Malformation of the Extremity in the Mouse Foetus

J. Embryol. exp. Morph., Vol. II, Part 3, pp. 549-569, September 1963
Printed in Great Britain
Malformation of the Extremity in the Mouse Foetus
Caused 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
producing malformations in the mouse foetus with X-rays, the authors
recognized an abnormal shortening of the extremities in a group treated on the
11th day of pregnancy. Such shortenings were caused by the absence, shortening, curvature, flexure or synostosis of the long bones of the extremities.
Furthermore, in foetuses treated on the 12th day of pregnancy, numerous cases
of ectrodactylism were found.
Recently it has been noted that thalidomide taken during the early stages of
pregnancy is liable to produce congenital malformations of the extremities such
as phocomelia, defects of radii and thumbs, ectrodactylism, etc. in human
beings. Lenz & Knapp (1962) pointed out that there is a critical stage when
such serious malformations of the extremities caused by the use of the drug
become manifest. The critical stage for the onset of serious malformation of
extremities in the mouse foetus resulting from X-radiation corresponded well
with the critical stage of the reduction malformations of the extremity caused
by thalidomide. This paper describes an experimental attempt, using laboratory animals, to determine the critical stage for certain other malformations
known in human beings. It also describes a mechanism responsible for
ectrodactylism.
WHILE
MATERIALS AND METHODS
Animals used were the ddN strain of mice, originally supplied by the
Zikkendobutsu 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 to
be in the 1st day of pregnancy. The pregnant mice were subjected to a single
dose of whole body X-radiation of 300 r., 200 r. or 150 r. between the 8th
and 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
1. Incidence of malformations of the extremities
caused by maternal X-radiation during pregnancy.
TEXT-FIG.
groups, i.e., group A corresponding to the 8th, group B to the 9th, group C
to the 10th, group D to the 11th, group E to the 12th, and group F to the 13th
day of pregnancy.
Each group treated with a 300 r. X-radiation, contained ten to twelve mice
whereas 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).
85
181
10
10
12
12
12
20
9th
10th
11th
12th
13th
C
D
E
F
116
76
81
81
89
B
12
8th
52
162
19
10-50%
9-05
83
36*
33
38-82%
33
28-45%
45
59-21%
34
9-08
9-67
6-07
47
58-02%
21
60
74-07%
8-10
810
9
80
89-89%
Foetuses
examined
7-42
Mean
litter
size
Foetal
deaths
including
placental
remnants
* Five dead ones are included.
Total
number of
foetuses
A
No. of
mice
treated
Day of
pregnancy
when
treated
Groups of
animals
classified
by the day of
treatment
19
90-48%
2
9-52%
162
100-00%
23
44-23%
0
18
50-00%
0
29
55-77%
83
100-00%
18
50-00%
25
73-53%
7
77-78%
2
22-22%
9
26-47%
Foetuses
with gross
malformations
Foetuses
wit/tout gross
malformations
H
H-1
O
XA
H
o
m
m
tn
i_
* THE MOl
CONTROL
TABLE 1
Pregnancy status of mice treated with 300 r. X-radiation at different stage of pregnancy
MALFOR1
UJIHIRO MURAKAMI et al
552
TABLE 2
Groups of
animals
classified
by the day of
treatment
Day of
pregnancy
when
treated
Brain
hernia
Anophthalmos
Hydrocephalus
Microcephalia
Microophthalmos
0
0
1
1111%
1
H-11%
A
8th
2
22-22%
B
9th
1
4-76%
8
38-10%
0
4
19-05%
11
52-38%
C
10th
0
18
52-94%
1
2-94%
4
11-76%
4
11-76%
D
11th
0
9
25-00%
6
16-67%
2
5-56%
5
13-89%
E
12th
0
43
51-81%
5
6-62%
75
90-36%
11
13-25%
F
13th
0
0
0
0
0
0
0
0
0
0
CONTROL
After sacrificing the mothers on the 19th day of pregnancy, the foetuses
were removed and studied under a low-power microscope. All foetuses were
examined once more under the same microscope after being cleared according
to the Dowson method, i.e., clearing tissues with 1 per cent potassium hydroxide
solution and staining the bone with alizarin red S solution.
