/ . Embryol. exp. Morph., Vol. 16, 3, pp. 487-496, December 1966
Printed in Great Britain
487
Thyroid growth and its relationship to
metamorphosis in Rana temporaria
By HAROLD FOX 1
Department of Zoology and Comparative Anatomy, University College, London
INTRODUCTION
In a previous work on pronephric degeneration, larvae of Rana temporaria
from practically the whole range of the metamorphic cycle were studied (Fox,
1962). Metamorphosis in anurans is dependent upon the presence of a functional
thyroid gland (Kollros, 1951; Lynn & Wachowski, 1951; Etkin, 1964), and the
same specimens have now been used to investigate thyroid growth and development quantitatively and qualitatively, especially in relation to those major
events which occur at the metamorphic climax between stages 49 and 54 (Cambar
& Marrot, 1954).
It will be shown that in larvae from 16 mm long (stage 41; see Fox, 1962) until
practically the end of the climax (stage > 53), thyroid cells are of stable volume
(about 700 /A3), and thyroid enlargement is by cellular proliferation and vesicular expansion. The maximum rate of thyroid growth occurs between 16 mm
and 28 mm (stages 41-45).
MATERIAL AND METHODS
Thyroid growth was investigated from stages 29 to 54 inclusive in Rana
temporaria (the stages were judged by those described for R. dalmatina, Cambar
& Marrot, 1954). Specimens were serially sectioned transversely at 10/* and
prepared by standard methods. The thyroid of an additional animal of stage 52
(almost identical with the other same-numbered stage) was also measured, and
other animals of stages 29 and 54 were used. The rate of development is altered
by such factors as crowding, feeding and temperature, and the following information on time/stage relations is consequently only approximately true. At
temperatures between 16 and 20 °C stage 26 is achieved by 7 days, stage 30 by 10
days, stage 33 by 15 days, stage 40 by 26 days, stage 42 by 31 days, stage 45 by 42
days, stage 48 by 50 days, stage 49 by 60 days and stage 54 by 70 days.
Quantitative methods were the same as those used for the pronephros (Fox,
1956, 1962). Mean and standard error of the length of 150 thyroid nuclei
(obtained from a horizontally serially sectioned specimen at stage 41, 15 mm
1
Author's address: Department of Zoology, University College London, Gower Street,
London, W.C. 1, England.
488
H. FOX
long) were 6-9 ±013/4, and 7 /i was therefore used to correct the crude thyroid
nuclear count from the transverse sections (see Abercrombie, 1946). The
nuclear length of thyroid cells therefore is about 64% of that of the pronephros
Transverse sections were projected at a magnification of x440, the most
satisfactory one for use throughout the series.
RESULTS
{a) Qualitative
At stage 29 the incipient thyroid anlage is a median, elongated, anteroposteriorly directed pharyngeal backgrowth, rounded in cross-section and
terminating behind at the region of the front of the heart. Thyroid lengths of
two specimens were 210 [i and 230 fi respectively. At stage 31 in general the
thyroid is similar in length (180 fi) and appearance to that of the previous stage,
but somewhat convex-shaped at its hind end with the convexity facing downwards, a prelude to its later division into paired lobes. In both stages the
granular tissue includes numerous yolk globules and pigment granules; cell
boundaries are hard to distinguish and nuclei are lightly stained. Many of them
include recognizable paired nucleoli and, as in all stages, only a few mitotic
figures are present. The young thyroid is situated between incipient geniocoracoidei and interhyoidei muscles, and in stage 31, below the front of the
medianly fused hyoid cartilages, behind and below the pars reuniens.
In stage 41 paired thyroid lobes (probably situated farther back than in the
previous stage) share a common medio-anterior origin (30 /<• long) below the
front of the post-copula. Behind, each separate lobe is situated lateral to
the post-copula. Incipient follicles, whose walls are of flattened epithelia, have
developed (though somewhat obscured by pigment), but there is no colloid
within them.
Thyroid lobes of stage 45 terminate anteriorly in common territory below
the free hind end of the post-copula, which is joined to the branchial plate 100 /i
farther forward. They now comprise large well-developed rounded follicles,
whose vacuoles contain conspicuously stained red colloid. A clear gap—which
may be an artifact—often exists between the periphery of the colloid and the
columnar epithelium of the follicle wall. In stage 47 each lobe is larger than in
the previous stage, and is situated medio-ventral to the hyoid; in front each lobe
ends freely against the lateral hind end of the post-copula. At the periphery
and within the colloid there are chromophobe droplets, but some vesicles are
empty.
