- Email: [email protected]
Morphological and functional maturation of the thyroid during early development of anuran larvae
GENERAL
AND
COMPARATIVE
Morphological during
21, 410423
ENDOCRINOLOGY
(1973)
and Functional Early Development
Maturation of the Thyroid of Anuran Larvae
YOICHI HANAOKA, SAKUJI MIYASHITA YOICHI KONDO, YASUO KOBAYASHI AND KIYOSHI YAMAMOTO Institute
of Endocrinology,
Gunma
Received
University,
November
Maebashi,
KOYA,
Japan
371
12, 1972
In Bufo bufo jnponicus, synthesis of thyroid hormones, as measured by ‘3iI-incorporation, developed very early, and small but significant amounts of iodothyronines were formed as early as stage 26, at which time the thyroid was recognized only as a simple thickening of the pharyngeal epithelium. The thyroid appeared in its typical form, and 13’1-labeled thyroglobulin was formed in an appreciable amount at st,age 33. Before this stage, soluble Y-proteins were formed only in small amounts, and the molecular sizes of these proteins were smaller than that of thyroglobulin. The changes in iodoamino acid formation were also quite critical between stages 32 and 33. The main iodotyrosine was monoiodotyrosine and the main iodothyronine was triiodothyronine before stage 32. After stage 33, diiodotyrosine was the main iodotyrosine and thyroxine the main iodothyronine. Similar sequential maturation of the thyroid function was observed also in Xenopus Zaevis.
The involvement of the thyroid in anuran metamorphosis is well known. It is generally agreed that the rate of 1311uptake by the thyroid is low during premetamorphic stages, increases during prometamorphic stages, and reaches a maximum at the beginning of the metamorphic climax (Saxkn et al., 1957; Kaye, 1961; Hanaoka, 1966). In Xenopus Zaevis, the rate of biosynthesis of thyroglobulin increases gradually during prometamorphic stages and reaches its peak approximately at the beginning of the climax, as observed by incorporation of labeled amino acid (Regaud and Mauchamp, 1971). These earlier investigations, however, were concerned with the thyroid after completion of its morphogenesis. Flickinger (1964) has reported that monoiodotyrosine (MIT) and diiodotyrosine (DIT) are formed earlier in development (body length, 11 mm) than thyroxine (T4), which is detectable only in premetamorphic stages (16 mm) of the frog larvae. Similar sequential appear410 Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reserved.
ante of these thyroidal products is also demonstrated in Senopus larvae by Shellabarger and Brown (1959), in chick embryos by Trunnell and Wade (1955)) and in rabbit fetuses by Waterman and Gorbman (1953). However, Shepard (1971) did not observe such a sequence in human fetuses. At present, little information is available with respect to the initiation period of biosynthesis of thyroid hormones and thyroglobulin, in relation to morphogenesis of the thyroid. Olin et al. (1970) have reported that thyroglobulin is formed in the thyroid of human fetuses (body length, 60 mm). At this stage, colloid-containing intercellular spaces are detectable by electron microscopy. Suzuki and Kondo (1973) have found thyroglobulin synthesis in the endostyle of ammocoetes before structural transformation of the tissue into the thyroid. These workers, however, did not give data on the initiation of thyroid hormone formation. The present work was carried out in order to clarify correlations between
THYROlD
MATURATION
IN
AND
Bufo
METHODS
The embryos of Bufo bufo jnponicus were obtained from a batch of eggs collected in the vicinity of Maebashi, and grown at 21°C. Developmental stages were determined after Tahara and Ichikawa (1965). The eggs of Xenopus laevis were obtained by induced ovulation after injection of 200 IU of human chorionic gonadotropin. Developmental stages of Xerwpus were classified according to the “Normal Table” of Nieuwkoop and Faber (1956). Table 1 indicates some morphological and physiological characteristics of
STAGES IN THF NORMAL I~VBLOPMENT ST.ir,es
411
LARVrlE
at each stage, and the stages of Xenopus approximately corresponding to those of Bufo. For morphological observation, the animals were fixed in Bouin’s fixative. After fixation, the ventral skin and the muscles covering thyroid area were removed under a dissecting microscope, and the thyroid was exposed for photography. Further, the thyroid was investigated by light microscopy after staining with hematosylin and eosin. For biochemical analysis of 13’I-laheled iodocompounds, the animals were immersed in a 0.04% Niu-Twitty solution containing carrier-free 1311- (5 &i/ml per animal) at 18 + 0.5”C for 24 hr. After immersion, the animals were washed thoroughly with tap water to remove ?I- adhering on the body surface, and the developmental stages were determined. Before stage 32, the whole cranial part or the cranioventral part of t,he body was taken, since it was difficult to isolate the thyroid at these early stages. After
the morphogenesis of the thyroid and the course of its functional development, employing Bufo larvae of very early developmental stages. The functional development was followed by the changes in biosynthesis of 13’1-labeled iodoproteins and iodoamino acids. MATERIALS
ANURAN
TABLE 1 OF Bufo bufo japonicus 0F Xmopus
AND CORRESPONDING
laeris
Developmental (Tahara and Ichikawa, 1965)
Bufo
Morphological
and functional
characteristics of
Bu,fo
larvae
