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Changes in thyroidal and plasma lodine compounds during and after metamorphosis of the bullfrog, Rana catesbeiana
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
45,
74-81
(1981)
Changes in Thyroidal and Plasma Iodine Compounds during after Metamorphosis of the Bullfrog, Rana catesbeiana SHINTARO Institute
SUZUKI
of Endocrinology,
AND Gunmu
MITSUO University,
and
SUZUKI Muebushi,
Japun
Accepted September 22, 1980 Thyroidal thyroxine (TJ, 3,5,3’-triiodothyronine (T:,), diiodotyrosine (DIT), monoiodotyrosine (MIT), and iodide (II) in metamorphosing tadpoles, froglets, and adult frogs were measured chemically by cation-exchange resin chromatography. Plasma Tq, T,, DIT, and 3,3’S’-triiodothyronine (rT,J were also measured by radioimmunoassay. Plasma proteinbound iodine (PBI) was determined by a modified ultramicro PBI method. Tq, T,, MIT, and DIT levels in thyroids tended to increase both during and after metamorphosis. Plasma T, incr,ased at the end of prometamorphosis, and a sharp increase was found at the onset of the stamorphic climax. At stage XXI, the T, level reached a peak of about 500 ng/lOO ml. I hc.:eafter the amount of circulating T1 decreased slowly, but even at 7 and 14 days after metamorphosis it remained at a higher level than in the premetamorphic stage. The level of plasma T, in adult frogs was only 40 ng/lOO ml and was undetectable in 4-month-old froglets. Similar changes were found in circulating plasma Tis and its level was less than one-third of T,, especially at the metamorphic climax. Throughout all metamorphic stages, plasma T, and T, levels fluctuated remarkably only at the metamorphic climax. Plasma DIT was at the low level of about 200 ng/lOO ml and did not change during metamorphosis. Moreover, a small amount of rT,, less than T, values, was detectable only in tadpoles during the metamorphic climax and in adult frogs. PBI values were at the level of about 2.0 pg/lOO ml from stage XV to stage XXII, and thereafter decreased gradually. The PBI level was much higher than the sum of plasma T,. T,, and DIT, and did not correlate with the circulating hormone levels. From these results a relationship between thyroidal and plasma hormone levels and an extrathyroidal monodeiodination of T, were discussed.
Since Gudernatsch (1912) found that the feeding of horse thyroid induces precocious metamorphosis in tadpoles, it has been shown that amphibian metamorphosis is dependent on thyroid hormone. In particular, from the result of immersion experiments with thyroiodectomized tadpoles in thyroxine, and from the data on the level of plasma protein-bound iodine in metamorphosing tadpoles, it has been thought that the level of circulating thyroid hormone rises remarkably as metamorphosis proceeds (Etkin, 1935; Just, 1972). Recently, several authors have reported plasma thyroid hormone levels in metamorphosing tadpoles, using radioimmunoassay procedures (Miyauchi et al., 1977; Leloup and Buscaglia, 1977; Regard et al., 1978; Suzuki and Suzuki, 1978; Mondou and Kaltenbach, 1979). However, plasma levels of thyroid 00 16-6480/8 l/090074-08$0 1.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
hormones during metamorphosis still remain to be elucidated in relation to thyroida1 hormone levels. In order to clarify comprehensive features of iodine metabolism during and after metamorphosis this paper deals with changes in thyroid and plasma iodoamino acids and PBI in tadpoles, froglets, and adult frogs. MATERIALS
AND METHODS
More than 500 tadpoles and adults frogs of Runu were collected from the same pond near Maebashi. The tadpoles were reared at 22 ? 2” in well water and were fed boiled spinach, continuously available. Metamorphic stages were determined by the criteria of Taylor and Kollros (1946). After metamorphosis, froglets were kept at the same temperature for a few months and fed on crickets during that period. Chemicul determination of iodine compounds. For
cutesbeiunu
IODINE
COMPOUNDS
analyses of iodide (I-), monoiodotyrosine (MIT), diiodotyrosine (DIT), thyroxine (T,), and triiodothyronine (T3), thyroid glands were dissected from tadpoles at various stages and from adult frogs. As shown in Table 1, these thyroid glands from 5 to 20 animals were pooled. The pooled thyroids were homogenized with 0.1 M phosphate buffer solution (pH 7.4) in a small Potter-Elvehjem homogenizer. The homogenates (about 2 to 8 mg of thyroid proteins) were hydrolyzed by incubation with Pronase E (Kaken Co., Tokyo; 5 mg to each homogenate) for 24 hr at 37” after the addition of 5 ~1 of 0.5 M I-methyl-2-mercaptoimidazole (Chugai Co., Tokyo) and 5 ~1 of toluene. The incubation was continued for 24 hr after the addition of a small amount of aminopeptidase M (Sigma Chemical Co., St. Louis, MO.). The hydrolyzed homogenates were centrifuged at 7OOg for 10 min and the supernatants were collected for analyses of iodoamino acids (Sorimachi and Ui, 1974). An aliquot of each supernatant was diluted with starting buffer solution (0.04 M ammonium acetate containing 30% (v/v) ethanol, pH 7.4). The final samples were applied to a column (cation-exchange resin, AG 5OW-X4, Bio-Rad Lab., Richmond, Calif.) equilibrated with the starting buffer solution. Iodoamino acids were eluted by the application of a linear gradient of the starting buffer solution and 1 N NH,OH containing 30% ethanol. The total protein concentration in thyroid homogenates was determined by the method of Lowry rt (11. (1951). To measure the thyroid protein-bound iodine (PBI) the proteins in homogenates were precipitated by the addition of an equal volume of 10% trichloroacetic acid solution (TCA). The solution was centrifuged at 7OOgfor 10 min and the supernatant was discarded. The precipitate was resuspended with 5% TCA and centrifuged again. After dissolving the precipitate with a drop of I N KOH and I ml of distilled water, and the iodine content was measured with a Technicon equipped with a digestor for the determination of PBI. Rtrdioimmunoussuy
of plasma
iodoamino
aids.