In stage 49 and succeeding stages the thyroid progressively becomes larger.
More droplets are present in the colloid, which now in most cases completely
fills the vesicle. The columnar epithelia of the follicles are slightly deeper, and
profuse nuclei are located in their outer regions. In many cases follicles have
Thyroid growth and metamorphosis
489
become separated during development, and delicate interfollicular blood vessels
vascularize the gland.
In stage > 53 large elongated follicles extend either dorso-ventrally or
horizontally; others are partially collapsed. Of three specimens at state 54 two
possessed thyroids with colloidal droplets. One of them included typical, but
independently situated, thyroid follicles (without droplets) medio-ventrally to
the paired lobes.
Table 1. Various measurements of thyroid components in different
stages o/Rana temporaria
9
31
11
41
45
47
49
50
51
52(1,2)
>53
54
16
28
33
33
33
32
25-6
14
12-5
0-21
(median)
018
(median)
0-34
0-55
0-89
0-68
0-68
0-72
0-70
107
0-75
129
160
508
4016
7450
6992
15710
14900
12746
23543
14738
—
0035
1186
2-611
2-339
3-849
4-505
2-888
6-242
3054
X
. M C
0-400
0-400
3101
0-446
0-446
2787
0-372
2-880
5-799
4-728
12191
11-691
9-843
17-647
6-445
0-407
4066
8-410
7067
16040
16196
12-731
23-881
9-499
732
111
778
676
116
785
757
749
437
;>oT
3
Calcu!
cell vc
t;
me of col
of thyroi
oo
ual
i
Total verall vol
of the hyroid (a
and v< icles) (mn
29
-24 T> Xt
1—1
Total ol. of the
tissue ells of the
thyroi (mm3 x 1
et
o
o
Total ol. of the
colloi< vesicles o
the th roid (mm1
<-> et
Total mgth of
larvae mm)
^s o
Total uclear po ulation o thyroid
03
Total mgth of
thyroi (right an
left lo es) (mm)
Stage Jo.
(equiv lent to R. almatina,
Camb r & Marr t, 1954)
From stage 41 onwards measurements of both lobes are combined, and individual
cell volume is calculated from the totals of the nuclear populations (corrected) and
tissue volume of the whole thyroid.
GO
—
8-6
29-2
310
33-1
240
27-8
22-7
261
321
(b) Quantitative
The data obtained are summarized in Table 1, from which it will be seen that
the calculated individual cell volume of the thyroid (volume of tissue/nucleus) of
stages 29 and 31 (3100 and 2787/A3 respectively) are similar. Thereafter cell
volume is about one-quarter of this, and remains practically constant (range
676-842 /i3) until stages > 53 (Fig. 3). Thyroid cell volume is therefore considerably less than that of the pronephros of amphibians and Neoceratodus
(Fox, 1963), and of liver in practically all adult amphibians studied (Szarski &
Czopek, 1965). Diploid pronephros and duct cell volumes of young larvae of
490
H. FOX
Xenopus are about 2400 and 1350/A3 respectively, and thyroid cell volume of
Rana is about the same as that of the haploid pronephric duct of Xenopus (Fox &
Hamilton, 1964). Small cell size throughout the metamorphic cycle and thus a
high surface area per unit volume may be of importance in thyroid function.
As total thyroid tissue volume hardly changes between stages 29 and 41,
while the nuclear population of the thyroid increases by about four times, this
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Larval stages
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47 50 52 54
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37
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49 51 53
50 52 54
Larval stages
Fig. 2
Fig. 1
Fig. 1. Regression line of the total nuclear population of the combined lobes of the
thyroid (ordinate) against numbered larval developmental stages (stages 45-54
inclusive; Cambar & Marrot, 1954) (abscissa).
Fig. 2. Regression lines of the total volumes of the colloid vesicles (A), cellular
tissue (B), and of the overall volume (vesicles and cells together) (C), of the combined lobes of the thyroid (ordinate) against numbered larval developmental stages
(stages 45-54 inclusive) (abscissa).
period is predominantly concerned with the attainment of the typical larval
thyroid individual cell volume (Table 1, Figs. 1, 2, 6). Thereafter thyroid tissue
volume increase is by cell multiplication with stable cell volume.