Formation of t,wo gill rudiment#s Branches of the second external gill distinct Fingerlike external gill becoming transparent; beginning of circulation Epidermis becoming transparent; stomodeal invagination much deeper Mouth open; anlage of larval teeth beginning to develop Cornea transparent; beginning of formation of opercular folds; hind-limb bud visible for the first time External gill beginning to degenerate; larval teeth developed Opercular folds covered with right external gill; cloaca open Premefamorphic stage Operculum completely covered with external gill Hind-limb bud semicircular Hind-limb bltd longer than broad; first indication struction First and second toe prominences distinct Four toes distinct Pronwtamorphir
of ankle con-
Xenopus (Nieuwkoop and Faber, 1956)
26 27 28
3,5-36 37-3x 39
29
39
30 31
40 41-42
32
43-44
33
45
34 35 36
46-48 49-50 51
37 38
52 53-54
39 40 41
55 56-57 58
42
59-60
stage
All five toes distinct Five toes elongating: femur, cubitus, and crus distinct Cloaca1 tail piece disappeared Metamorphic
st,ages
climax
Forelegs broken through --
412
HANAOKA
stage 33, the thyroid became easily recognizable under a dissecting microscope (Fig. l), and the gland, together with a small piece of the hyoid cartilage, was dissected without damage. Similar tissue samples were also taken from Xempus. These tissue samples were homogenized and used for biochemical studies. In some experiments, hypophysectomized animals were employed. Hypophysectomy was carried out by extirpation of the hypophysial anlage at the tail bud stage, after Allen (1916) and Smith (1916). Successfully hypophysectomizrd animals were distinguished by their light body color, caused by the lack of thus pars intermedia as t,he source of melanocytc-stimulating hormone. In these animals, the metamorphic process delayed and was arrested at, stage 40, caused by the lack of t,he pars distalis as the POUIW of thyroid-stimulating hormone,. ThitAayer chromntog~aphy of iodoamino acids. The thyroid-containing tissue samples, taken from individual animals at t,lre sxne stage. were pooled and homogenized with 0.3 ml of 0.1 111 phosphate buffer (pH 8.0) containing 0.2 mg Pronase E and 0.05 mg aminopeptidase M. The homogenate was incubatctl at 37°C for 48 hr to secure almost completr hydrolysis of iodinated proteins. The hydrolyzatcx was crntrifupctl at 3000 rpm for 15 min. and 0.1 ml of the supernatant, after adding authentic, MIT, DlT, 3.5.3’triiodothyroninc (T1) a.nd Ta. was applied to the plate for thin-layer chromatography (TLC) with 0.25 mm thick silica g(ll G. The proccdurcs of TLC were essentially the same as desrribed by Faircloth et crl. (1965) and Sofianidrs et al. (1966). as detailed by Soya (1968). TLC was run first with ethyl a,cetate: methanol:4N NH,OH (7:2:1. v/v) for 1 hr and successively with PI-butanol: acetic acid: water (78:5: 17, v/v) for 3 hr. Bftrr chromatography. the plate was dried and authentic iodoamino acids were made visible by spraying with ninhydrin. Silica gel at the visualized spots corresponding to each compound was scraped off, and the radioactivity was counted wit,h a liquid scintillation spectrometer, Beckman Model LS 2OOB, using 0.5% PPO and 10% naphthalene in dioxane as a scintillation mixture. After TLC of another 0.1 ml of the supernatant, radioautography was carried out by exposing the plate, usually for 10 days, to X-ray films (Fuji Film Co.). s ucrose density gradient centrijugation of iodoproteins. The thyroid-containing tissue samples were homogenized with 0.3 ml of 0.1 M phosphate buffer (pH 7.5). The homogenate was centrifuged at 10.000 rpm for 10 min, and 0.2 ml
ET
AL.
of the supernatant was layered on 4.7 ml of a 5 to 20% linear density gradient sucrose solution, prepared with the same buffer used for homogenization. Centrifugation was performed at 38,000 rpm for 4-5 hr at 5°C. After the cent,rifugation, the centrifuge tube was punctured with a needle at the bottom, and 5-drop fractions were taken in test tubts. Each fraction was applied to a paper strip and chromatographed with butanol: rthanol:2AV ammonia (5:1:2, v/v) for 2448 hr, in order to cause nonprotein iodocompounds to migrate away from the origin. After drying the paper, the origin arca was cut, and the radioactivity of ‘“‘I-labeled proteins was counted with a Nuclear Chicago well-type scintillation spectrometer. In the presc>nt study, since only the radioactivity of labeled iodoamino acids and iodoproteins was measured, the results do not show absolute amounts of any- iodocompounds, though the radioactivity can rrpresent the amounts of newly formed iodo~om~rounda. Carrier-frrr Nn’“‘I \vas purchased from Japan Radioisotope Association. Tokyo. Human chorionic gonadotrollin was obtained from TeikokuZoki Co., Tokyo. Pronase E and aminopeptidase M were obtained from K&n Co., Tokyo and R&m & Haas (“o., Germany (distributed by Tanpakushitsu Krnkyu Shoreikai, Osaka), respectively. Silica gel G was purchased from Merck Co., (Germany. All othrr chemicals were of commc,rcial source (reagent grade). RESULTS
Morphogenesis of the Thyoid Bufo bufo jnponicus
in
The thyroid first appears as a single median thickening of the pharyngeal epithelium, which then evaginates from the floor of the pharynx between the base of the second pair of the visceral arches at stage 26. The cvagination becomes flask-shaped at stage 27, and gradually elongates and moves away from the pharynx at stages 28 and 29 (Fig. 1, A). A split appears at the caudal end of the structure at’ stage 30 (Fig. 1, B), and the rudiment gradually becomes divided into two lobes at stages 31 and 32 (Fig. 1, C and D). Finally, at stage 33, the two lobes are completely separated and take their final position on each side of the hyoid cartilage (Fig. 1, E). At this stage, follicular structure is not observed by light
*
’
*
L
THYROID
FIG. 1. Configuration and 3X (F). The arkqe H, heart: G, gill. )(#I.