Tadpoles and froglets were anesthetized with MS 222 (tricane methanesulfonate, Sankyo Co., Tokyo), and blood samples were collected into heparinized capillaries after cutting the ventral aorta. Within a few days after being collected, adult frogs were also anesthetized and blood samples were obtained by heart puncture with heparinized syringes. Plasma T,. T:,. and DIT were measured by appropriate radioimmunoassay (RIA) methods. viz., Larsen (1972). Larsen et al. (1973). and Nelson et al. (1974). respectively. Plasma reverse triiodothyronine (rT:,) was measured with the use of a commercially available RIA kit made by Dainabot. Japan. [“;I]T, (718 ,&i/fig) and [‘““IIT:, (470 PCilpg) were purchased from Daiichi Isotope Company, while [‘,“I]DIT was labeled in our laboratory by the method of chloramine T oxidation with carrier-free ‘.‘lI- (NEN-0.35H) (Matsuzaki and
75
IN TADPOLES
Suzuki, 1967). [‘:“I]DIT was separated by the method of Pitt-Rivers and Sacks (1962). Antisera for Tq, TS, and DIT were prepared by immunizing rabbits with their conjugates with bovine serum albumin (BSA) and used at a final dilution of 1:3000, 1:2500, and 2000, respectively (Nishikawa et ul., 1979). The crossreactivities of these antisera with other iodoamino acids are as follows: T, antiserum to T:, was O.lO%, T:, antiserum to T, was 0.14%, and DIT antiserum to MIT, T,, and T, were 5.12, 0.10, and 0.20%, respectively. These results indicate that there was no significant interference by any of these. The sensitivities of these RIA systems were as follows when expressed as picograms per tube: T, 12; T,, 12; DIT, 10; and rT:,, 1.5, respectively. For duplicate RIA determination, T, and T, in 100 ~1 plasma were extracted with 1 ml of ethanol (Miyauchi et ul., 1977), while to measure an extremely low concentration of DIT, 250 to 450 ~1 of plasma was used for a single extraction. On the other hand, rT, in 100 or 200 ~1 of plasma was measured directly with the RIA kit. Extraction efficiencies for T,, T,, and DIT were more than 60, 80, and nearly lOO%, respectively. Thus the minimal detective ranges for T,, T,. DIT, and rT:, were more than 20, 15, 2, and 0.75 ng/lOO ml, respectively. Plasma PBI was determined in duplicate by the micromethod of Malkin (1965).
RESULTS Distribution of Iodoarnino Acids and Iodide in Tadpole and Adult Frog Thyroids
As shown in Table 1, the thyroid protein content tended to increase up to stage XXIII and then to slightly decrease at stage XXV, though abrupt changes in body weight were noted. The protein-bound iodine content in tadpole thyroids did not change markedly throughout metamorphosis. The distribution of iodoamino acids and iodide in hydrolyzed thyroids of tadpoles and adult frogs is shown in Table 2. The T, content increased continuously during metamorphosis and reached 574 ng at stage XXV. The T, content in adult thyroids was at most 1.6 times that found in stage XXV froglets. The T, level rose gradually to stage XXIII and slightly decreased at stage XXV, though the level was extremely low. The T,/T, ratio in the thyroid gland was 0.01 to 0.04 in tadpole and 0.06 in adult frog. DIT content in tadpole thyroids tended to in-
76
SUZUKI
BODY
WEIGHT,
AMOUNT
Stage
Number of animals
XV xx XXIII xxv Adult
20 10 10 10 5
OF THYROID
Body
” Thyroid gland means two ’ The results are expressed
PROTEINS,
weight
33.9 36.4 27.6 23.7 335.2
+ k IL t k
AND TABLE
1
AND
THYROIDAL
Thyroid weight”
(g)
1.1” 0.8 0.8 0.6 67.4
1.3 4.3 4.8 4.3 10.5
lobes in tadpoles as mean + SE.
T4 and T3 Levels in Plasma from Tadpoles and Adult Frogs
Plasma T4 and T3 levels in metamorphosing tadpoles, froglets, and adult frogs are shown in Table 3. At stage V, T, and T3
DISTRIBUTION
2
OF IODOAMINO
TADPOLE
AND ADULT ENZYMATIC
ACIDS
AND
IODIDE
FROG THYROIDS HYDROLYSIS
rig/Thyroid
wet (mg) k It k + +
and adult
crease gradually throughout metamorphosis, though MIT and iodide content reached the highest level at stage XXIII and slightly decreased at stage XXV. A major portion of thyroid iodine was found in protein-bound form (95.8% at stage XV; 93.1% at stage XX; 97.9% at stage XXIII; 97.9% at stage XXV; 97.0% in adult frogs). Accordingly, iodide content figures listed in Table 2 were probably produced by deiodination which might occur during enzymatic digestion of thyroid homogenates.
TABLE
IN
AFTER
gland”
Stage
I-
MIT
DIT
T,
T:,
T,/T,
xv xx XXIII xxv Adult
313 1781 2332 1982 9695
267 1458 2481 2278 8976
1987 7354 9876 11182 22654
116 310 567 574 901
2 4 25 14 58
0.01 0.01 0.04 0.02 0.06
SUZUKI
Note. For analyses of iodoamino acids and iodide the same thyroids as shown in Table 1 were used. II, MIT, DIT, and T, were measured by cation-exchange resin chromatography. T:, was measured by RIA method because of its low level. ” Thyroid gland means two lobes per animal.
IODINE
IN TADPOLES
Thyroid protein” Oakland)
0.1” 0.2 0.4 0.4 2.7
AND
ADULT
Iodine thyroid
0.10 0.46 0.65 0.61 1.66
FROGS
content protein
in (%)
0.9 1.1 0.9 1.0 1.2
frogs.
were detectable in some cases, namely a half and one-sixth, respectively. However, the number of animals in which the hormones were detected increased toward the metamorphic climax. The level of circulating T4 was only 26.6 ng/lOO ml (3.4 x lo-lo M) at stage V. Thereafter, there was a marked rise at stage XVIII and the T4 level reached a peak of 498.3 ng/lOO ml (6.4 x 10eg M) at stage XXI. After this stage, the T4 level fell gradually to the level 188.3 ng/ 100 ml (2.4 x 10eg M) at the end of metamorphosis. The higher levels of T, con-
PLASMA
TABLE 3 Tzs LEVELS IN TADPOLES,
T, AND
AND Stage V XI xv XVII XVIII XIX xx XXI XXII XXIII XXIV xxv xxv + 7 days xxv + 14 days xxv + 4 months xxv + 1 year Adult
ADULT
T, (r&100
FROGLETS,
FROGS”
ml)
+ 7.1 -c 7.9 t 4.1 rt 2.8 + 31.2 2 24.7 ” 61.9 k 38.5 k 34.6 r 36.0 + 27.1 + 31.8
140.1
+ 29.8 (14/14)
40.9 +
7.2
154.3
c 28.3 (16/16)
26.5 r
4.8 (10/16)
17.0 +
2.6 (10/19)
(O/19)
42.2, 108.6 (2/2) 39.4 k 4.6 (16/18)
” The results are expressed b Numbers in parentheses number assayed. ” ND, not detectable.
