/ . Embryol exp. Morph. Vol. 25, 3, pp. 331-338, 1971
Printed in Great Britain
331
Age factor in the induced metamorphosis
of thyroidectomized tadpoles
By C. Y. HSU1, N. W. YU 1 AND H. M. LIANG 1
From the Department of Biomorphics, National Defence Medical Centre, Taipei
SUMMARY
Three age-groups of thyroidectomized tadpoles of Rana catesbeiana were treated with a
series of the same graded increasing concentrations of thyroxin. Normal froglets were formed
from the three groups at the end of the induced metamorphosis. Growth and metamorphosis
were compared. It was found that while the growth rate was a function of ageing, the pattern
and rate of metamorphosis were not affected by senile processes. Therefore a case of differentiation independent of age was presented.
INTRODUCTION
Thyroidectomized tadpoles were found to be capable of growing to a huge
size (Allen, 1918; Hoskins & Hoskins, 1919; Etkin, 1964). They exhibited
accelerated and prolonged growth after the onset of metamorphosis of intact
controls (Hsu & Yii, 1967). However, thyroidectomy did not prevent them from
growing old, for young thyroidectomized tadpoles showed a greater growth rate
than old ones and the young adapted themselves better than the aged in adverse
conditions (Hsu, Yii & Liang, 1964). Therefore senile processes continue
inevitably in thyroidless tadpoles.
Nevertheless, tissue differentiation of these tadpoles stops at the limb-bud
stage because metamorphosis never proceeds beyond that limit (Hsu et al.
1964; Hsu & Yu, 1967). This intriguing condition poses two questions yet to be
answered: are tissues of the aged thyroidless tadpoles still capable of differentiation if treated with thyroxin? how does the competence of old tissues compare
with that of the young when reacting to the hormone? The present study aims to
answer these questions by comparing growth and metamorphosis of three agegroups of thyroidless tadpoles induced by thyroxin treatment.
MATERIALS AND METHODS
Larvae of Rana catesbeiana raised from fertilized eggs produced by induced breeding were surgically thyroidectomized at the gill-circulation stage.
The successfully thyroidectomized tadpoles appeared normal and remained at
1
Authors' address: Department of Biomorphics, National Defence Medical Centre,
Taipei, Taiwan, Republic of China.
B
16
15
15
15
15
15
12
10
10
10
10
10
38
A
20
20
20
20
20
20
20
17
17
17
17
17
15
A
B
Average metamorphic stage
,
C
12
III 0 ± 0
III0±0
III0 ± 0
10
X 7±0-16f
IX-6 ± 0-30
X-5±012
7
XI •8 ±007
XI-1 ± 0-36
XII0±019
5
XV •7 ±019
XV-0 + 0-05 XIV-6 ± 0-25
5 XVII •7 ±017 XVII-4±0-28 XVII-0 ± 0-75
5 XVIII •4 ±0-21 XVIII-3±0-07 XVIII-5±041
5 XIX •2 ±008 X I X 0 ± 0 0 7 XIX-0 ± 0-40
XX-0 + 0-10
4
XX •2±016
XX 0 ±015
4 XXII 3 ±016 XXII0±0-29 XX1I-2 ± 0-30
XXIII0±0
XXllI-0 ± 0
4 XXIII 0 ± 0
XXIV0±0
4 XXIV 0 ± 0
XXIV-0±0
XXV0±0
4 XXV 0 ± 0
XXIV-8 ± 0-28
67
c*
No. of tadpoles
Appearance of hind-limb bud
Growth of hind-limb bud
Growth of hind limb
Growth of hind limb
Growth of hind limb
Cloacal tail piece gone
Clear skin window
Emergence of forelegs
Widening of mouth and tail resorption
Widening of mouth and tail resorption
Tympanum recoenizable
Tail gone
Metamorphic pattern
(Similar for all groups)
* A, 6-month-old tadpoles; B, 18-month-old tadpoles; C, 30-month-old tadpoles.
t Standard error of the mean.
T4 cone.
(/*g/I.)
Onset of treatment
1
20-3 0
2
3-5
3
40
4
50
5
50
5
5-5
6
60
6
6-5
7
70
8
8-10
10-20
9
Mortality %
T 4 treatment
(months)
Table 1. Progress of induced metamorphosis in three age-groups of thyroidless tadpoles treated with thyroxin
a
X
o
U)
Age and induced metamorphosis in thyroidectomized tadpoles 333
the hindlimb-bud stage (stage III) after the intact controls had metamorphosed.