MATURATION
IN
ANURAS
LARVAE
413
of the t,hyroid of Bzrfo hlcfo japonicus at, stage 29 (.4), 30 (B), 31 (C), 32 (II), 33 (E), of the thyroid shown ill -4, B. alld C is sllrmlmded by white dashed lines. T, thyroid;
microscopy, and thyroid cells are laden with yolk. At stage 38 (Fig. 1, F), typical thyroid follicles are observable under a light microscope. Formation and Distribution 1311-Amino Acids
of
Table 2 shows the results of a series of experiments utilizing Bufo between stages 26 and 37. Considerable formation of labeled iodoamino acids, especially MIT and T,, was commenced very early (stage
26-32). After stage 33, formation of iodoamino acids, especially DIT and T,, increased rapidly with advancing stage. The values for incorporation of Ia11 into all iodoamino acids were highest at stage 35 and decreased at stage 37. This decrease might reflect an increased utilization of iodotyrosines for iodothyronine synthesis, and an increased release of synthesized iodothyronines. The ratios of labeled MIT: DIT and T, :T, (Table 3) were generally high before &age 32 and markedly low
414
HASAOKA
TABLE I)ISTRIBUTION
OF ACCUMULATED COMPOUNDS IN
AL.
ET
I~DIOIODINE Bufo TIIDPOLE:S
2 AMONG DUHIXG
Cpm Developmental stages 26 27 SO 32 33 34 35 37
No. of animals 40 40 40 20 36 23 20 10
O~b~cThyroid-containing cartilage (T), respectively.
Organic iodocompounds
Tissue samples
Cn c C c-v*
424 407 246 680 486 1022 22184 6303
TC T T T cranial
body
part
(C),
FIG. 2. Radioautographs 27 and stage 35. Solid-line iodoamino acids.
of lalI-
OF IODO-
per animal
MIT
DIT
T3
T,
287 224 36 314 161 300 7480 1914
36 20 21 31 Yll 616 14020 3897
33 133 147 272 s 44 53 ‘2s
6X SO 42 63 11 62 631 469
cranioventral
after stage 33. On the other hand, the ratio of labeled iodotyrosine : iodothyronine increased between stages 32 and 33, indicating that the increase of iodotyrosine formation exceeds that of iodothyronine formation. Figure 2 shows representative radioautographs of chromatographed iodoamino acids. At stage 27, a dense spot of 1311MIT and a distinct spot of lS11-T3 were seen, but the spots corresponding to
V.~RIOUS ~wTIONS DEVELOPMGKT
body
part,
(C-V),
and
thyroid
wit,h
hyoid
labeled DIT and T, were faint. At stage 35, the spots of labeled DIT and T, became relatively denser and only a faint spot of labeled T, was seen. These results correspond to those listed in Tables 2 and 3, and indicate very early formation of iodoamino acids, especially of MIT and T,. Although the same concentration of 13*1was used for immersion of the animals, the absolute radioactivity counts differed remarkably among animals of different
of thin-layer chromatographed iodoamino acids obtained from circles indicate the spots corresponding to I- and ninhydrin-positive
Bufo
at st,age authentic
THYROID
TABLE RATIOS
MATURATION
IN
Bujo
Developmental stages
Iodotyrosines iodothyronines
26 27 30 32 Mean 33 34 35 37 Mean
T.DPOLES
: MIT:
DIT
3.2
8.0
1.5
11.2 1.7 10.1 7.7 0.5 0.5 0.5 0.5 0.5
0.3 1.0 1.5
33.7 6.6 31.4 11.8 21.3
Hypophysectomized 40
animals 9.0
Ta : T, 0.5 4.4 3.5 4.3 3.1 0.3 0.7 0.08 0.05 0.3
0.4
415
LARVAE
ences in absorption and excretion rates of iodide among animals at different stages. Therefore, the course of the functional development of the thyroid was clearer when the experimental results were expressed in percentage distribution of radioactivity of iodoamino acids. As shown in Fig. 3, the changes in DIT and T, were especially marked. DIT promptly increased between stages 32 and 33. T, increased from stage 26 and was high until stage 32, at stages corresponding to low DIT. T, then dropped suddenly at stage 33, corresponding to increased formation of DIT. The changes in MIT and T, were less clear. However, MIT was high at stages 26 and 27, and lou- at stages 29-31, which appeared to correspond to an increased formation of T,. With decreasing formation of T, and still low formation of DIT at stage 32, MIT increased again, thereafter decreasing gradually probably due to increased formation of DIT. The percentage of T, did not change throughout the
3
BETWEEN VARIOUS IODOCOMPOUNDS LIBELED WITH R~DIOIODINE IN DEVELOPING
ANURAN
0.5
groups (Table 2). The reasons are unknown, but this might be due to differences in lz71- content in the animal body and the immersion medium, and differ-
b all,
T’ 8 ..
60.
. a
40'
.
. .
l
i
. i
20’
0
.
. 0 -
o,
.
.
.
.
.
.
(
o
-
-
_
_
_
.
29
30
31
i1.l
.
0
0
20. l ' 0
: 0-’ 26
. 27
20. 0
0 0
; 4
28
29
30
31
32
33
_
_
_
l
34
35
36
37
Developentrl
FIG. values
3. Percentage were calculated
distribution from data
0,
; -
-
-
26
27
26
it
32
33
34
.?
