. 2. A part of the section of the hand plate shown in Fig. 1 at a larger magnification.Many nuclei are involved in pyknosis and karyorrhexis.

FIG. 3. A similar section of the right-hand plate of a mouse foetus 6 hr. after 200 r.X-radiation. Foci of necrosis are in similar places to those in Fig. 1.

FIG. 4. A part of the section of the hand plate shown in Fig. 3 at a larger magnification.FIG. 5. Various stages during digital reduction are shown (a, b and c).

(a) Twelve hours after 200 r. X-radiation. Digital radiations corresponding to the partsof the first or second digit are clearly defective.

(b) Twenty-four hours after 200 r. X-radiation (13th-day stage). The digits are defectivein similar parts to those shown in (a).

(c) Seventy-two hours after 200 r. X-radiation (15th-day stage). Ectrodactylism isevident.

(d) A hand plate of a control foetus on the 13th-day.(e) A hand plate of a control foetus on the 15th-day.

/ . Embryol. exp. Morplt. Vol. 11, Part 3

5c

UJIHIRO MURAKAMI et al.PLATE 1

{Facing page 562)

TA

BL

E

7

Pre

gnan

cy

stat

us

of m

ice

trea

ted

with

150

r.

X-r

adia

tion

at d

iffer

ent

stag

e of

pre

gnan

cy

Gro

ups

ofan

imal

scl

assi

fied

by t

he d

ay o

ftr

eatm

ent

A B C D E

CO

NT

RO

L

Day

of

preg

nanc

yw

hen

trea

ted

8th

9th

10th

11th

12th

No.

of

mic

etr

eate

d

20 20 20 20 20 20

Tot

alnu

mbe

r of

foet

uses

173

164

162

154

162

181

Mea

nli

tter

size

8-65

8-2

8-1

7-7

81

9 05

Foe

tal

deat

hsin

clud

ing

plac

enta

rem

nant

s

40 23-1

2%

36 21-9

5%

23 14-2

0%

16 10-3

9%

12 7-41

%

19 10-5

0%

* So

me

dead

one

s ar

e in

clud

ed.

Foe

tuse

sex

amin

ed

134*

136*

143*

139*

151*

162

Foe

tuse

sw

ithou

tgr

oss

mal

-fo

rmat

ions

87 64-9

3%

100 73

-53%

103 72

-03%

109 78

-42%

102 67

-55%

162

1000

0%

Foe

tuse

sw

ithgr

oss

mal

-fo

rmat

ions

47 35-0

7%

36 26-4

7%

40 27-9

7%

30 21-5

8%

49 32-4

5%

0

<*" o i^ H i—

i O z 1—1 H X PI o c in PI Hrl o PI H


TABLE 8Groups ofanimalsclassified


A

B

C

D

E

CONTROL

Day ofpregnancy

whentreated

8th

9th

10th

11th

12th

Ex-encephaliaAcephalia

64-48%

42-94%

0

0

0

0

Brainhernia

139-70%

21-47%

0

0

0

0

Hydro-cephalus

118-21%

42-94%

1510-49%

1812-95%

0

0

Micro-cephalia

10-75%

0

0

0

0

0

Micro-ophthalmos

2216-42%

64-41%

42-80%

53-6%

106-62%

0

An-ophthalmc

1712-69%

1410-29%

0

0

0

0

In another series of experiments the authors (1963) gave an injection of0-1 cc. of a 2 per cent, trypan blue aqueous solution to mice in early stages ofpregnancy. This produced only one case of shortening of humerus, with absenceof the radius and ulna and ectrodactylism, in the group treated on the 10th dayof pregnancy, while in a second series of experiments (1962) mice in their 10thday of pregnancy, exposed to an hypoxia of about 225 mm. Hg for 5 hr.,showed a similar kind of limb malformation in approximately 20-42 per cent,of all cases. Humero-radial synostosis, however, were limited to the grouptreated with X-rays. The critical stage for malformations of the limbs occurs1 day, or for digital malformations 2 days, later when trypan blue injection orhypoxia are used. The effect of X-radiations upon the anlage of the extremitymay be considered as direct in contrast to that of agents such as trypan blue orhypoxia. It may be presumed that the critical stages for certain malformationsmay differ according to the agent employed.