as mean indicate
24.4 18.2 28.1 55.3 87.9 144.2 114.6 75.1 65.3 48.5
20.2 8.0 17.7 r 3.2 e 5.2 + 10.2 ST 8.1 f 11.7 k 13.1 + 10.4 + 10.1 k 5.8
ml)
26.6 27.1 31.2 47.7 168.7 198.4 480.6 498.3 361.4 317.8 310.5 188.3
ND”
(3/6)” (7/13) (8/12) (12/14) (16/17) (15/15) (16/16) (15/15) (14/14) (16/16) (15/15) (15/15)
T, (ng/lOO
2
28.4, 27.6 21.6 +- 3.5 C SE. number
(l/6) (6/13) (l/12) (6/14) (7/17) (9/15) (16/16) (15/15) (14/14) (16/16) (14/15) (11115) (8/14)
(2/2) (6/18)
detectable/
IODINE
COMPOUNDS
tinued for at least 2 weeks after metamorphosis. However, circulating T4 in 4month-old froglets was undetectable. In both adult frogs and l-year-old frogs the plasma T4 level was very low and almost the same as those found in stage XV tadpoles. On the other hand, in a large number of tadpoles circulating T3 was not found before the metamorphic climax. A distinct increase in circulating T3 was found at stage XVIII. Thereafter, the T, level rose suddenly and reached its maximum of 144.2 ng/lOO ml (2.2 x 10Pg M) at stage XXI. T, levels decreased toward the end of metamorphosis. Detectable levels of T, were still found in froglets kept for a few weeks and 4 months after metamorphosis. A low level of T, was also detected in both lyear-old frogs and adult frogs. The T,/T, ratio for young frogs was much higher in sera than those in the thyroids of tadpoles and adult frogs. In Fig. 1, circulating T, values in tadpoles during and after metamorphosis are plotted. T4L(ng/dl)
77
IN TADPOLES
The T, levels fluctuated remarkably only in the metamorphic climax. Such fluctuations were also observed in T3 levels in climactic tadpoles. T, and T3 levels were also measured in each tadpole at the metamorphic climax. As shown in Fig. 2, there was a positive significant correlation between T4 and T, values (Y = 0.492, P < 0.001). DIT Level in Plasma of Metamorphosing Tadpoles
Circulating DIT in metamorphosing tadpoles was measured by RIA. As shown in Fig. 3, DIT levels were low at about 20 ng/ 100 ml. No significant changes in DIT levels could be found throughout metamorphosis; namely 18.3 + 6.2 (stage V), 21.5 + 10.3 (stage XV), 22.9 t 7.7 (stage XX), and 18.9 + 11.1 (stage XXV) (mean + SE, ng/lOO ml), respectively. rT, Level in Plasma of Tadpoles, Froglets, and Adult Frogs
Circulating rT, was undetectable in tadpoles during pre- and prometamorphic stages (Table 4). At the metamorphic climax, especially at stage XXIII and stage XXV, rT, was measured in a small number
. T3bq/dl)
.
500 1 400
. .
l -
.
.
-
Xi’ 200 1 100 ro
.
loo 0 0
0
l e .
f
metamorphic
l
.
stage
1. Plasma T, concentration in tadpoles and froglets during and after metamorphosis. The stages marked on the abscissa were determined by the criteria of Taylor and Kollros (1946). Stages XXV7 and XXV14 indicate the froglets at 7 and 14 days after metamorphosis, respectively.
s
.
0
.
.
I*
.
100
ZOO
v.
.
. .
l
%
.
. l **
.
’
e
.
.’
.
‘/
FIG.
.
s
.
300
l
0 .
400
-
.
.
.
r=O 492 P
500
600
700
$,(ngldl)
FIG. 2. The correlation between Td and T,, values observed in climactic tadpoles (stages XX-XXV). Correlative coefficient was 0.492. A positively signiticant correlation was found in this experiment (P < 0.001).
78 -
SUZUKI 80
r
40
-
AND
0
k
0 2
0
3Q20-
n
O iT
8
’ V
I
0
I
0
01
0
,911
X
L
xv
xxv
xx
metamorphic
DISCUSSION
of tadpoles. In 4-month-old froglets plasma rT, was not detected at all. However, rT, was found in a few adult frogs. Plasma PBI in Tadpoles, and Adult Frogs
Froglets,
In tadpoles, froglets (1 week, 2 weeks, and 4 months after metamorphosis) and adult frogs used for thyroid hormone measurements, the amounts of PBI were measured by micromethod. As shown in Fig. 4, higher PBI values were found at prometamorphosis (stage XV, 2.9 + 0.7 &lo0 ml; stage XVIII, 1.9 + 0.9 &lo0 ml). However, higher PBI values were also observed at climactic stages, especially at stage TABLE rT,, LEVELS
Stage
XXII. Throughout metamorphosis, PBI levels tended to decrease as metamorphosis proceeded. In both 4-month-old froglets and adult frogs the PBI levels were less than 1.4 ,q/lOO ml. Moreover, there was no correlation between thyroid hormones and PBI levels in metamorphosing tadpoles, froglets, and adult frogs. The PBI level was much higher than the sum of plasma Tq, T3, and DIT.
stage
FIG. 3. Plasma DIT level in metamorphosing tadpoles. Metamorphic stage on the abscissa is Taylor and Kollros stage. Horizontal bars indicate mean values.
PLASMA
SUZUKI
Up to the present, several authors have studied the biosynthesis of thyroid hormones in tadpole thyroids during metamorphosis, using only radioiodine and chromatography (Shellabarger and Brown, 1959; Nataf et al., 1975; Dodd and Dodd, 1976). They found that the rate of thyroxine formation in Xenopus tadpoles increased from the prometamorphic stage to the beginning of the climactic stage. However, the rate decreased postclimax. By the present direct measurements it was shown that thyroidal T, and T, levels rose gradually throughout metamorphosis, although a slight drop in the T, level was found at stage XXV. Moreover even in adult frogs it was demonstrated that the larger amounts of
7r.
l
4
IN TADPOLES, ADULT FROGS
FROGLETS,
rT,, (ng/lOO
ml)
.
. . :
AND
-8
l .
. . . .
l . 0;
XV XVII XIX xx XXI XXIII xxv XXV Adult
+ 4 months
” ND, not detectable. o Number detectable/number
ND” ND ND 4.7 1.3 1.8 ? 0.5 3.4 2 1.0 ND 3.6 + 0.5
assayed
(O/9)” (O/9) (019) (l/9) (l/10) (519) W9) (O/9)
WV in paren-
4iAl’ .
.
:
c;*
,f;‘*.
:
x0 xx
metamorphic
.
rt*t
:
l
l . :
,:
XXV XXV7 XXV14 froglet stage
.2adult
FIG. 4. Plasma PBI level in metamorphosing tadpoles, froglets, and adult frogs. Metamorphic stage is according to Taylor and Kollros stage. Stages XXV7 and XXV14 indicate the froglets at 7 and 14 days after metamorphosis. Froglet means those kept in the laboratory for 4 months after metamorphosis. Horizontal bars indicate mean values.
IODINE
COMPOUNDS
hormones were stored in the thyroid gland. On the other hand, changes in circulating thyroid hormones during metamorphosis were examined by immersion experiments with thyroidectomized tadpoles in solutions of known T, concentrations (Etkin, 1935; 1964). Etkin concluded that low concentrations (3 x lo-lo M- 10eg M) resulted in good leg growth during metamorphosis, while the concentration increased during the prometamorphic stage (3 x 10eg M-2 x lo+ M). The highest level (2 x lo-’ M) was required at about the time of foreleg emergence, and remained until tail resorption was complete. Recently, plasma thyroid hormone levels in metamorphosing tadpoles have been measured by RIA (Miyauchi et al., 1977; Leloup and Buscaglia, 1977; Regard et al., 1978; Suzuki and Suzuki, 1978; Mondou and Kaltenbach, 1979). However, it is difficult to draw general conclusions because generally only a very limited number of tadpoles were used for the measurements. Present results showed that circulating thyroid hormone levels were very low prior to stage XVIII and thereafter increased suddenly. The highest level of these hormones was found at stages XX-XXI, just at the beginning of the metamorphic climax. After these stages the hormone level fell gradually. This pattern of changes in thyroid hormone levels during metamorphosis was very different from those found by Miyauchi et ul. (1977) and Regard et ul. (1978). However, Mondou and Kaltenbach (1979) reported a similar pattern of changes only in the T4 level. Circulating T, levels found in the present paper were very much lower than concentrations postulated by Etkin, though the pattern of changes in both hormone levels was similar to that shown by Etkin up to the metamorphic climax. Even 7 to 14 days after metamorphosis, circulating T, and T, levels in froglets were higher than those at the middle of prometamorphosis. It is likely that these hormones are still necessary for internal or physiological conditions which were unaccompanied by external changes.