In another series of experiments, meant to examine the process leading to
ectrodactylism, 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, the
foetuses were taken out at 1-day intervals until the 19th foetal day. These
embryos and foetuses were fixed with Bouin's solution, and their paraffin
sections were stained with haematoxylin and eosin for histological examination.
For the X-radiation a therapeutic X-ray machine was used. The beam was
produced 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 concentrated
in two groups, i.e., mice treated on the 11th (group D) and those treated on the
12th day of pregnancy (group E). Further, such malformations appeared mainly
in the two experimental series treated with 300 r. and 200 r. of X-radiation
(Tables 3, 6 & 9; Text-fig. 1).
M A L F O R M A T I O N IN THE MOUSE FOETUS
553
Classification of malformations (300 r.)
Tapered
snout
0
10
47-62%
0
0
Abnormal
maxilla and
mandible
0
11*
52-38%
0
8
22-22%
Harelip
Cleft
palate
Abdominal
hernia
Tail
abnormality
1
1M1%
1
11-11%
4
44-44%
1
H-11%
0
0
0
1
4-76%
0
2
5-88%
0
0
15
41-17%
Extremity
Vertebral
malformation malformatic
2
22-22%
9
100-00%
0
15
71-43%
20
58-82%
1
2-94%
24
70-59%
0
14
38-89%
17
47-22%
19
52-78%
0
0
0
75
90-36%
0
81
97-59%
81
97-59%
83
100-00%
0
0
0
4
7-69%
0
30
57-69%
7
13-46%
13
25-00%
0
0
0
0
0
0
* Fused premaxillae.
0
9
5-56%
Groups treated with 300 r. of X-radiation
In group D, scapular malformations appeared in nineteen cases (52 • 78 per
cent). Humero-radio-ulnar synostosis was observed in sixteen cases (44-44 per
cent), 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 observed
in fifteen cases (41-69 per cent.). Cases in which the bones constituting the
pelvic 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 of
the digits. In the fore-limbs, ectrodactylism occurred in eighty-seven cases (97 • 59
per cent.), while in the hind-limbs, there were seventy-three cases (87-95 per
cent.). Bone defects were rare in the fore-limb, while these were twenty-seven
cases (32-53 per cent.) in the hind-limbs.
In other groups, malformations of the extremities were markedly reduced in
number. In group B none were found (Table 3).
Other malformations were also detected. There were nine cases of hydrocephalus (25-07 per cent.) in group D, while in group E, there were forty-three
cases (51-81 per cent.). There were only two cases (5-56 per cent.) of microphthalmia in group D, while in group E, there were seventy-five cases (90 • 36
per 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
0
19
52-78%
1
1-21%
0
10th
11th
12th
13th
C
D
E
2-78%
Syndactylia
16 (44-44%)
1 ( 2-78%)
1-92%
81
97-59%
19-44%
0
0
0
Ectrodactylia
Fore-limbs
,,. fSynostosis humero-radio-ulnaris
[Radius defect
t Synostosis humero-radio-ulnaris.
0
9th
B
Scapula
malformation
,
0
Day of
pregnancy
when
treated
8th
Groups of
animals
classified
by the day of
treatment
3-85%
3
3-61%
61-11%
22
0
0
0
3-85%
4
4-82%
15
41-69%
2-94%
1
2
22-22%
Volar
flexion of
the foot
2
2-41%
5-56%
Polydactylia
1
1-92%
Syndactylia
Hind-limbs
. rFibula defects
* 1 Femur abnormalities with fibula defects
fibulae #
8 /Abnormal or defective
\Abnormal or defective tibiae with that of
If
1-21%
47-22%
17*
0
0
Bone
abnormali-
Pelvic
girdle
malfor mat ion
Classification of extremity malformations (300 r.)
TABLE 3
0
0
13 (36 • 11 %)
12 (33-33%)
27
fibulae
24
6
11-54%
73
87-95%
0
0
27§
32-53%
13t
33-33%
0
Bone
abnormalities
0
Ectrodactylia
o
<—i
i—i
c!
MALFORMATION IN THE MOUSE FOETUS
555
tail abnormalities were shortenings with curvatures or flexures, only fourteen
cases (38-89 per cent.) of them in group D, and eighty-one cases (97-59 per
cent.) in group E. The above malformations were usually associated with
malformations of the extremities (Table 2).