In Russell & Russell's experiments (1954), employing X-radiations upon themouse, the findings were quite similar to those revealed in the authors' presentexperiments. The experiments conducted by Warkany & Schraffenberger (1947)also showed similar results as to the pattern of extremity malformations,especially synostosis of the long bones in the fore-limb. The latter investigatorssubjected rats to X-radiations on the 13th day of pregnancy, a stage corres-ponding to that of the 11th or 12th day of pregnancy in the mouse.

The present experiments show the mechanism forming reductions of digits.Similar findings were also described by Hicks, Brawn & D'Amato (1957).They observed that after 4 hr. of X-radiation at the 13th-day stage densemesenchyme in the bases and at the distal end of the limb buds was severely



Taperedsnout

3*2-24%

21*15-44%4f2-94%

10-71

0

0

0

Abnormalear

1

32-21%

0

0

0

0

Abnormalmaxilla

andmandible

10-75%

It

0

21-44%

0

0

85

42

0

0

0

0

Harelip

•97%

•94%

Cleftpalate

107-46%

64-41%

21-40%

42-88%

149-27%

0

Abdominalhernia

64-48%

21-47%

0

0

0

0

Tailabnormality

10-75%

21-47%

2618-18%

53-6%

117-28%

0

Extremitymal-

formation

32-24%

42-94%

53-50%

64-32%

3523-18%

0

Vertebralmal-

formations

8563-43%

7353-68%

4027-97%

2719-42%

117-22%

0

* Fused premaxillae. t Abnormal nasal bone. J Agnathia.

damaged in rat embryos. However, in the authors' present findings, a cysticdefect in the centre of the limb bud could not be detected, as shown in theirresults.

As the formation of digital reductions was followed up, no tendency torepair, but only a regressive change, was observed. Hicks et al. also statedthat after severe destruction of the mesenchyme the part involved could not becompensated for. This is in evident contrast to the findings in neural tissue inwhich an active process of repair takes place. Therefore it may be assumedthat the behaviour of the mesenchymal tissue after exposure to X-radiations isdifferent from that in the neural tissue.

SUMMARY

1. Different groups of the ddN strain mice were exposed to X-radiation of300 r., 200 r. and 150 r. from the 8th to 13th day of pregnancy. In offspringfrom mothers treated on the 11th day, malformations of the extremitieswere observed in groups treated with over 200 r. Scapular hypoplasia andshortening of the fore-limb, as a result of absence, synostosis, shortening,flexure or curvature of the limb bone, etc., were typical malformations. How-ever, in the group treated with 150 r. of X-radiations the incidence of suchmalformations was very low.

2. In offspring from mothers treated on the 12th day, digital reductions, onaccount of ectrodactylias, were the typical malformation in the fore-limb.Ectrodactylia was dominant in the second digit of the fore-limbs, but involve-

TA

BL

E

9

Cla

ssif

icat

ion

of e

xtre

mit

y m

alfo

rmat

ions

{15

0 r.

)