IN TADPOLES
79
However, we could not explain why the T, level was lower in 4-month-old froglets. Moreover, we observed a marked fluctuation in these hormone levels only at the climactic stage during metamorphosis. Such a fluctuation is probably explained by differences in the amount and timing of the hormone surge among tadpoles. In adult frogs and toads some variations in circulating thyroid hormone levels have been reported by many authors (Packard et al., 1976; Miyauchi et ul., 1977; Leloup and Buscaglia, 1977; Regard et al., 1978; Rosenkilde and Jorgensen, 1977; Mondou and Kaltenbach, 1979). We found very low levels of thyroid hormones while Miyauchi et al. (1977) and Regard et al. (1978) showed undetectable levels of these hormones in the same species. However, it is most probable that these variations found in adult frogs and toads depend on species and physiological conditions. The T,/T, ratio in thyroid glands was only 0.02 to 0.03, although the ratio in circulating hormone levels was 0.2 to 0.9 throughout metamorphosis. This finding confirms the theory that extrathyroidal monodeiodination of T, giving rise to T3 takes place in metamorphosing tadpoles (Leloup and Buscaglia, 1977). In addition, a small amount of rT, was detected only at the climactic stage. It seems that monodeiodination from T, to rT, occurs at least at the metamorphic climax. These monodeiodinations including the metabolism of thyroid hormones during metamorphosis should be clarified in a future study. It is well known that the PBI level is an index of circulating thyroid hormones in mammals. In order to clarify the changes in circulating levels of thyroid hormones during metamorphosis Just (1972) measured the levels of PBI in tadpoles of Rum pipiens by the micromethod of Malkin (1965). He found that the levels rose gradually to stage XVIII and reached their maximum at stage XIX (average 10.52 lug/100 ml). Thereafter, the PBI levels dropped precipitously toward the end of metamorphosis. By the
80
SUZUKI
AND
same method we measured PBI levels in R. catesbeiana tadpoles. In our experiments PBI levels were less than 7 pg/lOO ml throughout metamorphosis. Also, we failed to find a sharp increase in the PBI values at the onset of the metamornhic climax. Finally we concluded that there was no correlation between PBI values and the hormone levels. The PBI values which represented iodothyronines or iodinated proteins were much higher than the sum of plasma T4 and T3, even including DIT. This discrepancy may be ascribed to the presence of considerable amounts of iodoprotein( s) in tadpole plasma. We have to refer to the relation between thyroidal and plasma thyroid hormone levels. As mentioned already, we demonstrated that thyroid hormone levels in tadpole thyroids attained the plateau at stages XXIII-XXV and increased even after metamorphosis. However, plasma hormone levels which are an index of the hormone secretion from thyroid gland showed the maximum surge at stages XX- XXII. These results seem to show that the rate of thyroid hormone secretion decreases before late prometamorphosis (XVIII) and after completion of metamorphosis (XXV) to the adult, though thyroid hormones are still synthesized and/or stored in these thyroids. Thyroid hormone levels are certainly controlled by the secretion of thyroid-stimulating hormone during metamorphosis, coupled with hypothalamic factors (Etkin, 1964; Dodd and Dodd, 1976). However, at present the mechanism for thyroid hormone secretion remains unknown in tadpoles, especially in adult frogs. ACKNOWLEDGMENTS
SUZUKI
(B. Lofts, ed.), Vol. 3, pp. 467-599. Academic Press, New York. Etkin, W. (1935). The mechanisms of anuran metamorphosis. I. Thyroxine concentration and the metamorphic pattern. J. Exp. Zool. 71, 317-340. Etkin, W. (1964). Metamorphosis. In “Physiology of the Amphibia” (J. A. Moore, ed.), Vol. 1, pp. 427-468. Academic Press, New York. Gudernatsch, J. F. (1912). Feeding experiments on tadpoles. 1. The influence of specific organs given as food on growth and differentiation. A contribution to the knowledge of organs with internal secretion. Arch. Entwicklungsmech. Orgunismen 35, 457-483. Just, J. J. (1972). Protein-bound iodine and protein concentration in plasma and pericardial fluid of metamorphosing anuran tadpoles. Physiol. Zool. 45, 143- 152. Larsen, P. R. (1972). Direct immunoassay of triiodothyronine in human serum. J. C/in. Znvest. 51, 1939- 1949. Larsen, P. R., Dockalova, J., Sipula, D., and Wu, F. M. (1973). Immunoassay of thyroxine in unextracted human serum. J. Clin. Endocrinol. Metah. 37, 177- 182. Leloup, J., and Buscaglia, M. (1977). La triiodothyronine, hormone de la metamorphose des amphibiens. C.R. Acad. Sci. Paris 2842261-2263. Lowry, N., Rosenbrough, J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin reagent. J. Biol. Chem. 193, 265-275. Malkin. H. M. (1965). An ultramicro protein-bound iodine method on capillary blood. J. Clin. Endocrinol.
Metab.
25, 28-38.
Matsuzaki, S., and Suzuki, M. (1967). Deiodination of iodinated amino acids by pig thyroid microsomes. J. Biochem.
62, 746-755.
Miyauchi, H., LaRochelle, F. T., Jr., Suzuki. M., Freeman, M., and Frieden, E. (1977). Studies on thyroid hormones and their binding in bullfrog tadpole plasma during metamorphosis. Gen. Comp.
Endocrinol.
33, 254-266.
Mondou, P. M., and Kaltenbach, J. C. (1979). Thyroxine concentrations in blood serum and pericardial fluid of metamorphosing tadpoles and of adult frogs. Gen. Comp. Endocrinol. 39, 343349. Nataf, B., Regard, E., and Hourdry, J. (1975). Metabolisme du radio-iode dans la thyroi’de du Xenope au tours de la vie larvaire et de la metamorphose. C.R.
Acad.
Sci. Puris
280, 657-660.
The authors are indebted to Mr. M. Saito, Mr. T. Kakegawa, and Mr. T. Narita, who provided excellent technical assistance.
Nelson, J. C.. Weiss, R. M.. Lewis, J., Wilcox, R. B., and Palmer, F. J. (1974). A multiple ligand binding radioimmunoassay of diiodotyrosine. J. Clin.
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Invest.
Dodd, M. H., and Dodd, J. M. (1976). The biology of metamorphosis. III “Physiology of the Amphibia”
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IODINE
measured Japon.
by radioimmunoassay.
COMPOUNDS Endocrinoi.
26, 731-735.