2. A line drawing of
the outline of the skeleton of
a 19-day mouse foetus whose
mother was treated with 200 r.
X-radiation on the 1 lth day of
pregnancy. Hypoplasia of the
scapula, humero-radio-ulnar
synostosis, hypoplastic tibia and
absence of the fibula are shown.
The ilium also is malformed.
Hands and feet are also in
malposition, s, the scapula; h,
the humerus; r, the radius; u,
the ulna; i, the ilium; f, the
femur; t, the tibia; fi.the fibula.
TEXT-FIG.
3. A line drawing of the
outline of the skeleton of a 19-day
mouse foetus whose mother was
treated with 200 r. X-radiation on
the 11 th day of pregnancy. Scapula
and other long bones of the forelimb are involved in malformation
but no synostosis is seen. In the
hind-limb, the femur and tibia are
abnormal and the fibula is absent.
Hands and feet show malposition.
Abbreviations as in Text-fig. 2.
TEXT-FIG.
Groups treated with 200 r. of X-radiation
In group D, there were forty-eight cases (37 • 8 per cent.) of hypoplasia of
the scapula (Text-figs. 2 and 3). Malformation of the fore-limbs appeared in
thirteen cases (11-02 per cent.). Shortening, curvature, flexure, etc. of the bone
existed in nine cases (7-09 per cent.). In four cases (3-15 per cent.) the shortening 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.) of
malformation 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
556
U J I H I R O M U R A K A M I et al
malformations due to the absence, flexures or curvatures of the tibia or fibula
were detected in three cases (2 • 36 per cent.) (Text-fig. 2). Further, there were
eleven cases with volar flexure of the foot with or without extension of the hip
joint, many of them along with the above bone malformations.
In the group treated on the 11th day of pregnancy, there were some cases
with digital malformations. Only one case of polydactylia and two cases of
polysyndactylia could be found in the fore-limbs. In the hind-limbs, a tendency
to 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 were
detected.
TEXT-FIG. 4. A line drawing of the outline of the
skeleton of a 19-day mouse whose long bones of the
extremity 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 the
most frequent and typical malformation, and the incidence in the fore-limb
was very high (101 cases or 63 -33 per cent.), whereas in the hind-limb the same
malformation occurred only in thirty-three cases (22 per cent.). In the forelimbs, there were eight cases (5 • 33 per cent.) of syndactylia and one case of
polysyndactylism (Table 6). Reductions in the number or size of the digits and
the mechanism will be discussed later.
In group D there were twenty cases of microcephalia (15-75 per cent.) and
fourteen cases (11 -02 per cent.) of hydrocephalic conditions, while in group E
the cases of microcephalia were very few, the incidence of hydrocephalus,
however, was increased (forty-nine cases or 32-67 per cent.). In group D, few
cases of micro- or anophthalmoses were detected, in group E, however, cases
CONTROL
9 05
181
20
19
10-50%
16
9-41%
26
14-86%
39
24-38%
162
157*
150*
127*
109*
137*
42
23-6%
45
30-20%
143
Foetuses
examined
26
15-38%
Foetal
deaths
including
placental
remnants
* Some dead ones are included.
8-50
170
20
13th
F
8-75
175
20
12th
E
800
160
20
11th
D
7-45
20
10th
C
8-45
149
20
9th
B
169
Mean
litter
size
8-90
20
8th
A
Total
number of
foetuses
178
No. of
mice
treated
Day of
pregnancy
when
treated
Groups of
animals
classified
by the day of
treatment
162
100-00%
124
78-98%
19
12-67
64
50-39%
45
41-21%
61
44-53%
114
79-72%
Foetuses
without
gross
malformations
Pregnancy status of mice treated with 200 r. X-radiation at different stage of pregnancy
TABLE 4
0
33
21-02%
131
87-33%
63
49-61%
64
58-72%
76
55-47%
29
20-28%
Foetuses
with
gross
malformations
CO
C
O
W
H
W
00
*—1
(-1
o
w
H
X
%
»—i
•25
o
H
o
UJIHIRO MURAKAMI et a!