In stage 54 cell volume of the thyroid is 437 /t3, which could represent a significant post-climatic reduction. In larval Rana temporaria whose development
was inhibited by phenyl-thiourea, thyroid individual cell volume of one control
and two experimental animals (stage 49) were 917, 920 and 920 /*3 respectively.
Thyroid growth and metamorphosis
491
At stage 54 two control and two experimental animals, which after developmental inhibition were returned to water in which they finally metamorphosed,
had thyroid cell volumes of 607, 736, 529 and 630 /iz respectively. It is possible
therefore that cell volume is slightly reduced after metamorphosis, which agrees
with the conclusion of Szarski & Czopek (1965), who found post-metamorphic
cell shrinkage as a result of decrease of water.
3200 3100 - •
3000 2900 2800
2700 2600 2500 -
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1500
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1100
900 -
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i i
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i i i i t
33
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i i 1 1 iiii
45
43
49 51 53
47 50 52 54
Larval stages
Fig. 3
i
29
i i i i
i i i
i i i 111
11
33 37 41 45 49 51 53
31 35 39 43 47 50 52 54
Larval stages
Fig. 4
Fig. 3. Regression line of the calculated individual cell volume of the thyroid
(ordinate) against numbered larval developmental stages (stages 41-54) inclusive
(abscissa).
Fig. 4. Percentage increase of nuclear population of the thyroid (ordinate) against
numbered larval developmental stages (stages 29-54 inclusive) (abscissa).
Maximum growth rate of the thyroid (expressed as percentage change) occurs
between stages 41 and 45, especially in relation to the thyroid vesicles. Thereafter the graph tends to flatten, and the rise is more gradual (Figs. 4, 5). Maximum percentage decrease in cell volume occurs between stages 31 and 41, owing
to cell multiplication with little cytoplasmic growth (Fig. 6).
In R. temporaria the end of premetamorphosis is probably equivalent to
stages 41/42 (tiny hind-limb buds visible). In the succeeding prometamorphic
stage, or actual start of the metamorphic cycle, there is extensive growth and
differentiation of the fore- and externally visible hind-limbs. The metamorphic
climax of Etkin (1964) would commence at stage 51 (emergence of both forelimbs), and ends at stage 54 with the typical frog.
The thyroid data have been expressed graphically as regression lines, whose
abscissae are the stage numbers (especially stages 45-54 inclusive, when the
492
H. FOX
most dramatic changes occur), and ordinates are the component measurements in question (Figs. 1-3).
At stage 47 the pronephric system has reached its maximum size. Thereafter
degeneration ensues—to be first recognized at stage 49—and forms part of the
programme of late prometamorphosis and climax, under thyroid control
4000
3800
3600
3400
3200
3000
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2800
I 2600
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a 1800
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Larval stages
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90
80
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40
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20
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-20
-30
-40
-50
i
29
i i i i i i i i i i
i i i
33
37
41
45
49 51 53
31
35
39
43
47 50 52 54
i
Larval stages
Fig. 5
Fig. 6
Fig. 5. Percentage increase in size of the vesicular (A), tissue (B) and overall
volumes (vesicles and cells together) (C), of the thyroid (ordinate) against numbered
larval developmental stages (stages 29-54 inclusive) (abscissa).
Fig. 6. Percentage variation in the calculated individual cell volume of the thyroid
(ordinate) against numbered larval developmental stages (stages 29-54 inclusive)
(abscissa).
(Fox, 1962, 1963, 1965). According to the regression lines at stage 49, the
thyroid nuclear population is about 10800, and vesicular, tissue and overall
thyroid volumes are 0-0028, 0-0079 and 0-0109 mm3 respectively. Thus in such a
larva of R. temporaria with a normally functioning thyroid of this size, pronephric degeneration is initiated.
At stage 50 (left fore-limb emerged) thyroidal nuclear population is about
12500, and vesicular, tissue and overall volumes are 0-0032, 0-0089 and
0-0122 mm3 respectively. At stage 51 these components measure 14180, 0-0036,
0-0099 and 0-0135 mm3.
