J. Embryol. exp. Morph. Vol. 23, 1, pp. 213-218, 1970
213
Printed in Great Britain
The effect of trypan blue on the growth of the
rat embryo in vivo
By C O L I N L. BERRY 1
From the Department of Morbid Anatomy, Institute of Child Health,
University of London
In a previous paper (Berry, 1968) it has been demonstrated, by comparison of
in vivo and in vitro growth of the rat foetus, that it is possible to dissociate growth
and differentiation in the rat. In a series now exceeding 1000 embryos it is
evident that for a given somite number the protein content of an animal developing in vitro might be less than half that of the normal control, suggesting
that a considerable reduction in growth rate might be found without necessarily
inducing death or malformation. The rate of somite formation is not reduced
in vitro in the rat; it has been shown by Herrman & Schultz (1958) that the
rate of somite formation is not interfered with by different expiant conditions
in the chick embryo. These findings suggest that the increase in somite number is
more rigidly determined than the increase in growth rate.
In an attempt to determine whether other methods of interfering with growth
alter the rate of somite formation, the effects of trypan blue on the growth and
development of the rat embryo in vivo were examined.
The dye is localized in the yolk sac after maternal injection (Wilson, Shepard
& Gennaro, 1963) and at this site has an inhibitory effect on lysosomal hydrolytic
enzymes in the cells of this membrane (Beck, Lloyd & Griffiths, 1967). The
compound is ineffective after the 10th day of pregnancy (Wilson, Beaudoin &
Free, 1959)—counting the day on which a vaginal plug is found as day 1—
presumably due to the development of effective placentation at this stage in the
rat. Since foetal death following maternal trypan blue administration is secondary to malformation (Beck & Lloyd, 1963, 1966) it appears that trypan blue
will produce malformation and death by an effect on embryonic nutrition via
the yolk sac. This argument has been subsequently extended by Lloyd & Beck
(1968).
If embryonic death and malformation are related to defects in yolk sac
function, it seems likely that all animals exposed to the compound in the
susceptible period, whether morphologically abnormal or not, should show
evidence of growth retardation. The embryos should be examined soon after
1
Author's address: Department of Morbid Anatomy, Department of Child Health,
30 Guildford Street, London W.C. 1.
14-2
214
C. L. B E R R Y
exposure to prevent masking of any inhibition of growth by a subsequent spurt.
Comparison of the rate of protein synthesis in morphologically normal and
abnormal foetuses would indicate whether it is possible to recover from impairment of growth in early development, and whether severe reduction is associated
with maldevelopment.
MATERIAL AND METHODS
Samples of trypan blue (Hopkins & Williams (H & W), British Drug Houses
(B.D.H.), Gurr, Gurr 'Vital Stain') were desalted by dialysis against water. The
dialysed solution was then evaporated to dryness under reduced pressure. An
aqueous solution of 5 mg/100 g maternal body weight of samples from B.D.H.
and H & W was found to cause resorption and malformation in the offspring of
Wistar rats when injected subcutaneously on the 7th day of pregnancy (the
morning on which a vaginal plug was found being considered to be 'day 1 ').
Using these samples, further rats were injected as before, and on the 10th,
1 lth and 12th day of pregnancy were sacrificed. In this way, adequate numbers
of animals in the range of somite numbers from 5 to 45 would be obtained. The
number of somites of the embryos present was determined, and their protein
content found by the method of Lowry, Rosebrough, Farr & Randall (1951). As
each embryo is dissolved in a standard volume of alkali in this technique,
results are expressed as fig protein/ml. Non-viable embryos, including those
with sluggish or intermittent yolk sac circulation on microscopy, were noted.
Later, certain results suggested that further animals should be examined on
day 13 of pregnancy (vide infra).
Animals were divided into 'normal' and 'abnormal' groups on the basis of
observable defects, i.e. failure of closure or abnormal shape of the neural tube,
hydrocephalus, abnormal enlargement of the pericardium, etc. However, it is
probable that animals with unobserved defects have been included in the normal
group.
RESULTS
After administration of the B.D.H. sample, 331 conceptuses were examined:
75 (22-5 %) were dead, 87 (26-4%) abnormal, and 169 (501 %) were normal.
Results for the H & W sample were 310 examined: 91 (29-4%) were dead,
58 (18-7%) were abnormal, and 161 (51-9%) were normal. A more detailed
presentation of these figures is seen in Table 1. In addition, 62 embryos were
obtained from animals examined on day 13 of pregnancy : 21 of these were dead,
three abnormal, and the remainder normal. In this group all pregnant females
had received the B.D.H. sample on day 7.
The somite numbers of morphologically normal and abnormal embryos were
compared with values derived from a control series of the same strain (see
Berry, 1968). Morphologically normal animals exhibit various somite numbers
within the normal range for days 10 and 11 of pregnancy, with some animals of
Trypan blue and embryonic growth
215
a lower than expected somite number on day 12. The impaired development of
these apparently normal embryos led to examination of animals on day 13, in
order to obtain sufficient embryos between somite numbers 40 to 45 for comparison with normals. Somite number was then plotted against protein content
for the apparently normal animals (Fig. 1—control data from Berry, 1968). The
figures for B.D.H. and H & W samples are pooled.