558
TABLE 5
Groups of
animals
classified
by the day of
treatment
Day of
Expregnancy
encephalia
when
Acephalia
treated
Brain
hernia
Hydrocephalus
9
6-29%
3
2-1%
9
6-57%
Microophthalmos
Anophthalmos
0
1
0-7%
13
9-09%
2
1-46%
0
35
25-55%
Microcephalia
A
8th
2
1-4%
B
9th
10
7-3%
C
10th
0
1
0-92%
17
15-6%
1
0-92%
5
4-59%
9
8-26%
D
11th
0
4
3-15%
14
11-02%
20
15-75%
4
3-15%
9
7-09%
E
12th
0
1
0-67%
49
32-67%
2
1-33%
49
32-67%
29
19-33%
F
13th
0
0
0
0
1
0-64%
0
0
0
0
0
0
0
CONTROL
10
7-3%
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 were
detected. Tail abnormalities, such as curvature or flexures with or without
shortening, existed in twenty cases (15-75 per cent.) in group D, as compared
to 106 cases (70-67 per cent.) in group E. Malformations of the extremities
were sometimes associated with micro- or hydrocephalic conditions or cleft
palates. In addition to the peculiar appearance of the head, due to a microor hydrocephalic condition, the shape of the snout was also abnormal, the
neck was short and thick, and the whole external appearance of these
foetuses was peculiar. The above malformations were often associated with
cleft palates and reductions of the eye and tail, abnormalities due to X-radiation
at the same critical stage (Table 5).
Groups treated with 150 r. of X-radiation
As shown in Table 9, there were only two cases (1 -44 per cent.) in group D
in which the fibula was absent, while in group E there were several cases of
reduction in the number or size of the digits. However, the incidence was
lower than in the 300 r. and 200 r. experimental series. All the other malformations were also less frequent, as shown in Tables 8 and 9.
M A L F O R M A T I O N IN THE MOUSE FOETUS
559
Classification of malformations (200 r.)
Extremity
malformation
Harelip \
Cleft
palate
Abdominal
hernia
Tail
abnormality
2
1-4%
5
3-5%
6
4-2%
8
5-59%
3
2-1%
6
4-2%
81
56-64%
2
1-46%
8
5-84%
13
9-49%
1
0-73%
0
6
4-38%
84
61-31%
0
0
0
5
4-59%
0
53
48-62%
12
11-01%
64
58-72%
4
3-15%
6
4-72%
0
37
29-13%
0
20
15-75%
22
17-32%
60
47-24%
3t
0
0
44
29-33%
0
106
70-67%
117
78-00%
65
43-33%
0
0
0
8
5-10%
0
24
15-29%
13
8-28%
10
6-37%
0
0
0
0
0
Tapered
snout
Abnormal
mandible
3
2-1%
34*
24-82%
2-0%
* Fused premaxillae 44 (32-12%).
0
0
Vertebral
malformation
9
5-56%
f Abnormal nasal bone.
Now, some interesting points on reduction in number and size of digits
and the mechanism by which X-radiation acts will be made. In the forelimbs, ectrodactylism most frequently affected the second digit, many of which
lacked ossification in part of the metacarpal bone. The group treated on the
13th day of pregnancy showed only syndactyly and none had osseous
syndactylies.
In both fore- and hind-limbs, ectrodactylias were the predominant digital
abnormalities, in the hind-limb, however, the predominant site was in the third
digit. There were few cases of polydactylias, polysyndactylias and the so-called
'annular grooves'. The fourth digit was the most resistant to malformation
both in fore- and hind-limbs.