35
36
31
stages
of W-labeled MIT, DIT, T3, and T1 in developing Hzljo. The plotSted shown in Table 2 and those obtained in some additional experiments.
416
HANAOKA
ET
observation period presumably due to an equilibration between formation and seof the hormone, though the cretion amount of formed 1311-Tq increased markedly between stage 34 and 35 (Table 2). Formation and Distribution of 1311-Proteins Incorporation of 1311 into proteins was evident as early as at stage 26 (Fig. 4).
From the position of the radioactivity peak, the S values of the iodinated proteins were estimated as 3-8. The SDG (sucrose density gradient) profiles of labeled proteins did not change substantially during development from stage 26 to 29, indicating no marked functional development of the thyroid with respect to iodoprotein synthesis. After stage 30, radioactive peaks appeared at a region of higher S values. At stage 32, a peak at a
4 4,
stage
02 : B
\ ‘\.
I
\ .._
-.-r-
'
10
. FRACTIONS
20
30
1
-I-,
4
0
10
FRACl TOidS 20
/../
10
20
30
0
“.
0.
\
20 FRACTIONS
31
iJ*~~~.'\i.P, .f I '.-.A.,.\.,.-.,
10
20
30
FRACTIONS
FRACTIONS
10
30
stage
1
0
30
1
7
\
"I
I
; .F s
‘r / 2'
r t
stage 26
I’!
AL.
IO
FRACTIONS
20
proteins in developing Bufo beFIG. 4. Sucrose densit,y gradient centrifugation profiles of 1311-labeled tween &ages 26 and 32. The last fraction shown in the right, end of each pattern was obt’ained from t.he bottom 1311 expressed in cpm per animal. The arrow of the centrifuge tube. The plott,ed values are the protein-bound in the figure indicat,es 19 S position determined wit,h pig 19 S t.hyroglobulin as a reference st,andard. The number of animals used was 43, 38, 72, 40, 45, and 40, respect,ively, at each stage.
THYROID
MATURATION
IN
15-17 S region became quite clear, though slight elevations of radioactivity at a similar region were already seen at stages 30 and 31. These findings indicated that formation of new iodoproteins with higher S values was initiated after stage 30. Figure 5 shows SDG profiles of labeled proteins obtained from animals at stages between 33 and 38. At stage 33, striking differences were observed from stage 32 in
10
0
FRACTIONS
10
FRACTIOYS
ANURAN
the iodination pattern. The level of incorporated 1311 into proteins increased by a factor of 10. The radioactivity peak, seen at a 15-17 S region at stage 32 (Fig. 4), shifted to a 17-18 S region. A slight shoulder on the left slope of this peak might be a remnant of the 15-17 S peak. The peaks of radioactivity of smaller proteins lowered markedly. Based on its S value, the sharp peak at a 17-18 S region
20
10
30
"
417
LARVAE
0
10
stage
FRiCTlONS
FRACTIONS
stage
37
stage
38
20
2030
35
'-4 E 2 i 7 22
82
.I
I 0
10
‘I. ...)
FRACTIONS
20
30
FIG. 5. Sucrose density gradient centrifugation profiles of W-labeled protein in developing stages 33 and 38. Indications are the same as in Fig. 4, except that an additional arrow shows tion. The number of animals was 38, 24, 24, 30, and 4, respectively, at each &age.
Bufo between the 27 S posi-
41s
HANAOKA
ET
corresponds to “immature thyroglobulin” or newly synthesized and poorly iodinated thyroglobulin. In other words, the process of thyroglobulin formation was almost completed at stage 33. After stage 33, further maturation of the thyroid protein synthetic function took place. The iodinated small proteins practically disappeared after stage 34. The S values of the main peak increased slightly to 19 S at stage 35, indicating molecular maturation of thyroglobulin. Finally, a peak appeared at a 27 S region after stage 37, possibly indicating formation of thyroglobulin dimers. The peaks of small iodoproteins, which were high before st,age 32, were sometimes found even at later stages (Fig. 5). This suggested a possibility that the changes in the SDG profiles with advancing stage were clue to differences in the rate of aggregation of small iodoproteins. If such were the case, animals at later stages,
AL.
when immersed for a short time, would show SDG profiles similar to those observed at earlier stages. In order to examine this possibility, time course study was performed using animals at stage 41. As shown in Fig. 6, all SDG profiles obtained after immersion for l-24 hr were the same in their typical features. This result indicated that the changes in SDG profiles following development were ascribed to the changes in the species of iodoproteins, ruling out the possibility of changes in aggregative processes. The SDG analysis mentioned above was carried out only with the soluble iodoproteins. It should be noted here that less than 20% of the total labeled iodoprotein was soluble in animals before stage 32, while 60-80s was soluble after stage 33. This difference in solubility suggests another feature in the developmental changes in iodoprotein synthesis between stage 32 and 33. The properties of the insoluble
1
A
‘1 II
1
0
.i
j-i.\
lo
FRACTIONS
C
.--.-.-. 2o
30
0
__.A. 10
I
I ‘...p.
FRACTIONS
2o
30
1
10 FRACTIONS
20
FIG. 6. Time dependence of sucrose density gradient Bufo. Animals at stage 41 were immersed in ‘slI-containing 24 hr (I)), respectively.