Packard, G. C., Packard, M. J., and Gorbman, A. (1976). Serum thyroxine concentrations in the pacific hagfish and lamprey and in the leopard frog. Gen. Comp. Endocrinal. 28, 365-367. Pitt-Rivers, R., and Sacks, B. I. (1962). The separation of iodinated tyrosines and thyroxine from serum. B&hem. J. 82, 111-112. Regard, E., Taurog, A., and Nakashima, T. (1978). Plasma thyroxine and triiodothyronine levels in spontaneously metamorphosing Rana cutesbeiunu tadpoles and in adult anuran amphibia. Endocrinology
102, 674-684.
Rosenkilde, P., and Jorgensen, I. (1977). Determination of serum thyroxine in two species of toads:
IN
TADPOLES
81
Variation with season. Gen. Cnmp. Endocrinol. 33, 566-573. Shellabarger, C. J., and Brown, J. R. (1959). The biosynthesis of thyroxine and 3:5:3’-triiodothyronine in larval and adult toads. J. Endocrinol. 18, 98- 101. Sorimachi, K., and Ui, N. (1974). An improved chromatographic method for analysis of iodoamino acids in thyroglobulin. J. B&hem. (Tokyo) 76, 39-45. Suzuki, S., and Suzuki, M. (1978). On the synthesis and secretion of thyroid hormones during metamorphosis in Runu cutesbeiunu. Zool. Mug. 87, 465. Taylor, A. C., and Kolh-os, J. J. (1946). Stages in the normal development of Runu pipiens larvae. Anut.
Rec.
94, 7-23.
AND
COMPARATIVE
ENDOCRINOLOGY
45,
74-81
(1981)
Changes in Thyroidal and Plasma Iodine Compounds during after Metamorphosis of the Bullfrog, Rana catesbeiana SHINTARO Institute
SUZUKI
of Endocrinology,
AND Gunmu
MITSUO University,
and
SUZUKI Muebushi,
Japun
Accepted September 22, 1980 Thyroidal thyroxine (TJ, 3,5,3’-triiodothyronine (T:,), diiodotyrosine (DIT), monoiodotyrosine (MIT), and iodide (II) in metamorphosing tadpoles, froglets, and adult frogs were measured chemically by cation-exchange resin chromatography. Plasma Tq, T,, DIT, and 3,3’S’-triiodothyronine (rT,J were also measured by radioimmunoassay. Plasma proteinbound iodine (PBI) was determined by a modified ultramicro PBI method. Tq, T,, MIT, and DIT levels in thyroids tended to increase both during and after metamorphosis. Plasma T, incr,ased at the end of prometamorphosis, and a sharp increase was found at the onset of the stamorphic climax. At stage XXI, the T, level reached a peak of about 500 ng/lOO ml. I hc.:eafter the amount of circulating T1 decreased slowly, but even at 7 and 14 days after metamorphosis it remained at a higher level than in the premetamorphic stage. The level of plasma T, in adult frogs was only 40 ng/lOO ml and was undetectable in 4-month-old froglets. Similar changes were found in circulating plasma Tis and its level was less than one-third of T,, especially at the metamorphic climax. Throughout all metamorphic stages, plasma T, and T, levels fluctuated remarkably only at the metamorphic climax. Plasma DIT was at the low level of about 200 ng/lOO ml and did not change during metamorphosis. Moreover, a small amount of rT,, less than T, values, was detectable only in tadpoles during the metamorphic climax and in adult frogs. PBI values were at the level of about 2.0 pg/lOO ml from stage XV to stage XXII, and thereafter decreased gradually. The PBI level was much higher than the sum of plasma T,. T,, and DIT, and did not correlate with the circulating hormone levels. From these results a relationship between thyroidal and plasma hormone levels and an extrathyroidal monodeiodination of T, were discussed.
Since Gudernatsch (1912) found that the feeding of horse thyroid induces precocious metamorphosis in tadpoles, it has been shown that amphibian metamorphosis is dependent on thyroid hormone. In particular, from the result of immersion experiments with thyroiodectomized tadpoles in thyroxine, and from the data on the level of plasma protein-bound iodine in metamorphosing tadpoles, it has been thought that the level of circulating thyroid hormone rises remarkably as metamorphosis proceeds (Etkin, 1935; Just, 1972). Recently, several authors have reported plasma thyroid hormone levels in metamorphosing tadpoles, using radioimmunoassay procedures (Miyauchi et al., 1977; Leloup and Buscaglia, 1977; Regard et al., 1978; Suzuki and Suzuki, 1978; Mondou and Kaltenbach, 1979). However, plasma levels of thyroid 00 16-6480/8 l/090074-08$0 1.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
hormones during metamorphosis still remain to be elucidated in relation to thyroida1 hormone levels. In order to clarify comprehensive features of iodine metabolism during and after metamorphosis this paper deals with changes in thyroid and plasma iodoamino acids and PBI in tadpoles, froglets, and adult frogs. MATERIALS
AND METHODS
More than 500 tadpoles and adults frogs of Runu were collected from the same pond near Maebashi. The tadpoles were reared at 22 ? 2” in well water and were fed boiled spinach, continuously available. Metamorphic stages were determined by the criteria of Taylor and Kollros (1946). After metamorphosis, froglets were kept at the same temperature for a few months and fed on crickets during that period. Chemicul determination of iodine compounds. For
cutesbeiunu
IODINE
COMPOUNDS
analyses of iodide (I-), monoiodotyrosine (MIT), diiodotyrosine (DIT), thyroxine (T,), and triiodothyronine (T3), thyroid glands were dissected from tadpoles at various stages and from adult frogs. As shown in Table 1, these thyroid glands from 5 to 20 animals were pooled. The pooled thyroids were homogenized with 0.1 M phosphate buffer solution (pH 7.4) in a small Potter-Elvehjem homogenizer. The homogenates (about 2 to 8 mg of thyroid proteins) were hydrolyzed by incubation with Pronase E (Kaken Co., Tokyo; 5 mg to each homogenate) for 24 hr at 37” after the addition of 5 ~1 of 0.5 M I-methyl-2-mercaptoimidazole (Chugai Co., Tokyo) and 5 ~1 of toluene. The incubation was continued for 24 hr after the addition of a small amount of aminopeptidase M (Sigma Chemical Co., St. Louis, MO.). The hydrolyzed homogenates were centrifuged at 7OOg for 10 min and the supernatants were collected for analyses of iodoamino acids (Sorimachi and Ui, 1974). An aliquot of each supernatant was diluted with starting buffer solution (0.04 M ammonium acetate containing 30% (v/v) ethanol, pH 7.4). The final samples were applied to a column (cation-exchange resin, AG 5OW-X4, Bio-Rad Lab., Richmond, Calif.) equilibrated with the starting buffer solution. Iodoamino acids were eluted by the application of a linear gradient of the starting buffer solution and 1 N NH,OH containing 30% ethanol. The total protein concentration in thyroid homogenates was determined by the method of Lowry rt (11. (1951). To measure the thyroid protein-bound iodine (PBI) the proteins in homogenates were precipitated by the addition of an equal volume of 10% trichloroacetic acid solution (TCA). The solution was centrifuged at 7OOgfor 10 min and the supernatant was discarded. The precipitate was resuspended with 5% TCA and centrifuged again. After dissolving the precipitate with a drop of I N KOH and I ml of distilled water, and the iodine content was measured with a Technicon equipped with a digestor for the determination of PBI. Rtrdioimmunoussuy
of plasma
iodoamino
aids.