As to the mechanism forming digital reductions, no digital radiations could
be observed in the hand plate at the 12th-day stage. Twelve hours after
X-radiation, findings that suggested digital abnormalities were already present,
mostly in the parts corresponding to the second digit and sometimes also in
the parts corresponding to the fifth digit. After 24 hr., a marked absence of
the second or fifth digital radiation was noticed. In preparations taken at the
15-day stage, i.e. 72 hr. after X-radiation, absence or reduction in size of the
second 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-hand
plate of a mouse embryo 2 hr. after X-radiation of 200 r. The arrow indicates
an evident focus of cellular necrosis localized in the radial side. In other
UJIHIRO MURAKAMI et al
560
TABLE 6
Groups of
animals
classified
by the day of
of treatment
Day of
pregnancy
when
treated
8th
Fore-limbs
Scapula
Volar
malflexion
of
formation
the hand
0
0
Split
hand-like
state
0
Syn' dactylia
Ectrodactylia
0
0
9th
0
1
0 •92%
0
0
0
0
4
3 •15%
0
0
3
2-36%
9
7-09%
1
0-67%
0
0
8
5-33%
0
0
7
0
0
0
0
0
0
0
0
0
c
10th
D
11th
48
37-80%
E
12th
F
13th
CONTROL
Bone
abnormalities
0
101
67-33%
0
cases, foci of cellular necrosis are evident on both the radial and ulnar sides
of the hand and foot plate. In some cases, besides the above findings, there is
also a focus of cellular necrosis at the base of the hand and foot plate corresponding to the third phalanx and foreshadowing its shortening. In a similar
preparation, taken 6 hr. after X-radiation of 200 r., such necrotic cells were
detected in profusion (Plate 1, Figs. 3 and 4). After 24 hr., the concentration of
pre-cartilage cells to form the digital radiation became evident everywhere
except in those parts involved in necrosis. At this stage, nuclei involved in
pyknosis or karyorrhexis were almost eliminated.
In other cases, various transitional forms of developing ectrodactylias
were present as shown in the figures indicating the condition on the 17th and
19th day. The distal part of the second digit, for example, moves towards the
radial side, until it fuses with the first digit and, by resorption, is gradually
reduced to a rudimentary condition. The detail of the whole process will be
reported by Nogami elsewhere.
COMMENT
In this paper some malformations of the extremities in foetuses of female
mice exposed to X-radiation, at certain developmental stages, are discussed.
However, some hereditary reduction malformations of the extremities in the
mouse are also known. Rabaud (1914) described a stock of mice called 'luxate',
and Carter (1951) gave a detailed description of the trait. Landauer (1953) reported a malformed hereditary condition in mice called' brachypodia'. Recently,
Gleucksohn-Waelsch et al. (1956) described a hereditary strain in mice having
peculiar shortening of the extremity called 'phocomelic'.
MALFORMATION IN THE MOUSE FOETUS
561
Classification of extremity malformations (200 r.)
Hind-limbs
Pelvic
girdle
malformation
Volar
flexion of
the foot
Malformed
foot
Split
foot like
state
Polydactylia
Syndactylia
Ectrodactylia
Bone
abnormalities
0
3
2-10%
0
0
3
2-10%
1
0-7%
0
0
0
4
2-92%
0
0
1
0-73%
0
1
0-73%
0
0
8
7-34%
1
0-92%
0
0
0
0
0
0
0
1
0-79%
2*
1-58%
0
8
3-6%
23
1811%
11
8-66%
6
3-97%
7
4-67%
3
2-00%
0
3
2-00%
1*
0-67%
0
1
0-64%
1
0-64%
4
2-55%
0
0
0
0
0
0
0
0
0
0
0
0
* Polysyndactylia.
33
22-00%
4
2-67%
In luxates, in addition to limb malformations, a urogenital syndrome is seen
in homozygous individuals. In phocomelics, the head is small and the palate
is usually cleft, in association with malformations of the nasal and premaxillary bones, in almost all individuals.
It is noteworthy that cases of X-ray malformations of the extremities are
associated with an abnormally shaped head, a microcephalic or hydrocephalic
condition, peculiarly shaped snout or cleft palate, etc. Such multiple malformations produced by X-rays can be explained by the common onset of these
malformations at the same critical stage.
Experimental production of extremity malformations, other than by Xradiation, was carried out in pregnant rats or mice by means of a rachitogenic
diet (Warkany & Schraffenberger, 1944) and by riboflavin deficiency in
pregnant rats (Warkany, Nelson & Schraffenberger, 1943, Giroud, Lefebvres
& Prost, 1953). Kalter & Warkany (1957) produced such malformations
in mouse foetuses from mothers fed with a galactoflavin-containing, riboflavin
deficient, diet. Asling, Nelson, Wright & Evans (1955) also described a similar
kind of malformation in rats induced by pteroylglutamic acid deficiency. Other
extrinsic factors also interfered with the differentiation of the extremities in
embryos and caused malformations whose details were discussed by Millen
(1962).
Recently, it has been reported that thalidomide taken during early stages of
pregnancy caused reduction malformations of the extremities in human beings.