10
centrifugation medium
for
FRACTIONS
20
profile of 1311-labeled proteins in 1 hr (A), 3 hr (B), 6 hr (C), and
THYROID
MATURATION
iodoproteins are unknown at present and further detailed studies are in progress. Effects of Hypophysectomy Functional Development
on the of the Thyroid
The results mentioned in the previous sections show that distinct functional changes during the morphological development of the thyroid. However, the relation of these changes to the pituitary is unsettled since animals can develop up to stage 40 in the absence of the pituitary. When measured in hypophysectomized animals at stage 40, the labeled MIT:DIT ratio was 0.4 and labeled T, :T, ratio 0.5. Similar ratios were obtained in normal animals after stage 33 (Table 3). The ratio of labeled iodotyrosine : iodothyronine (9.0) was much higher in hypophysectomized animals than normal animals before stage 32, and close to that of normal animals after stage 33 (Table 3).
0
10
20
30
FRACTIONS
FIG. 7. Effect of protein profiles of as in Fig. 4. (A) stage 40; (B) normal ber of animals w&s
hypophysectomy on ‘3’1-labeled Bufo. Indications are the same Hypophysectomized animals at animals at stage 41. The num20 and 8, respectively.
IN
ANURAN
419
LARVAE
The SDG profile of the 1311-labeled proteins obtained from hypophysectomized animals was the same as that of normal animals, though the total amount of the labeled proteins was only one fortieth of that of the normal animals (Fig. 7). These similarities and differences indicated that, even in the absence of the pituitary, essentially normal functional development of the thyroid took place until stage 40 in the qualitative sense t.hough the iodination efficiency dropped markedly. Maturation of Thyroid Developing Xenopus
Function
in
The question whether the results obtained with Bufo are also observable in other species of anura, was examined employing Xenopus larvae. The morphological development of the Xenopus between stages 40 and 50 was approximately the same as that of Bufo between stages 30 and 35 (Table 1). The tendency of the functional changes in Xenopus was found similar to those seen in Bufo as follows: In developing Xenopus, the percentage distribution of 13’1-labeled DIT increased, that of labeled T, decreased and those of labeled MIT and T, did not, change notably (Fig. 8). The ratios between newly formed iodoamino acids shown in Table 4 generally decreased with advancing stage. The reversal of the values for MIT:DIT and for T,: T, took place between stages 40 and 46 (corresponding to stages 30 and 34 in Bufo). These changes were qualitatively the same as in Bufo. However, a critical period of rapid and extensive changes was not apparent in this study. Figure 9 shows the changes in SDG profiles of 1311-labeled iodoproteins in Xenopus. At stage 46, a distinct peak of labeled 17-18 S immature thyroglobulin appeared, together with peaks of iodinated smaller proteins. With advancing stages, labeled thyroglobulin formation increased and the proportion of smaller iodoproteins decreased. At stage 53, a stage corresponding to stage 38 in Bufo, labeled 19 S thyroglobulin was clearly demonstrated.
420
HANAOKA
RATIOS
BETWEEN
Developmental stages
No.
a,b Same c Thyroid
of animals
Tissue
10 10 10 20 20 10 5 5 5 2.5
symbols only.
as in Table
AL.
TABLE 4 IODOCOMPOUNDS LABELED ?ienopus TADPOLES
VARIOUS
40 45 -46 46 48 48 N 49 49 N 50 49 - 51 52 - 54 53 - 55 60
ET
WITH
Iodotyrosines iodothyronines
samples co C-V* F T T T T T T T
RADIOIODINE
IN
DEVELOPING
: MIT:
0.4 0.5 1.2 0.9 0 3 0 s 0.5 0. :3 0 ,5 0.3
DIT
Ts:Tr
3.9 1.1 0.7 0.4 0.7 0 .4 0.6 0 6 0.1 0.3
2.4 2.3 0. 3 0 7 0.4 0.7 0 3 0 3 0.6 0.04
2.
These results indicated the same tendency of the developmental changes as in Bufo.
results, other investigators reported that the synthesis of thyroid hormones (T, and T,) was detectable after formation of the thyroid follicles in later developmental stages (Table 1). Shellabarger and Brown (1959)) studying on Xelzopus Zaevis, were able to detect T, in the thyroid at the beginning of the prometamorphic stage. Flickinger (1964) showed that T, is detectable in premetamorphic larvae (16
DISCUSSION
The present results have provided clear evidence for very early formation of thyroid hormones, especially of T,, in Bufo bufo japonicus at stage 26, when the thyroid appears only as a thickening of the pharyngeal epithelium. In contrast to our
l
20t
0
l
40
l
l
. .
l 0
8
b
l 0
40
45
50
55
60 0eve10pmenta1
FIG.
8. Percentage
distribution
of 1311-labeled
MIT,
stages
DIT,
T,,
and Tb in developing
Xempus.
THYROID
MATURATION
IN
ANURAN
421
LARVAE
C
0 10 -
lo-
20
FRACTIklNS
30
0
10
FRACTIONS
20
B
30
0 2. ":
Y ;
2 2 e
5'
5
1'
0
FIG.