Tadpoles and froglets were anesthetized with MS 222 (tricane methanesulfonate, Sankyo Co., Tokyo), and blood samples were collected into heparinized capillaries after cutting the ventral aorta. Within a few days after being collected, adult frogs were also anesthetized and blood samples were obtained by heart puncture with heparinized syringes. Plasma T,. T:,. and DIT were measured by appropriate radioimmunoassay (RIA) methods. viz., Larsen (1972). Larsen et al. (1973). and Nelson et al. (1974). respectively. Plasma reverse triiodothyronine (rT:,) was measured with the use of a commercially available RIA kit made by Dainabot. Japan. [“;I]T, (718 ,&i/fig) and [‘““IIT:, (470 PCilpg) were purchased from Daiichi Isotope Company, while [‘,“I]DIT was labeled in our laboratory by the method of chloramine T oxidation with carrier-free ‘.‘lI- (NEN-0.35H) (Matsuzaki and
75
IN TADPOLES
Suzuki, 1967). [‘:“I]DIT was separated by the method of Pitt-Rivers and Sacks (1962). Antisera for Tq, TS, and DIT were prepared by immunizing rabbits with their conjugates with bovine serum albumin (BSA) and used at a final dilution of 1:3000, 1:2500, and 2000, respectively (Nishikawa et ul., 1979). The crossreactivities of these antisera with other iodoamino acids are as follows: T, antiserum to T:, was O.lO%, T:, antiserum to T, was 0.14%, and DIT antiserum to MIT, T,, and T, were 5.12, 0.10, and 0.20%, respectively. These results indicate that there was no significant interference by any of these. The sensitivities of these RIA systems were as follows when expressed as picograms per tube: T, 12; T,, 12; DIT, 10; and rT:,, 1.5, respectively. For duplicate RIA determination, T, and T, in 100 ~1 plasma were extracted with 1 ml of ethanol (Miyauchi et ul., 1977), while to measure an extremely low concentration of DIT, 250 to 450 ~1 of plasma was used for a single extraction. On the other hand, rT, in 100 or 200 ~1 of plasma was measured directly with the RIA kit. Extraction efficiencies for T,, T,, and DIT were more than 60, 80, and nearly lOO%, respectively. Thus the minimal detective ranges for T,, T,. DIT, and rT:, were more than 20, 15, 2, and 0.75 ng/lOO ml, respectively. Plasma PBI was determined in duplicate by the micromethod of Malkin (1965).
RESULTS Distribution of Iodoarnino Acids and Iodide in Tadpole and Adult Frog Thyroids
As shown in Table 1, the thyroid protein content tended to increase up to stage XXIII and then to slightly decrease at stage XXV, though abrupt changes in body weight were noted. The protein-bound iodine content in tadpole thyroids did not change markedly throughout metamorphosis. The distribution of iodoamino acids and iodide in hydrolyzed thyroids of tadpoles and adult frogs is shown in Table 2. The T, content increased continuously during metamorphosis and reached 574 ng at stage XXV. The T, content in adult thyroids was at most 1.6 times that found in stage XXV froglets. The T, level rose gradually to stage XXIII and slightly decreased at stage XXV, though the level was extremely low. The T,/T, ratio in the thyroid gland was 0.01 to 0.04 in tadpole and 0.06 in adult frog. DIT content in tadpole thyroids tended to in-
76
SUZUKI
BODY
WEIGHT,
AMOUNT
Stage
Number of animals
XV xx XXIII xxv Adult
20 10 10 10 5
OF THYROID
Body
” Thyroid gland means two ’ The results are expressed
PROTEINS,
weight
33.9 36.4 27.6 23.7 335.2
+ k IL t k
AND TABLE
1
AND
THYROIDAL
Thyroid weight”
(g)
1.1” 0.8 0.8 0.6 67.4
1.3 4.3 4.8 4.3 10.5
lobes in tadpoles as mean + SE.
T4 and T3 Levels in Plasma from Tadpoles and Adult Frogs
Plasma T4 and T3 levels in metamorphosing tadpoles, froglets, and adult frogs are shown in Table 3. At stage V, T, and T3
DISTRIBUTION
2
OF IODOAMINO
TADPOLE
AND ADULT ENZYMATIC
ACIDS
AND
IODIDE
FROG THYROIDS HYDROLYSIS
rig/Thyroid
wet (mg) k It k + +
and adult
crease gradually throughout metamorphosis, though MIT and iodide content reached the highest level at stage XXIII and slightly decreased at stage XXV. A major portion of thyroid iodine was found in protein-bound form (95.8% at stage XV; 93.1% at stage XX; 97.9% at stage XXIII; 97.9% at stage XXV; 97.0% in adult frogs). Accordingly, iodide content figures listed in Table 2 were probably produced by deiodination which might occur during enzymatic digestion of thyroid homogenates.
TABLE
IN
AFTER
gland”
Stage
I-
MIT
DIT
T,
T:,
T,/T,
xv xx XXIII xxv Adult
313 1781 2332 1982 9695
267 1458 2481 2278 8976
1987 7354 9876 11182 22654
116 310 567 574 901
2 4 25 14 58
0.01 0.01 0.04 0.02 0.06
SUZUKI
Note. For analyses of iodoamino acids and iodide the same thyroids as shown in Table 1 were used. II, MIT, DIT, and T, were measured by cation-exchange resin chromatography. T:, was measured by RIA method because of its low level. ” Thyroid gland means two lobes per animal.
IODINE
IN TADPOLES
Thyroid protein” Oakland)
0.1” 0.2 0.4 0.4 2.7
AND
ADULT
Iodine thyroid
0.10 0.46 0.65 0.61 1.66
FROGS
content protein
in (%)
0.9 1.1 0.9 1.0 1.2
frogs.
were detectable in some cases, namely a half and one-sixth, respectively. However, the number of animals in which the hormones were detected increased toward the metamorphic climax. The level of circulating T4 was only 26.6 ng/lOO ml (3.4 x lo-lo M) at stage V. Thereafter, there was a marked rise at stage XVIII and the T4 level reached a peak of 498.3 ng/lOO ml (6.4 x 10eg M) at stage XXI. After this stage, the T4 level fell gradually to the level 188.3 ng/ 100 ml (2.4 x 10eg M) at the end of metamorphosis. The higher levels of T, con-
PLASMA
TABLE 3 Tzs LEVELS IN TADPOLES,
T, AND
AND Stage V XI xv XVII XVIII XIX xx XXI XXII XXIII XXIV xxv xxv + 7 days xxv + 14 days xxv + 4 months xxv + 1 year Adult
ADULT
T, (r&100
FROGLETS,
FROGS”
ml)
+ 7.1 -c 7.9 t 4.1 rt 2.8 + 31.2 2 24.7 ” 61.9 k 38.5 k 34.6 r 36.0 + 27.1 + 31.8
140.1
+ 29.8 (14/14)
40.9 +
7.2
154.3
c 28.3 (16/16)
26.5 r
4.8 (10/16)
17.0 +
2.6 (10/19)
(O/19)
42.2, 108.6 (2/2) 39.4 k 4.6 (16/18)
” The results are expressed b Numbers in parentheses number assayed. ” ND, not detectable.