Lenz & Knapp (1962) noticed amelia, phocomelia, absence of the radius and
562
U J I H I R O M U R A K A M I et al
thumb, ectrodactylias etc. in the upper-limbs, and frequent amelia, polydactyalia etc. in the lower-limbs. However, they stated that it was not clear
whether there were also cases of clubfoot or congenital dislocation of the hip,
as such malformations were not specifically listed. Since 1961 many other
investigators have reported such cases too.
Lenz & Knapp stated that in human embryos the critical stage for severe
malformations of the extremities caused by thalidomide, such as micromelia
or 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 and
33rd day of pregnancy, malformations were localized in the upper limbs, while
if it had been taken at a later stage, the malformation occurred in the upperlimbs 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 the
27th to the 40th day after conception. In the authors' present experiments,
X-radiation induced extremity malformations in mouse foetuses, such as
shortenings, became manifest after treatment on the 11th day, while digital
malformations, the reduction of number and size of the digits, became manifest
after 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 33rd
or 35th day in human embryos, a fact that is in accordance with the critical
stage for thalidomide extremity malformations indicated by Lenz & Knapp.
It was said that the pattern of malformations caused by X-radiation resembles
the one produced by riboflavin deficiency but with some additional skeletal
changes, such as humero-radial synostosis which were not found in the riboflavin deficiency cases (Warkany, Nelson & Schraffenberger, 1943; Warkany
& Schraffenberger, 1947). Another interesting fact is that thaUdomide also
interferes 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-radiation
parallel to the palmar plane. At the base of the hand plate there are three foci of cellular
necrosis, 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 parts
of the first or second digit are clearly defective.
(b) Twenty-four hours after 200 r. X-radiation (13th-day stage). The digits are defective
in similar parts to those shown in (a).
(c) Seventy-two hours after 200 r. X-radiation (15th-day stage). Ectrodactylism is
evident.
(d) A hand plate of a control foetus on the 13th-day.
(e) A hand plate of a control foetus on the 15th-day.
Vol. 11, Part 3
/ . Embryol. exp. Morplt.
5c
PLATE 1
UJIHIRO MURAKAMI et al.
{Facing page 562)
8th
9th
10th
11th
12th
A
B
C
D
E
CONTROL
Day of
pregnancy
when
treated
Groups of
animals
classified
by the day of
treatment
20
20
181
162
154
162
20
20
164
173
9 05
81
7-7
8-1
8-2
8-65
Mean
litter
size
10-50%
19
7-41%
12
10-39%
16
14-20%
23
21-95%
36
40
23-12%
* Some dead ones are included.
Total
number of
foetuses
20
20
No. of
mice
treated
Foetal
deaths
including
placenta
remnants
162
151*
139*
143*
136*
134*
Foetuses
examined
10000%
162
67-55%
102
78-42%
109
72-03%
103
73-53%
100
64-93%
87
Foetuses
without
gross
malformations
Pregnancy status of mice treated with 150 r. X-radiation at different stage of pregnancy
TABLE 7
0
49
32-45%
30
21-58%
40
27-97%
36
26-47%
47
35-07%
Foetuses
with
gross
malformations
H
oPI
Hrl
in
PI
c
o
PI
X
H
1—1
z
H
i—i
O
i ^
o
<*"
UJIHIRO MURAKAMI et al
564
TABLE 8
Groups of
animals
classified
by the day of
treatment
Day of
pregnancy
when
treated
A
Exencephalia
Acephalia
Brain
hernia
Hydrocephalus
Microcephalia
Microophthalmos
Anophthalmc
8th
6
4-48%
13
9-70%
11
8-21%
1
0-75%
22
16-42%
17
12-69%
B
9th
4
2-94%
2
1-47%
4
2-94%
0
6
4-41%
14
10-29%
C
10th
0
0
15
10-49%
0
4
2-80%
0
D
11th
0
0
18
12-95%
0
5
3-6%
0
E
12th
0
0
0
0
10
6-62%
0
0
0
0
0
CONTROL
0
0
In another series of experiments the authors (1963) gave an injection of
0-1 cc. of a 2 per cent, trypan blue aqueous solution to mice in early stages of
pregnancy. This produced only one case of shortening of humerus, with absence
of the radius and ulna and ectrodactylism, in the group treated on the 10th day
of pregnancy, while in a second series of experiments (1962) mice in their 10th
day 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 group
treated with X-rays. The critical stage for malformations of the limbs occurs
1 day, or for digital malformations 2 days, later when trypan blue injection or
hypoxia are used. The effect of X-radiations upon the anlage of the extremity
may be considered as direct in contrast to that of agents such as trypan blue or
hypoxia. It may be presumed that the critical stages for certain malformations
may differ according to the agent employed.