Perhaps the most striking external climactic change is tail degeneration. In
Thyroid growth and metamorphosis
493
stage 52 it is half-degenerated. Thyroid nuclear population is about 15800 and
vesicular, tissue and overall thyroid volumes are 0-0040, 00109 and 0-0148 mm3
respectively. Finally, with the complete loss of the tail (stage 54), the same components measure 19160, 0-0045, 0-0129 and 0-0174 mm3.
DISCUSSION
In larvae of R. temporaria the thyroid increases substantially in size from
stage 41 onwards. The most rapid rate of thyroid growth occurs between stages
41 and 45, and then growth decreases gradually towards the climax, when the
gland reaches its maximum size. These results in general agree with those of
previous workers on R. temporaria and R.pipiens (Etkin, 1930; Clements, 1932;
D'Angelo & Charipper, 1939; see also Lynn & Wachowski, 1951). In Pseudacris
triseriata, Rana palustris and R. catesbeiana there is a marked acceleration of
growth at the beginning of prometamorphosis, and thyroid enlargement continues
to the climax (Etkin, 1936). In anuran tadpoles {Rana pipiens) during premetamorphosis thyroid hormone concentration in the blood is extremely low (Etkin,
1964), and the sharp rise which occurs thereafter in prometamorphosis would
seem to be related to the period of stable thyroid individual cell volume and
intense cellular proliferation, seen in R. temporaria in the present work.
In R. pipiens, from prometamorphosis to climax, the concentration of thyroid
hormone in the blood rises about 70 times (Etkin, 1964). Iodine binding increases from premetamorphosis (late Shumway stage 25, hind-limb buds present;
probably equivalent to stage 39 in R. temporaria) to climax by 40 times (Kaye,
1961), though in Xenopus from 2 to 6 weeks (beginning of climax) it increases
by only 20 times (Saxen et al. 1957). In a comparable period in R. temporaria
(stage 41 to the highest recorded tissue volume at stage > 53) thyroid tissue
volume increases by nearly 50 times. At similar stages of the cycle in Bufo bufo
thyroidal epithelial volume increases by 26 times (Morita, 1932). Pseudacris
showed similar and R. palustris and R. catesbeiana smaller increases in epithelial
volume (Etkin, 1936). Thus in various genera increases in thyroid tissue volume
and iodine utilization, through prometamorphosis to climax, differ. Likewise
thyroid size is variable, for in a number of different larval anurans, the largest
thyroids are found in the largest genera (Morita, 1932; Etkin, 1936). Furthermore, as it is well known that in different amphibian genera similar tissues may
show different degrees of responsiveness to thyroid hormone (Lynn & Wachowski, 1951), it may well be that the circulatory concentration of thyroid hormone
in different genera at similar stages is also variable. However, in most anurans,
as in R. pipiens, presumably there is a marked and rapid increase in circulatory
concentration of thyroid hormone during the metamorphic cycle. This no
doubt coincides with an increasing potential hormonal output of the thyroid,
which probably reaches its peak when the gland is at maximum size during
climax.
31
JEEM l6
494
H. FOX
Peripheral colloidal chromophobe droplets are first seen in stage 47. At the
height of the climax droplets are at their largest and most plentiful. Presumably
there is a relationship between the follicular histological appearance and the
high climactic thyroidal activity. However, follicle histology, and consideration
of the measurements of the percentage volume of the colloid vesicles of the
thyroid, do not support the results of previous workers on R. temporaria and
R.pipiens, who described colloid shrinkage at climax (Clements, 1932; D'Angelo
& Charipper, 1939). Vesicles are not collapsed and colloid is present at all stages
in larvae of R. temporaria, though during growth, follicles become more closely
packed together. The colloid vesicle percentage volume is fairly constant at
about 30 % from stage 45 to the end of climax, and if there had been a pronounced collapse of follicles and marked loss of colloid, then this measurement would be expected to fall noticeably. The results support those of Etkin
(1936), that there is no specific stage of colloidal evacuation.
During larval development the output of thyroid hormone presumably
rises steadily, as the thyroid enlarges under the influence of adenohypophyseal
TSH. At prometamorphosis and climax, the circulatory thyroid hormone
concentration is maintained through a thyrostatic mechanism or 'feed back'
system, operating on the pituitary and hypothalamus, via the thyroxine level
(see Etkin, 1964). If the control mechanism merely effects the release of limited
amounts of hormone from the colloid into the circulation, to maintain or raise
the circulatory concentration, and if thyroid hormone is synthesized continuously to replace the deficit from the vesicles, then this would account for
the failure to observe pronounced vesicular contraction at climax.