Indications
10
FRACTIONS
20
9. Sucrose density gradient are the same as in Fig.
mm) of the
30
00
10
20
30
FRACTIOWS
centrifugation profiles of W-labeled prot,eins in developing Xenopus. 4. A, stage 46; B, stages 47 and 48; C, &ages 49-51; D, stage 53.
frog (presumably Rana pipiens, though he did not show the species). In these cases, at the time of thyroid hormone formation, the thyroid was found to be filled with the follicles containing colloid. However, a recent report on the chick by Shain et al. (1972) is in agreement with our finding. They clearly demonstrated that T, and T, are formed at a very early stage of thyroid development, just when the thickening of the pharyngeal epithelium occurs. The discrepancy in the time of appearance of thyroid hormones may be resulted from different analytical procedures. Concerning the successive appearance of iodoamino acids, some investigators support the view that MIT, DIT, T,, and T, are formed in this sequence in rabbit (Waterman and Gorbman, 1953), chick (Trunnell and Wade, 1955)) Xenopus (Shellabarger and Brown, 1959) and probably in Ranu (Flickinger, 1964). Our data indicate that these iodoamino acids do not appear in this sequence, since all iodo-
amino acids are formed early at stage 26 in Bufo. However, the relative proportions of these compounds at different developmental stages indicates that the production increases in the order of MIT, T,, DIT, and T,. The early relative increase of labeled T, production can be accounted for by the facts that MIT is the first iodotyrosine amply formed, and the efficiency of coupling of iodotyrosines is unexpectedly high, as shown by a low ratio of iodotyrosines : iodothyronines, at early developmental stages. Another interesting finding is the presence of a critical period of rapid functional changes and morphological transformation of the thyroid between stages 32 and 33. While formation of labeled MIT exceeds that of DIT and formation of labeled T, exceeds that of T, before stage 32, labeled DIT is formed more than MIT and T, more than T, after stage 33. The pattern of labeled iodoprotein formation also strikingly changes between stages 32 and 33. While 3-8 protein(s) and
422
HANAOKA
then 15-17 S protein(s) are mainly labeled with 1311 before stage 32, 17-18 S 1311-labeled protein is amply formed for the first time at stage 33 and simultaneously smaller iodoproteins practically disof appear. At the same time, solubility 1311-labeled protein increases strikingly. The protein synthetic functional maturation of the thyroid is thus almost completed at stage 33. It is surprising that all these changes occur rapidly in only 20 hr during development from stage 32 to 33. Along with these functional changes, the thyroid first appears in its typical form of two separated lobes and takes the final anatomical position at both sides of the hyoid cartilage at stage 33. Colloid-containing intercellular spaces are first observed at stage 33 in our preliminary electron microscopy study. Olin et nl. (1970) and Lissitzky et al. (1971)) studying the thyroid of the human fetus and the rabbit fetus, respectively, have shown that 17-18 S iodoprotein appears at the period of formation of the intercellular colloid spaces. Since the 17-18 S iodoprotein is assumed to be immature thyroglobulin, it appears that the formation of immature thyroglobulin occurs synchronously with the formation of the thyroid follicles. On the other hand, however, such synchronization is not observed in the ammocoetes endostyle (Suzuki and Kondo, 1973). This tissue produces labeled thyroglobulin before intercellular spaces are formed. In such lower vertebrates, thyroglobulin may be synthesized without follicle formation. In Bufo, large molecules of 15-17 S 1311-protein(s) are formed during the early stages of 30-32, just before the appearance of 17-18 S immature thyroglobulin. The 15-17 S molecules do not seem to be immature thyroglobulin based on their lower S values than those of immature thyroglobulin ever reported, though the relationship between these two iodoproteins remains to be investigated. In relation to the finding of 15-17 S iodoprotein(s), it is noted that the branchial sac of tunicates, which can accumulate iodide and store organic-bound iodine, produces similar sized iodoprotein (16 S)
ET
AL.
in addition to smaller iodoproteins (Suzuki and Kondo, 1971). The formation of these various sized iodoproteins is a possibly significant fact for understanding evolution of t,hyroid functions. Before stage 29, 3-8 S iodoprotein(s) is the main component among labeled iodoproteins. Protein(s) of this size is not labeled with Ia11 in the well-developed normal thyroid of higher vertebrates but is iodinated in some thyroid tumors (Salabe and Robbins, 1970) and in the ammocoetes endostyle (Suzuki and Kondo, 1973). At present, the properties of this small iodoprotein(s) and its relationship to noniodinated 3-8 S precursors of thyroglobulin arc obscure. The possibility of extrathyroidal origin cannot be excluded, since our preliminary studies indicated the formation of similar iodoprotein(s) in nonthyroidal tissues of Bzhfo larvae. Detailed studies are in progress. The role of the anterior pituitary in thyroid morphogenesis and functional maturation of the thyroid is not clear at present. In external appearance, Bufo larvae develop up to stage 40 and thyroid functions mature even in the absence of the pituitary. The thyroid of hypophysectomized animals at stage 40 forms-labeled S 19 thyroglobulin, though in a very small amount, and the main iodotyrosine and iodothyronine are DIT and T, as in normal animals. Moreover, previous investigators have reported that the thyroid forms typical follicles in hypophysectomized anura (Allen, 1929; Etkin, 1964; Hanaoka, 1967). These observations appear to indicate that the pituitary is not necessary for the morphological and functional maturation of the thyroid during early development of anuran larvae. However, this may not necessarily exclude the possibility that the pituitary becomes involved at early developmental stages in normal animals to accelerate the observed rapid changes between stages 32 and 33, or to enhance quantitatively the functions of the mature thyroid after stage 33. The precise evidence will be obtainable when TSH assay becomes applicable to anuran larvae. In Rana pipiens, Kaye (1961) has
THYROID
IN
RIATURATION
reported that the first evidence of cir;ulating TSH is found at stage III, at which stage the thyroid contains follicles filled with colloid. REFEREXCES ALLEN, B. M. (1916). Extirpation experiments in 1 Rana pipiens larvae. Science 44, 755-757. ALLEN, B. M. (1929). The influence of the thyroid gland and the hypophysis upon growth and development of amphibian larvae. Quart. Rev, ETKIN,
Biol.