Three different age-groups of thyroidless tadpoles were thus maintained:
the first group, 6 months old, averaged 1-94 g in body weight and 5-5 cm in total
length; the second group, 18 months old, 6-80 g in body weight and 7-4 cm in
total length; the third group, 30 months old, 15-80 g in body weight and 11-9 cm
in total length. They were treated with a series of the same graded increasing
concentrations of L-thyroxin sodium (General Biochemical Inc.) for more
than 8 months until they became froglets (Table 1). The initial dose was 2/*g
of the hormone per litre of aquarium water. The dosage was increased gradually
to a final concentration of 20 /eg/1. A fourth group often 18-month-old thyroidless tadpoles without thyroxin treatment served as the controls.
All tadpoles were kept in an identical laboratory environment: water temperature at 20 ± 1 °C, same space factor, and 10 h of incandescent illumination each
day. The aquarium water was changed every other day and new thyroxin was
added. The tadpoles were fed thawed leaves of water-convolvulus.
The pattern of metamorphosis of the tadpoles, their progress in metamorphic
stage (Taylor & Kollros, 1946), total length, hind-limb length and body weight
were recorded monthly.
RESULTS
Growth
The 18-month-old thyroidless tadpoles without thyroxin treatment grew to a
huge size as usual (Hsu et al. 1964; Hsu & Yii, 1967). At the end of the 8th
month after the commencement of this study they attained 14-50 g in body weight
and 11-5 cm in total length.
However, this pattern of continued growth was not observed in the three
groups of thyroxin-treated tadpoles. They grew very slowly, and beginning from
the 5th month diminution was seen as indicated in Figs. 1 and 2. Because of the
unequal sizes in the different age-groups it was rather difficult to compare their
growth rates directly from the growth curves. Therefore the curves of Figs. 1
and 2 were transformed by graphical measurements of the values of the tangents
of all points on the curves divided by the body weight or total length. Thus the
curves of relative growth rate in Figs. 3 and 4 were obtained by dWjdt x 1/ W or
dL/dtx \\L, where W, L, and t stand for body weight, total length and unit of
time respectively.
Figs. 3 and 4 show a comparable tendency of decreased growth rate in the
body weight and total length for all three groups. However, the amount of
decrease varied proportionately with the age. The older tadpoles gained their
body weight and total length more slowly and lost their growth more quickly
than the young ones. Yet the zero growth rate for all groups was found at the
same time, after 4 months of hormone treatment.
The effect of age on growth was further observed in the ratio of hind-limb
334
C. Y. HSU, N. W. YU AND H. M. LIANG
length over body length (Fig. 5). It was obvious that the three groups of metamorphosing tadpoles had their own ratios, the magnitude of which was inversely
proportional to their ages.
The results indicate that, after thyroxin treatment, the pattern of growth was
comparable for the three groups but that the rates of growth were different due
to an effect of age.
2
2
3 4
5 6 7
Months of treatment
3 4
5 6 7
Months of treatment
Fig. 2
Fig. 1
Fig. 1. Growth of body weight in three age-groups of thyroidless tapdoles after
thyroxin treatment.
Fig. 2. Growth of total length in three age-groups of thyroidless tadpoles after
thyroxin treatment.
Metamorphosis
The control group of thyroidless tadpoles remained at stage III throughout
the experimental period, while the three thyroxin-treated groups showed normal
advances and the same time scale in metamorphosis, resulting in perfectly
normal froglets as shown in Table 1 and Fig. 6. The results indicate that no
discrepancy of tissue response to thyroxin was observed in spite of age differences.
This study therefore shows differences of the effect of age on growth and
differentiation during induced metamorphosis of thyroidless tadpoles. Growth
rate was slowed down while differentiation was not affected by senile processes.
Age and induced metamorphosis in thyroidectomized tadpoles 335
2
3 4
5
6 7
Months of treatment
0
1
Fig. 3
2
3 4
5 6 7
Months of treatment
Fig. 4
Fig. 3. Relative growth rate of body weight in three age-groups of thyroidless tadpoles after thyroxin treatment.
Fig. 4. Relative growth rate of total length in three age-groups of thyroidless
tadpoles after thyroxin treatment.