Gro

ups

ofan

imal

scl

assi

fied

by t

he d

ay o

ftr

eatm

ent

A B C D E

CO

NT

RO

L

Day

of

preg

nanc

yw

hen

trea

ted

8th

9th

10th

11th

12th

Scap

ulae

mal

-fo

rmat

ion

0 0 0 4 2-88

%

0 0

For

e-li

mbs

Syn-

dact

ylia

0 0 0 0 8 5-30

%

0

Ves

tigi

al1

st d

igit

0 0 1 0-70

%

0 9 5-96

%

0

Ect

ro-

dact

ylia

0 0 0 0 1* 0-66

%

0

Vol

arfl

exio

nof

the

foot

1 0-75

%

0 2 1-40

%

3 2-16

%

2 1-32

%

0

Pol

y-da

ctyl

ia

1 0-75

%

2 1-47

%

0 1 0-72

%

1 0-66

%

0

Hin

d-lim

bs

Ect

ro-

dact

ylia

0 0 0 0 2 1-32

%

0

Pos

ition

abno

rmal

i-tie

sof

the

dig

it

1 0-7

5%

0 0 0 0 0

Bon

eab

norm

ali-

ties

0 0 0 2t 1-44

%

0 0

UJIHIJ o URAKAMI t

* P

arti

al d

efec

t. t

Fib

ula

defe

ct.


ment of the fifth digit was also seen. In the hind-limbs, the incidence was alittle lower and the predominant site of involvement was the third digit. Bothin the fore- and hind-limbs, the fourth digit was the most resistant to malforma-tion. Other digital malformations occurred more often in the hind-limb thanin the fore-limbs, though not frequently.

3. The formation of ectrodactylias after X-radiation was studied in afollow-up experiment. The process began with cellular necrosis in the handand foot plate corresponding to the predominant site of ectrodactylias, i.e.chiefly in the second digit of the fore-limb and in the third digit of the hind-limb. The process may damage the precursor of the pre-cartilage. In contrastto the regenerative neural tissue, compensation does not take place in themesenchymal tissue once the destruction is severe, and the reduction of digitswill therefore persist.

4. The effect of X-radiation upon a developing digital primordium may be adirectly destructive process.

5. The critical stage for extremity malformations due to thalidomide inhuman beings corresponds with the critical stage for X-ray malformations ofthe extremities in mouse.

RESUME

Malformation de V extremite chez le foetus de la souris apres Virradiation de larneme en cours de gestation

1. Differents groupes de la souche de Souris ddN ont ete exposes a des dosesde rayons X de 300 r., 200 r. et 150 r., entre le 8e et le 13e jour de la gestation.Dans les portees de souris traitees le l ie jour, on observe des cas de malforma-tion de membres, quand la dose est superieure a 200 r. Les malformationstypiques de ce groupe ont ete l'hypoplasie de l'omoplate et le raccourcissementdes membres, resultant d'absences de synostoses, de raccourcissement, deflexion ou de courbure des os des membres. Par contre dans le groupe traitepar des radiations de 150 r., la proportion de malformations est tres basse.

2. Chez les meres traitees au 12e jour, les embryons montrent des reductionsde doigts, en rapport avec l'ectrodactylie, comme malformation typique dumembre anterieur. L'ectrodactylie affecte surtout le 2e doigt des membresanterieurs, mais elle peut aussi concerner le 4e doigt. Dans les membres poste-rieurs, la proportion de ces malformations est un peu plus faible et sa locali-sation affecte surtout le 3e doigt, aussi bien dans les membres anterieurs quedansles membres posterieurs, le 4e doigt est le plus resistant a la malformation.D'autres malformations digitales se manifestent plus souvent dans le membreposterieur que dans le membre anterieur, mais moins frequemment.

3. La genese de l'electrodactylie a ete etudiee au cours d'observations suivantde pres l'experience d'irradiation. Le premier processus est une necrose cellu-laire dans la palette de la main et du pied: elle correspond aux localisations


predominantes de l'electrodactylie, c'est-a-dire principalement dans le seconddoigt du membre posterieur. II peut en resulter une deficience du precurseurdu precartilage. En opposition avec la regeneration du tissu neural, il n'y apas de reparation du tissu mesenchymateux: la destruction est considerable etla reduction des doigts definitive.

4. L'effet de l'irradiation aux Rayons X sur l'ebauche d'un doigt en voie dedeveloppement est un processus de destruction directe.

5. Le stade critique pour les malformations des membres sous rinfluence dela thalidomide dans l'espece humaine correspond au stade critique permettantd'obtenir des malformations chez la Souris sous 1'influence des rayons X.