4, 32&352.
W. (1964). Metamorphosis. In “Physiology of Amphibia” (J. A. Moore, ed.), pp. 427-468. Academic Press, New York. FAIRCLOTH, M. A., WILLIAMS, A. D., AND FLORSHEIM, W. H. (1965). A thin-layer chromatographic method for the analysis of thyroidal ’ iodoamino acids. Anal. Biochem. 12, 437443. FLICKINGER, R. A. (1964). Sequential appearance of monoiodotyrosine, diiodotyrosine, and thyroxine in the developing frog embryo. Gen. Comp. HANAOKA,
Endocrinol.
4, 285-289.
Y. (1966). Uptake of 1311by the thyroid gland during metamorphosis in Xenopus laevis. J. Fat. Sci. Hokkaido Univ. 16, 106-112. BANAOKA, Y. (1967). The effects of posterior hypothalectomy upon the growth and metamorphosis of the tadpole of Rana pipiens. Gen. Comp. Endocrinol. 8, 417-431. <
Comp.
Endocrinol.
1, 1-19.
S. M. (1968). The application layer chromatography to the thyroid and analogues. Folia Endocrinol. 64%654.
LISSITZKY,
S.,
ROQUES,
M.,
of thinhormones Jap. 44,
TORRESANI,
J..
AND
A. (1971). Relationship between thyroglobulin biosynthesis, thyroid hormone formation and histogenesis of the fetal rabbit thyroid. In vivo studies. In “Further Advances in Thyroid Research” (K. Fellinger and R. Hofer, eds.), Vol. 1, pp. 189-199. Verlag der Wiener Medisinishen Akademie, Vienna. NIEUWKOOP, P. D., AND FABER, J. (1956). Normal JOST,
Table
of
Xenopus
laevk
Daudin.
North
Hol-
land Publ., Amsterdam. OLIN, P., EKHOLM. R., AND ALMQUIST, S. (1970). Biosynthesis of thyroglobulin related to the ultrastructure of the human fetal thyroid gland. Endocrinology 87, 1000-1014. REGAUD, E., AND MAUCHAMP, J. (1971). Ultra-
ANURAN
423
LARVAE
structure de la glande thyro’ide du Xknope larvaire normal et hypophysectomisC : corrClations avec la biosynthhse de la thyroglobuline. J. Ultrastruct. Res. 37, 664-678. SALABE, G. B., AND ROBBINS, J. (1970). The nature of particulate thyroid proteins in an experimental rat thyroid tumor. Biochim. Biophys. Acta 214, 198-206 SAXON, L., SAXI~N, E., TOIVONEN, S., AND SALIM~KI, K. (1957). Quantitative investigation on the anterior pituitary-thyroid mechanism during metamorphosis. Endocrinology 61, 35-44. SHAIN, W. G., HILFER, R., AND FONTE, V. G. (1972). Early organogenesis of the embryonic chick thyroid. I. Morphology and biochemistry. Develop. Biol 28, 202-218. SHELLABARGER, C. J., AND BROWN, J. R. (1959). The biosynthesis of thyroxine and 3: 5:3’triiodothyronine in larval and adult toads. J. Endocrinol. SHEPARD, T.
18, 9%101.
H. (1971). Development of the human fetal thyroid. 11%“Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.), pp. 767-780. Appleton-CenturyCrofts, New York. SMITH, P. E. (1916). Experimental ablation of the hypophysis in the frog embryo. Science 44,
28&282.
T., MELONI, C. R., ALGER, E., AND J. J. (1966). Separation and quantitative recovery of iodinated amino acids and iodide by thin-layer chromatography. Proc.
SOFIANIDES, C.\N.~RY.
Sot.
Exp.
Biol.
Med.
123,
646650.
S., ASD KONDO, Y. (1971). Demonstration of thyroglobulin-like iodinated proteins in t,he branchial sac of tunicntes. Gen. Comp.
SUZUKI,
Endocrinol.
17, 402-406.
S.. AND KONDO, Y. (1973). Thyroidal morphogenesis and biosynthesis of thyroglobulin before and after metamorphosis in the lamprey- Lampetru reissneri. Gen. Comp.
SUZUKI,
Endocrkol. TAHARA,
21,
451460.
U.. AND ICHIKAWA, M. (1965). Stages in the normal development of Bufo bufo japonicus. Zn “Embryology of Vertebrate” (M. Kume, ed.), pp. 178-195. (in Japanese.) Baifukan Publ., Tokyo. TRUNNELL, J. B., AND WADE, P. (1955). Fact.ors governing the development of the chick embryo thyroid. II. Chronology of the synthesis of iodinated compounds studied by chromatographic analysis. J. Clin. Endocrinol. Metab. 15, 107-117. WATERMAN, A. J., AND G~RBMAN, A. (1953). Onset of function of the foetal rabbit thyroid gland. Anat. Rec. 117, 612-613.