20
1-8
16
1-4
•^ 1-2
j 10
.2 0-8
w
(S
0-6
0-4
0-2
2
3
4
5
6
7
Months of treatment
Fig. 5. Changes in the ratios of hind-limb length (H.L.L.) to body length
three age-groups of thyroidless tadpoles after thyroxin treatment.
(B.L.)
in
Fig. 6. Advances of metamorphosis in three age-groups of thyroidless tadpoles after thyroxin treatment (6-month-old
on the left, 30-month-old on the right and 18-month-old in between). Scale markings in both cm and in. for A-E.
(A) Before treatment
(D) After 7 | months of treatment, 8 jig/l
(B) After 4 months of treatment, 4-5 /tg/I.
(E) After 8 months of treatment, 10/fg/l.
(C) After 6 months of treatment, 6-5 /tg/1.
o
r
>
s
0
X
3
2
on
X
O
OJ
Age and induced metamorphosis in thyroidectomized tadpoles 337
DISCUSSION
The pattern of the relative growth rate of the induced metamorphosing tadpoles in this study was different from that of normal thyroidless ones in our
previous report (Hsu & Yii, 1967). In the present case the growth rate was
decreasing throughout metamorphosis, reaching negative values, whereas in
the previous study the growth rate was found to increase at first and then to
decrease, but it never reached a negative value. The difference was presumably
due to the occurrence of metamorphosis in the present case. Growth and
differentiation are always antagonistic; they seldom occur simultaneously at
the maximum rate. While differentiation is interrupted and growth dominates
in thyroidless tadpoles, the opposite is true in metamorphosing tadpoles.
However, there was a common phenomenon in the two cases: the effect of
ageing on growth. Age had an effect not only on normal growth (Hsu et al. 1964)
but also on diminution.
On the other hand the ability of tissues to differentiate was the same in all
groups; the young and the old metamorphosed alike: there was no effect of
age on differentiation.
The ability to differentiate resides in the activation of tissue genes which
control the synthesis of specific proteins. The genetic information is, in a way,
immortal; it is always there and correct unless depleted or mutated. During
development, genes are switched on and off. In thyroidless tadpoles the genes
for metamorphosis are not activated because of lack of thyroxin. Whenever the
cells are stimulated with the right inducer, the hormone, differentiation is
resumed. This study therefore presents a case of differentiation independent of
age.
Our finding on delayed differentiation agrees with the result of Prahlad &
DeLanney (1965). They found that all age-groups of the genetically thyroxindeficient Mexican Axolotl (Siredon mexicanwri), ranging from 3 to 571 days
old, showed the same capacity to metamorphose when treated with T3 or T4.
In the case of Drosophila melanogaster, Hadorn (1965, 1966) and Schubiger &
Hadorn (1968) demonstrated that imaginal discs of genitalia and leg, cultivated
for years without differentiation in the abdomens of adults, were able to form
normal adult structures when transplanted back into metamorphosing hosts.
Therefore it appears that the retention of the capacity to differentiate until
triggered by the right stimulus is common to all animals.
It was Etkin (1935) who first attempted to induce thyroidless tadpoles of
Rana cantabrigensis into metamorphosis with thyroxin administered in the
ambient water. By gradually increasing the hormone concentration from 1 /*g
to 1 mg per litre he obtained approximately normal froglets. However, his
experiment was terminated by death of the animals. Later, the same author
reported that thyroidectomized or hypophysectomized tadpoles of Rana pipiens
were able to metamorphose normally with a patterned sequence of increasing
338
C. Y. HSU, N. W. YU AND H. M. LIANG
thyroxin concentrations (Etkin, 1964) although he did not mention the exact
dosage.
The final concentration of thyroxin in this study was 20 /^g/1. at 20 °C, which
was far below that of Etkin's for normal tadpoles. The discrepancy was due
mainly to the duration of hormone treatment. Etkin's administration of thyroxin
was an acute treatment whereas ours was a chronic one.
The thyroidless froglets obtained in this study were not only normal morphologically but also physiologically. The 30-month-old group grew and attained
an average weight of 10-5 g with an increment of 34-5 % over the initial body
weight in a period of 2 months after the end of the induced metamorphosis.
In this experiment, some tadpoles could not stand the thyroxin treatment and
succumbed during the induced metamorphosis. As shown in Table 1, the mortality rate of the three groups varied according to their age. This was another
manifestation of senile processes.
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ALLEN,
{Manuscript received 25 June 1970)