In Russell & Russell's experiments (1954), employing X-radiations upon the
mouse, the findings were quite similar to those revealed in the authors' present
experiments. 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 investigators
subjected rats to X-radiations on the 13th day of pregnancy, a stage corresponding 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 dense
mesenchyme in the bases and at the distal end of the limb buds was severely
MALFORMATION IN THE MOUSE FOETUS
565
Classification of malformations (150 r.)
Tapered
snout
Abnormal
ear
Abnormal
maxilla
and
mandible
Harelip
Cleft
palate
Abdominal
hernia
Tail
abnormality
Extremity
malformation
Vertebral
malformations
1
1
0-75%
8
5 •97%
10
7-46%
6
4-48%
1
0-75%
3
2-24%
85
63-43%
3
2-21%
It
4
2 •94%
6
4-41%
2
1-47%
2
1-47%
4
2-94%
73
53-68%
1
0-71
0
0
0
2
1-40%
0
26
18-18%
5
3-50%
40
27-97%
0
0
2
1-44%
0
4
2-88%
0
5
3-6%
6
4-32%
27
19-42%
0
0
0
0
14
9-27%
0
11
7-28%
35
23-18%
11
7-22%
0
0
0
0
3*
2-24%
21*
15-44%
4f
2-94%
0
0
0
0
0
* Fused premaxillae. t Abnormal nasal bone. J Agnathia.
damaged in rat embryos. However, in the authors' present findings, a cystic
defect in the centre of the limb bud could not be detected, as shown in their
results.
As the formation of digital reductions was followed up, no tendency to
repair, but only a regressive change, was observed. Hicks et al. also stated
that after severe destruction of the mesenchyme the part involved could not be
compensated for. This is in evident contrast to the findings in neural tissue in
which an active process of repair takes place. Therefore it may be assumed
that the behaviour of the mesenchymal tissue after exposure to X-radiations is
different from that in the neural tissue.
SUMMARY
1. Different groups of the ddN strain mice were exposed to X-radiation of
300 r., 200 r. and 150 r. from the 8th to 13th day of pregnancy. In offspring
from mothers treated on the 11th day, malformations of the extremities
were observed in groups treated with over 200 r. Scapular hypoplasia and
shortening of the fore-limb, as a result of absence, synostosis, shortening,
flexure or curvature of the limb bone, etc., were typical malformations. However, in the group treated with 150 r. of X-radiations the incidence of such
malformations was very low.
2. In offspring from mothers treated on the 12th day, digital reductions, on
account of ectrodactylias, were the typical malformation in the fore-limb.
Ectrodactylia was dominant in the second digit of the fore-limbs, but involve-
0
4
2-88%
0
0
10th
11th
12th
C
D
E
CONTROL
8
5-30%
0
9th
B
0
0
8th
0
0
0
0
Syndactylia
A
Scapulae
malformation
Day of
pregnancy
when
treated
Groups of
animals
classified
by the day of
treatment
Fore-limbs
* Partial defect.
0
9
5-96%
0
1
0-70%
0
0
Vestigial
1 st digit
0
0
2
1-32%
3
2-16%
2
1-40%
0
0
0
0
2
1-32%
0
0
1-44%
1
0-66%
2t
0
0
1
0-72%
0
0
0
0
0
Bone
abnormalities
0
0
1
0-75%
Position
abnormalities
of the digit
0
2
1-47%
0
1
0-75%
1
0-75%
0
Ectrodactylia
Polydactylia
t Fibula defect.
1*
0-66%
0
0
0
0
Ectrodactylia
Hind-limbs
Volar
flexion
of the foot
Classification of extremity malformations {150 r.)