as mean indicate
24.4 18.2 28.1 55.3 87.9 144.2 114.6 75.1 65.3 48.5
20.2 8.0 17.7 r 3.2 e 5.2 + 10.2 ST 8.1 f 11.7 k 13.1 + 10.4 + 10.1 k 5.8
ml)
26.6 27.1 31.2 47.7 168.7 198.4 480.6 498.3 361.4 317.8 310.5 188.3
ND”
(3/6)” (7/13) (8/12) (12/14) (16/17) (15/15) (16/16) (15/15) (14/14) (16/16) (15/15) (15/15)
T, (ng/lOO
2
28.4, 27.6 21.6 +- 3.5 C SE. number
(l/6) (6/13) (l/12) (6/14) (7/17) (9/15) (16/16) (15/15) (14/14) (16/16) (14/15) (11115) (8/14)
(2/2) (6/18)
detectable/
IODINE
COMPOUNDS
tinued for at least 2 weeks after metamorphosis. However, circulating T4 in 4month-old froglets was undetectable. In both adult frogs and l-year-old frogs the plasma T4 level was very low and almost the same as those found in stage XV tadpoles. On the other hand, in a large number of tadpoles circulating T3 was not found before the metamorphic climax. A distinct increase in circulating T3 was found at stage XVIII. Thereafter, the T, level rose suddenly and reached its maximum of 144.2 ng/lOO ml (2.2 x 10Pg M) at stage XXI. T, levels decreased toward the end of metamorphosis. Detectable levels of T, were still found in froglets kept for a few weeks and 4 months after metamorphosis. A low level of T, was also detected in both lyear-old frogs and adult frogs. The T,/T, ratio for young frogs was much higher in sera than those in the thyroids of tadpoles and adult frogs. In Fig. 1, circulating T, values in tadpoles during and after metamorphosis are plotted. T4L(ng/dl)
77
IN TADPOLES
The T, levels fluctuated remarkably only in the metamorphic climax. Such fluctuations were also observed in T3 levels in climactic tadpoles. T, and T3 levels were also measured in each tadpole at the metamorphic climax. As shown in Fig. 2, there was a positive significant correlation between T4 and T, values (Y = 0.492, P < 0.001). DIT Level in Plasma of Metamorphosing Tadpoles
Circulating DIT in metamorphosing tadpoles was measured by RIA. As shown in Fig. 3, DIT levels were low at about 20 ng/ 100 ml. No significant changes in DIT levels could be found throughout metamorphosis; namely 18.3 + 6.2 (stage V), 21.5 + 10.3 (stage XV), 22.9 t 7.7 (stage XX), and 18.9 + 11.1 (stage XXV) (mean + SE, ng/lOO ml), respectively. rT, Level in Plasma of Tadpoles, Froglets, and Adult Frogs
Circulating rT, was undetectable in tadpoles during pre- and prometamorphic stages (Table 4). At the metamorphic climax, especially at stage XXIII and stage XXV, rT, was measured in a small number
. T3bq/dl)
.
500 1 400
. .
l -
.
.
-
Xi’ 200 1 100 ro
.
loo 0 0
0
l e .
f
metamorphic
l
.
stage
1. Plasma T, concentration in tadpoles and froglets during and after metamorphosis. The stages marked on the abscissa were determined by the criteria of Taylor and Kollros (1946). Stages XXV7 and XXV14 indicate the froglets at 7 and 14 days after metamorphosis, respectively.
s
.
0
.
.
I*
.
100
ZOO
v.
.
. .
l
%
.
. l **
.
’
e
.
.’
.
‘/
FIG.
.
s
.
300
l
0 .
400
-
.
.
.
r=O 492 P
500
600
700
$,(ngldl)
FIG. 2. The correlation between Td and T,, values observed in climactic tadpoles (stages XX-XXV). Correlative coefficient was 0.492. A positively signiticant correlation was found in this experiment (P < 0.001).
78 -
SUZUKI 80
r
40
-
AND
0
k
0 2
0
3Q20-
n
O iT
8
’ V
I
0
I
0
01
0
,911
X
L
xv
xxv
xx
metamorphic
DISCUSSION
of tadpoles. In 4-month-old froglets plasma rT, was not detected at all. However, rT, was found in a few adult frogs. Plasma PBI in Tadpoles, and Adult Frogs
Froglets,
In tadpoles, froglets (1 week, 2 weeks, and 4 months after metamorphosis) and adult frogs used for thyroid hormone measurements, the amounts of PBI were measured by micromethod. As shown in Fig. 4, higher PBI values were found at prometamorphosis (stage XV, 2.9 + 0.7 &lo0 ml; stage XVIII, 1.9 + 0.9 &lo0 ml). However, higher PBI values were also observed at climactic stages, especially at stage TABLE rT,, LEVELS
Stage
XXII. Throughout metamorphosis, PBI levels tended to decrease as metamorphosis proceeded. In both 4-month-old froglets and adult frogs the PBI levels were less than 1.4 ,q/lOO ml. Moreover, there was no correlation between thyroid hormones and PBI levels in metamorphosing tadpoles, froglets, and adult frogs. The PBI level was much higher than the sum of plasma Tq, T3, and DIT.
stage
FIG. 3. Plasma DIT level in metamorphosing tadpoles. Metamorphic stage on the abscissa is Taylor and Kollros stage. Horizontal bars indicate mean values.
PLASMA
SUZUKI
Up to the present, several authors have studied the biosynthesis of thyroid hormones in tadpole thyroids during metamorphosis, using only radioiodine and chromatography (Shellabarger and Brown, 1959; Nataf et al., 1975; Dodd and Dodd, 1976). They found that the rate of thyroxine formation in Xenopus tadpoles increased from the prometamorphic stage to the beginning of the climactic stage. However, the rate decreased postclimax. By the present direct measurements it was shown that thyroidal T, and T, levels rose gradually throughout metamorphosis, although a slight drop in the T, level was found at stage XXV. Moreover even in adult frogs it was demonstrated that the larger amounts of
7r.
l
4
IN TADPOLES, ADULT FROGS
FROGLETS,
rT,, (ng/lOO
ml)
.
. . :
AND
-8
l .
. . . .
l . 0;
XV XVII XIX xx XXI XXIII xxv XXV Adult
+ 4 months
” ND, not detectable. o Number detectable/number
ND” ND ND 4.7 1.3 1.8 ? 0.5 3.4 2 1.0 ND 3.6 + 0.5
assayed
(O/9)” (O/9) (019) (l/9) (l/10) (519) W9) (O/9)
WV in paren-
4iAl’ .
.
:
c;*
,f;‘*.
:
x0 xx
metamorphic
.
rt*t
:
l
l . :
,:
XXV XXV7 XXV14 froglet stage
.2adult
FIG. 4. Plasma PBI level in metamorphosing tadpoles, froglets, and adult frogs. Metamorphic stage is according to Taylor and Kollros stage. Stages XXV7 and XXV14 indicate the froglets at 7 and 14 days after metamorphosis. Froglet means those kept in the laboratory for 4 months after metamorphosis. Horizontal bars indicate mean values.