Abnormal embryos show a considerable reduction in the rate of development
of somites (Table 2). That growth was not entirely suspended in abnormal
embryos is shown by the finding that 10-somite animals obtained on days 10
Table 1
D u r a t i o n of
Pregnancy
(days)
Normal
Abnormal
\
Non-viable
10
11
12
68
65
36
26
23
38
21
21
33
55
69
37
24
24
10
52
16
23
169
(50-1%)
87
(26-4%)
75
(22-5%)
161
(51-9%)
58
(18-7%)
91
(29-4%)
Totals
B . D . H . Sample
H & W Sample
t
Normal Abnormal
Non-viable
2400
X
2200
x Control
0 Trypan-blue treated
2000 -
X I
1800
X
ƒ
_ 1600
|^
~|P 1400
c
'2 1200
o
/°
t.
c 1000
x
nj
<U
£
800
600
400 -
SrS>
yo
200
v**^
10
15
/
x/
x/
Yox
o0°
n0 O °
3
x
x
o
0 0
O
o
^o°°
1
o
/x
xx*x
1
1
20
25
30
Somite no.
1
1
1
35
40
45
Fig. 1. Relationship of protein content to somite number in control and test animals.
^
216
C. L. BERRY
(five animals), 11 (seven animals), and 12 (four animals) had a mean protein
content of 65, 105 and 145 /<g/ml respectively.
The change in the rate of development of somites associated with persistent
growth leads to the anomalous finding that certain abnormal animals have a
protein content greater than the normal for their somite number. This somite
number is, however, considerably lower than normal for the gestational age of
the animal concerned. In all instances where the somite number was within the
Table 2. Somite numbers of control and test animals
B.D.H.
Day
Controls
Normal
10
\l
12
13
5-20
21-34
34-45
31-52
5-15
20-31
21-43
45-52
H&W
Abnormal
2-8
3-26
3-36
26, 31, 34
Normal
Abnormal
6-15
19-30
28-31
—
4-14
2-23
15-31
Table 3. Protein content of abnormal animals within
normal range of somite numbers
Mean protein (/tg/ml)
Somite number
10
10
12
14
80
90
110
80
95
90
65(5)*
85 (4)
50(4)
20
21
23
24
26
36
230
260
400
410
460
945
160
175
220
295
380
420
155(1)
130(1)
200(1)
190(1)
185(1)
181 (1)
11
12
Controls
Morphologically
normal
Day
Abnormal
* Figures in parentheses indicate number of animals at each somite number examined.
normal range for the duration of pregnancy, the abnormal animals had a lower
protein content than controls or morphologically normal, trypan blue-treated
animals. Thus, of 50 abnormal animals found on day 10, 37 were of less than
five somites, of 47 found on day 11 only five had more than 20 somites, and of
48 abnormal animals found on day 12 only one animal had more than 34 somites.
The protein content of these animals is compared with controls and morphologically normal, trypan-blue treated animals in Table 3.
Trypan blue and embryonic
growth
217
DISCUSSION
It can be seen from Fig. 1 that for a given developmental stage morphologically normal animals exposed to trypan blue contain less protein than normal
controls. This effect becomes more marked until, around the 35 somite stage, a
rapid increase in the growth rate occurs. It is at this period that the placenta
becomes the principal source of nutrition for the rat foetus, and presumably a
defect in yolk-sac function is less significant. By the 45 somite stage the protein
content of the dye-treated animals is normal.
These findings indicate that all embryos of trypan blue-treated animals are
affected by the drug, whether morphologically abnormal or not. However, the
abnormal animals in addition show a fall in the number of somites present at a
particular time in pregnancy compared with normal controls (Table 2). This
suggests that at a certain level of growth retardation, development is interfered
with and malformation may ensue. Apparently, severe impairment of growth in
early pregnancy will not necessarily cause malformation and is, at least in part,
a reversible phenomenon.
SUMMARY
1. The effect of trypan blue on the growth of the rat embryo in vivo was
examined, using the protein content for a given somite number as an index of
growth.
2. In morphologically normal animals exposed to trypan blue, there is
evidence of growth retardation.
3. The reduction in protein content for a given somite number does not
permanently affect the capacity of the animal to grow, since when placentation
occurs the 'protein deficit' is repaired.
4. Beyond certain limits, impairment of growth in early pregnancy apparently
interferes with differentiation.
RÉSUMÉ
Action du bleu Trypan sur la croissance de
rembryon de rat in vivo
1. L'action du bleu Trypan sur la croissance de l'embryon de rat in vivo a
été étudiée en utilisant comme indice de croissance la teneur en protéines pour
un nombre donné de somites.
2. Chez les animaux morphologiquement nosaux traités au bleu trypan, on
observe un retard de croissance évident.
3. La diminution de la teneur en protéines pour un nombre donné de somites
n'affecte pas de façon permanente la capacité de croissance de l'animal, puisque,
au moment de la placentation, le 'deficit en protéines' est réparé.
4. Au-delà de certaines limites, l'altération de la croissance au début de la
gestation entrave apparemment la différenciation.
218
C. L. B E R R Y
This work is supported by the British Heart Foundation, the Central Research Fund of the
University of London, and the Mary Kinross Charitable Trust. C. L. Berry is the Giilson
Scholar of the Worshipful Society of Apothecaries of London.
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{Manuscript received 13 February 1969)