TABLE 9
o
UJIHIJ
URAKAMI t
MALFORMATION IN THE MOUSE FOETUS
567
ment of the fifth digit was also seen. In the hind-limbs, the incidence was a
little lower and the predominant site of involvement was the third digit. Both
in the fore- and hind-limbs, the fourth digit was the most resistant to malformation. Other digital malformations occurred more often in the hind-limb than
in the fore-limbs, though not frequently.
3. The formation of ectrodactylias after X-radiation was studied in a
follow-up experiment. The process began with cellular necrosis in the hand
and 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 hindlimb. The process may damage the precursor of the pre-cartilage. In contrast
to the regenerative neural tissue, compensation does not take place in the
mesenchymal tissue once the destruction is severe, and the reduction of digits
will therefore persist.
4. The effect of X-radiation upon a developing digital primordium may be a
directly destructive process.
5. The critical stage for extremity malformations due to thalidomide in
human beings corresponds with the critical stage for X-ray malformations of
the extremities in mouse.
RESUME
Malformation de V extremite chez le foetus de la souris apres Virradiation de la
rneme en cours de gestation
1. Differents groupes de la souche de Souris ddN ont ete exposes a des doses
de 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 i e jour, on observe des cas de malformation de membres, quand la dose est superieure a 200 r. Les malformations
typiques de ce groupe ont ete l'hypoplasie de l'omoplate et le raccourcissement
des membres, resultant d'absences de synostoses, de raccourcissement, de
flexion ou de courbure des os des membres. Par contre dans le groupe traite
par des radiations de 150 r., la proportion de malformations est tres basse.
2. Chez les meres traitees au 12e jour, les embryons montrent des reductions
de doigts, en rapport avec l'ectrodactylie, comme malformation typique du
membre anterieur. L'ectrodactylie affecte surtout le 2e doigt des membres
anterieurs, mais elle peut aussi concerner le 4e doigt. Dans les membres posterieurs, la proportion de ces malformations est un peu plus faible et sa localisation affecte surtout le 3e doigt, aussi bien dans les membres anterieurs quedans
les membres posterieurs, le 4e doigt est le plus resistant a la malformation.
D'autres malformations digitales se manifestent plus souvent dans le membre
posterieur que dans le membre anterieur, mais moins frequemment.
3. La genese de l'electrodactylie a ete etudiee au cours d'observations suivant
de pres l'experience d'irradiation. Le premier processus est une necrose cellulaire dans la palette de la main et du pied: elle correspond aux localisations
568
UJIHIRO MURAKAMI et al
predominantes de l'electrodactylie, c'est-a-dire principalement dans le second
doigt du membre posterieur. II peut en resulter une deficience du precurseur
du precartilage. En opposition avec la regeneration du tissu neural, il n'y a
pas de reparation du tissu mesenchymateux: la destruction est considerable et
la reduction des doigts definitive.
4. L'effet de l'irradiation aux Rayons X sur l'ebauche d'un doigt en voie de
developpement est un processus de destruction directe.
5. Le stade critique pour les malformations des membres sous rinfluence de
la thalidomide dans l'espece humaine correspond au stade critique permettant
d'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 of
Education of Japan.
REFERENCES
C. W., NELSON, M. M., WRIGHT, H. V. & EVANS, H. M. (1955). Congenital skeletal
abnormalities in fetal rats resulting from maternal pteroylglutamic acid deficiency during
gestation. Anat. Rec. 127, 775-99.
CARTER, T. C. (1951). The genetics of luxate mice. I. Morphological abnormalities of
heterozygotes and homozygotes. / . Genet. 50, 277-99.
GIROUD, A., LEFEBVRES, J. & PROST, H. (1953). Anomalies des membres chez les foetus de
rat par carence en riboflavine. Arch. Anat. micr. Morph. exp. 42, 41-8.
GLUECKSOHN-WAELSCH, S., HAGEDORN, D. & SISKEN, B. F. (1956). Genetics and morphology
of 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 in
the nervous system, eye, and mesenchyme of the mammalian embryo after radiation
injury. Amer. J. Path. 33, 459-81.
KALTER, H. & WARKANY, J. (1957). Congenital malformations in inbred strain of mice
induced 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 by
trypan 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 response
of the developing mouse embryo. /. cell. comp. Physiol. Supple. 1, May 1954, 104-47.
ASLING,
MALFORMATION IN THE MOUSE FOETUS
569
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.
RUSSELL,
{Manuscript received 13th February 1963)