IODINE
COMPOUNDS
hormones were stored in the thyroid gland. On the other hand, changes in circulating thyroid hormones during metamorphosis were examined by immersion experiments with thyroidectomized tadpoles in solutions of known T, concentrations (Etkin, 1935; 1964). Etkin concluded that low concentrations (3 x lo-lo M- 10eg M) resulted in good leg growth during metamorphosis, while the concentration increased during the prometamorphic stage (3 x 10eg M-2 x lo+ M). The highest level (2 x lo-’ M) was required at about the time of foreleg emergence, and remained until tail resorption was complete. Recently, plasma thyroid hormone levels in metamorphosing tadpoles have been measured by RIA (Miyauchi et al., 1977; Leloup and Buscaglia, 1977; Regard et al., 1978; Suzuki and Suzuki, 1978; Mondou and Kaltenbach, 1979). However, it is difficult to draw general conclusions because generally only a very limited number of tadpoles were used for the measurements. Present results showed that circulating thyroid hormone levels were very low prior to stage XVIII and thereafter increased suddenly. The highest level of these hormones was found at stages XX-XXI, just at the beginning of the metamorphic climax. After these stages the hormone level fell gradually. This pattern of changes in thyroid hormone levels during metamorphosis was very different from those found by Miyauchi et ul. (1977) and Regard et ul. (1978). However, Mondou and Kaltenbach (1979) reported a similar pattern of changes only in the T4 level. Circulating T, levels found in the present paper were very much lower than concentrations postulated by Etkin, though the pattern of changes in both hormone levels was similar to that shown by Etkin up to the metamorphic climax. Even 7 to 14 days after metamorphosis, circulating T, and T, levels in froglets were higher than those at the middle of prometamorphosis. It is likely that these hormones are still necessary for internal or physiological conditions which were unaccompanied by external changes.
IN TADPOLES
79
However, we could not explain why the T, level was lower in 4-month-old froglets. Moreover, we observed a marked fluctuation in these hormone levels only at the climactic stage during metamorphosis. Such a fluctuation is probably explained by differences in the amount and timing of the hormone surge among tadpoles. In adult frogs and toads some variations in circulating thyroid hormone levels have been reported by many authors (Packard et al., 1976; Miyauchi et ul., 1977; Leloup and Buscaglia, 1977; Regard et al., 1978; Rosenkilde and Jorgensen, 1977; Mondou and Kaltenbach, 1979). We found very low levels of thyroid hormones while Miyauchi et al. (1977) and Regard et al. (1978) showed undetectable levels of these hormones in the same species. However, it is most probable that these variations found in adult frogs and toads depend on species and physiological conditions. The T,/T, ratio in thyroid glands was only 0.02 to 0.03, although the ratio in circulating hormone levels was 0.2 to 0.9 throughout metamorphosis. This finding confirms the theory that extrathyroidal monodeiodination of T, giving rise to T3 takes place in metamorphosing tadpoles (Leloup and Buscaglia, 1977). In addition, a small amount of rT, was detected only at the climactic stage. It seems that monodeiodination from T, to rT, occurs at least at the metamorphic climax. These monodeiodinations including the metabolism of thyroid hormones during metamorphosis should be clarified in a future study. It is well known that the PBI level is an index of circulating thyroid hormones in mammals. In order to clarify the changes in circulating levels of thyroid hormones during metamorphosis Just (1972) measured the levels of PBI in tadpoles of Rum pipiens by the micromethod of Malkin (1965). He found that the levels rose gradually to stage XVIII and reached their maximum at stage XIX (average 10.52 lug/100 ml). Thereafter, the PBI levels dropped precipitously toward the end of metamorphosis. By the
80
SUZUKI
AND
same method we measured PBI levels in R. catesbeiana tadpoles. In our experiments PBI levels were less than 7 pg/lOO ml throughout metamorphosis. Also, we failed to find a sharp increase in the PBI values at the onset of the metamornhic climax. Finally we concluded that there was no correlation between PBI values and the hormone levels. The PBI values which represented iodothyronines or iodinated proteins were much higher than the sum of plasma T4 and T3, even including DIT. This discrepancy may be ascribed to the presence of considerable amounts of iodoprotein( s) in tadpole plasma. We have to refer to the relation between thyroidal and plasma thyroid hormone levels. As mentioned already, we demonstrated that thyroid hormone levels in tadpole thyroids attained the plateau at stages XXIII-XXV and increased even after metamorphosis. However, plasma hormone levels which are an index of the hormone secretion from thyroid gland showed the maximum surge at stages XX- XXII. These results seem to show that the rate of thyroid hormone secretion decreases before late prometamorphosis (XVIII) and after completion of metamorphosis (XXV) to the adult, though thyroid hormones are still synthesized and/or stored in these thyroids. Thyroid hormone levels are certainly controlled by the secretion of thyroid-stimulating hormone during metamorphosis, coupled with hypothalamic factors (Etkin, 1964; Dodd and Dodd, 1976). However, at present the mechanism for thyroid hormone secretion remains unknown in tadpoles, especially in adult frogs. ACKNOWLEDGMENTS
SUZUKI
(B. Lofts, ed.), Vol. 3, pp. 467-599. Academic Press, New York. Etkin, W. (1935). The mechanisms of anuran metamorphosis. I. Thyroxine concentration and the metamorphic pattern. J. Exp. Zool. 71, 317-340. Etkin, W. (1964). Metamorphosis. In “Physiology of the Amphibia” (J. A. Moore, ed.), Vol. 1, pp. 427-468. Academic Press, New York. Gudernatsch, J. F. (1912). Feeding experiments on tadpoles. 1. The influence of specific organs given as food on growth and differentiation. A contribution to the knowledge of organs with internal secretion. Arch. Entwicklungsmech. Orgunismen 35, 457-483. Just, J. J. (1972). Protein-bound iodine and protein concentration in plasma and pericardial fluid of metamorphosing anuran tadpoles. Physiol. Zool. 45, 143- 152. Larsen, P. R. (1972). Direct immunoassay of triiodothyronine in human serum. J. C/in. Znvest. 51, 1939- 1949. Larsen, P. R., Dockalova, J., Sipula, D., and Wu, F. M. (1973). Immunoassay of thyroxine in unextracted human serum. J. Clin. Endocrinol. Metah. 37, 177- 182. Leloup, J., and Buscaglia, M. (1977). La triiodothyronine, hormone de la metamorphose des amphibiens. C.R. Acad. Sci. Paris 2842261-2263. Lowry, N., Rosenbrough, J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin reagent. J. Biol. Chem. 193, 265-275. Malkin. H. M. (1965). An ultramicro protein-bound iodine method on capillary blood. J. Clin. Endocrinol.
Metab.
25, 28-38.
Matsuzaki, S., and Suzuki, M. (1967). Deiodination of iodinated amino acids by pig thyroid microsomes. J. Biochem.
62, 746-755.
Miyauchi, H., LaRochelle, F. T., Jr., Suzuki. M., Freeman, M., and Frieden, E. (1977). Studies on thyroid hormones and their binding in bullfrog tadpole plasma during metamorphosis. Gen. Comp.
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33, 254-266.
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