ACKNOWLEDGEMENT

This work was supported by a grant for fundamental research from the Ministry ofEducation of Japan.

REFERENCES

ASLING, C. W., NELSON, M. M., WRIGHT, H. V. & EVANS, H. M. (1955). Congenital skeletalabnormalities in fetal rats resulting from maternal pteroylglutamic acid deficiency duringgestation. Anat. Rec. 127, 775-99.

CARTER, T. C. (1951). The genetics of luxate mice. I. Morphological abnormalities ofheterozygotes and homozygotes. / . Genet. 50, 277-99.

GIROUD, A., LEFEBVRES, J. & PROST, H. (1953). Anomalies des membres chez les foetus derat par carence en riboflavine. Arch. Anat. micr. Morph. exp. 42, 41-8.

GLUECKSOHN-WAELSCH, S., HAGEDORN, D. & SISKEN, B. F. (1956). Genetics and morphologyof a recessive mutation in the house mouse affecting heart and limb skeleton. / . Morph.99,465-78.

HICKS, S. P., BRAWN, B. L. & D'AMATO, C. J. (1957). Regeneration and malformation inthe nervous system, eye, and mesenchyme of the mammalian embryo after radiationinjury. Amer. J. Path. 33, 459-81.

KALTER, H. & WARKANY, J. (1957). Congenital malformations in inbred strain of miceinduced by riboflavin deficient, galactoflavin-containing diets. / . exp. Zool. 136, 531-66.

LANDAUER, W. (1952). Brachypodism. / . Hered. 43, 293-8.LECK, L. M. & MILLER, E. L. M. (1962). Incidence of malformations since the introduction

of thalidomide. Brit. med. J. 7, 16-20.LENZ, W. & KNAPP, K. (1962). Thalidomid-Embryopathie. Dtsch. med. Wschr. 87,1232-42.LENZ, W. & KNAPP, K. (1962). Thalidomide embryopathy. Arch. Environ. Health. 5,100-5.MILLEN, J. W. (1962). Thalidomide and limb deformities. Lancet, 22, 599-600.MURAKAMI, U., KAMEYAMA, Y. & NOGAMI, H. (1962). Skeletal malformations in the mouse

fetus caused by maternal hypoxia during early stages of pregnancy. Ann. Rep. Environ.Med. Nagoya Univ. 10 (1961), 45-55.

MURAKAMI, U. & KAMEYAMA, Y. (1963). Skeletal malformations of mice fetuses caused bytrypan blue injection of the mother during early stages of pregnancy. Ann. Rep. Environ.Med. Nagoya Univ. 11 (to be published).

OTIS, E. & BRENT, R. (1954). Equivalent ages in mouse and human embryos. Anat. Rec.•20, 33-64.

RABAUD, E. (1914). Sur une anomalie hereditaire des membres posterieurs chez la souris.C. R. Soc. Bioi, Paris, 11, 411-12.

RUSSELL, L. B. & RUSSELL, W. L. (1954). An analysis of the changing radiation responseof the developing mouse embryo. / . cell. comp. Physiol. Supple. 1, May 1954, 104-47.

MALFORMATION IN THE MOUSE FOETUS 569RUSSELL, L. B. (1954). The effects of radiation on mammalian prenatal development.

Radiation Biology, II/l. N.Y., Toronto, London: McGraw-Hill.WARKANY, J., NELSON, R. C. & SCHRAFFENBERGER, E. (1943). Congenital malformations

induced in rats by maternal nutritional deficiency. / . Bone Jt. Surg. 25, 261-70.WARKANY, J. & SCHRAFFENBERGER, E. (1944). Congenital malformations induced in rats by

maternal nutritional deficiency. / . Nutr. 27, 477-89.WARKANY, J. & SCHRAFFENBERGER, E. (1947). Congenital malformations induced in rats by

roentgen rays. Amer. J. Roentgenol. 57, 455-63.

{Manuscript received 13th February 1963)

malformation of the extremity in the mouse foetus caused by x

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