In Eleutherodactylus where there is no marked stage of colloid storage or
release, it is assumed that hormone is released from the gland as quickly as it is
formed, in order to elicit the 'telescoping' of larval stages, which is characteristic
of its direct embryonic development (Lynn & Wachowski, 1951; Lynn & Peadon,
1955).
SUMMARY
1. Thyroid growth of larvae of Rana temporaria was investigated in relation
to specific stages (29-54 inclusive; see Cambar & Marrot, 1954) and events
in the metamorphic cycle.
2. Growth is by cellular multiplication and vesicular enlargement, and from
stage 41 (16 mm larva) to almost the end of metamorphosis cell volume is
constant.
3. Maximum rate of growth in vesicular, tissue and overall volumes occurs
between stages 41 and 45 (16 and 28 mm) respectively.
Thyroid growth and metamorphosis
495
RESUME
La croissance de la thyroide et ses rapports avec la metamorphose
chez Rana temporaria
1. On a examine la croissance de la thyroide de larves de Rana temporaria en
rapport avec les stades specifiques (29 a 54 inclus; voir Cambar et Marrot, 1954)
et les evenements du cycle metamorphotique.
2. La croissance a lieu par multiplication cellulaire et elargissement des
vesicules, et le volume cellulaire est constant depuis le stade 41 (larve de 16 mm),
presque jusqu'a la fin de la metamorphose.
3. Le taux de croissance volumetrique maximale pour les vesicules, le tissu
et l'ensemble, est atteint entre les stades 41 et 45 (16 et 28 mm) respectivement.
My thanks are due to Professor M. Abercrombie and Professor D. R. Newth for valuable
advice in the preparation of this work.
REFERENCES
M. (1946). Estimation of nuclear populations from microscopic sections.
Anat. Rec. 94, 239-47.
CAMBAR, R. & MARROT, Br. (1954). Table chronologique du developpement de la Grenouille
agile {Rana dalmatina Bon.). Bull. biol. 88, 168-77.
CLEMENTS, D. I. (1932). Comparative histological studies of the thyroids and pituitaries in
frog tadpoles in normal and accelerated metamorphosis. /. R. micr. Soc. 52, 138-48.
D'ANGELO, S. A. & CHARIPPER, H. A. (1939). The morphology of the thyroid gland in the
metamorphosing Rana pipiens. J. Morph. 64, 355-73.
ETKIN, W. B. (1930). Growth of the thyroid gland of Rana pipiens in relation to metamorphosis. Biol. Bull. mar. biol. Lab., Woods Hole 59, 285-92.
ETKIN, W. B. (1936). The phenomena of auran metamorphosis, III. The development of the
thyroid gland. /. Morph. 59, 69-89.
ETKIN, W. B. (1964). Metamorphosis. In Physiology of the Amphibia (ed. J. A. Moore), pp.
427-68. New York: Academic Press.
Fox, H. (1956). Compensation in a remaining pronephros of Triturus after unilateral pronephrectomy. /. Embryol. exp. Morph. 4, 139-51.
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Fox, H. (1963). The amphibian pronephros. Q. Rev. Biol. 38, 1-25.
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Fox, H. & HAMILTON, L. (1964). Pronephric system in haploid and diploid larvae of Xenopus
laevis. Experientia 20, 289.
KAYE, N. W. (1961). Interrelationships of the thyroid and pituitary in embryonic and premetamorphic stages of the frog {Rana pipiens). Gen. Comp. Endocr. 1, 1-19.
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LYNN, W. F. & PEADON, A. (1955). The role of the thyroid gland in direct development in the
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ABERCROMBLE,
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S. (1932). Quantitative Untersuchungen iiber die Schilddruese von Bufo in
Beziehung zur Metamorphose. /. Sci. Hiroshima 2, 1-20.
SAXIN, L., SAXEN, E., TOIVONEN, S. & SALIMAKI, K. (1957). Quantitative investigation on the
anterior pituitary-thyroid mechanism during frog metamorphosis. Endocrinology 61, 35-44.
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MORITA,
(Manuscript received 16 March 1966, revised 18 May 1966)