AND
COMPARATIVE
Morphological during
21, 410423
ENDOCRINOLOGY
(1973)
and Functional Early Development
Maturation of the Thyroid of Anuran Larvae
YOICHI HANAOKA, SAKUJI MIYASHITA YOICHI KONDO, YASUO KOBAYASHI AND KIYOSHI YAMAMOTO Institute
of Endocrinology,
Gunma
Received
University,
November
Maebashi,
KOYA,
Japan
371
12, 1972
In Bufo bufo jnponicus, synthesis of thyroid hormones, as measured by ‘3iI-incorporation, developed very early, and small but significant amounts of iodothyronines were formed as early as stage 26, at which time the thyroid was recognized only as a simple thickening of the pharyngeal epithelium. The thyroid appeared in its typical form, and 13’1-labeled thyroglobulin was formed in an appreciable amount at st,age 33. Before this stage, soluble Y-proteins were formed only in small amounts, and the molecular sizes of these proteins were smaller than that of thyroglobulin. The changes in iodoamino acid formation were also quite critical between stages 32 and 33. The main iodotyrosine was monoiodotyrosine and the main iodothyronine was triiodothyronine before stage 32. After stage 33, diiodotyrosine was the main iodotyrosine and thyroxine the main iodothyronine. Similar sequential maturation of the thyroid function was observed also in Xenopus Zaevis.
The involvement of the thyroid in anuran metamorphosis is well known. It is generally agreed that the rate of 1311uptake by the thyroid is low during premetamorphic stages, increases during prometamorphic stages, and reaches a maximum at the beginning of the metamorphic climax (Saxkn et al., 1957; Kaye, 1961; Hanaoka, 1966). In Xenopus Zaevis, the rate of biosynthesis of thyroglobulin increases gradually during prometamorphic stages and reaches its peak approximately at the beginning of the climax, as observed by incorporation of labeled amino acid (Regaud and Mauchamp, 1971). These earlier investigations, however, were concerned with the thyroid after completion of its morphogenesis. Flickinger (1964) has reported that monoiodotyrosine (MIT) and diiodotyrosine (DIT) are formed earlier in development (body length, 11 mm) than thyroxine (T4), which is detectable only in premetamorphic stages (16 mm) of the frog larvae. Similar sequential appear410 Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reserved.
ante of these thyroidal products is also demonstrated in Senopus larvae by Shellabarger and Brown (1959), in chick embryos by Trunnell and Wade (1955)) and in rabbit fetuses by Waterman and Gorbman (1953). However, Shepard (1971) did not observe such a sequence in human fetuses. At present, little information is available with respect to the initiation period of biosynthesis of thyroid hormones and thyroglobulin, in relation to morphogenesis of the thyroid. Olin et al. (1970) have reported that thyroglobulin is formed in the thyroid of human fetuses (body length, 60 mm). At this stage, colloid-containing intercellular spaces are detectable by electron microscopy. Suzuki and Kondo (1973) have found thyroglobulin synthesis in the endostyle of ammocoetes before structural transformation of the tissue into the thyroid. These workers, however, did not give data on the initiation of thyroid hormone formation. The present work was carried out in order to clarify correlations between
THYROlD
MATURATION
IN
AND
Bufo
METHODS
The embryos of Bufo bufo jnponicus were obtained from a batch of eggs collected in the vicinity of Maebashi, and grown at 21°C. Developmental stages were determined after Tahara and Ichikawa (1965). The eggs of Xenopus laevis were obtained by induced ovulation after injection of 200 IU of human chorionic gonadotropin. Developmental stages of Xerwpus were classified according to the “Normal Table” of Nieuwkoop and Faber (1956). Table 1 indicates some morphological and physiological characteristics of
STAGES IN THF NORMAL I~VBLOPMENT ST.ir,es
411
LARVrlE
at each stage, and the stages of Xenopus approximately corresponding to those of Bufo. For morphological observation, the animals were fixed in Bouin’s fixative. After fixation, the ventral skin and the muscles covering thyroid area were removed under a dissecting microscope, and the thyroid was exposed for photography. Further, the thyroid was investigated by light microscopy after staining with hematosylin and eosin. For biochemical analysis of 13’I-laheled iodocompounds, the animals were immersed in a 0.04% Niu-Twitty solution containing carrier-free 1311- (5 &i/ml per animal) at 18 + 0.5”C for 24 hr. After immersion, the animals were washed thoroughly with tap water to remove ?I- adhering on the body surface, and the developmental stages were determined. Before stage 32, the whole cranial part or the cranioventral part of t,he body was taken, since it was difficult to isolate the thyroid at these early stages. After
the morphogenesis of the thyroid and the course of its functional development, employing Bufo larvae of very early developmental stages. The functional development was followed by the changes in biosynthesis of 13’1-labeled iodoproteins and iodoamino acids. MATERIALS
ANURAN
TABLE 1 OF Bufo bufo japonicus 0F Xmopus
AND CORRESPONDING
laeris
Developmental (Tahara and Ichikawa, 1965)
Bufo
Morphological
and functional
characteristics of
Bu,fo
larvae
Formation of t,wo gill rudiment#s Branches of the second external gill distinct Fingerlike external gill becoming transparent; beginning of circulation Epidermis becoming transparent; stomodeal invagination much deeper Mouth open; anlage of larval teeth beginning to develop Cornea transparent; beginning of formation of opercular folds; hind-limb bud visible for the first time External gill beginning to degenerate; larval teeth developed Opercular folds covered with right external gill; cloaca open Premefamorphic stage Operculum completely covered with external gill Hind-limb bud semicircular Hind-limb bltd longer than broad; first indication struction First and second toe prominences distinct Four toes distinct Pronwtamorphir
of ankle con-
Xenopus (Nieuwkoop and Faber, 1956)
26 27 28
3,5-36 37-3x 39
29
39
30 31
40 41-42
32
43-44
33
45
34 35 36
46-48 49-50 51
37 38
52 53-54
39 40 41
55 56-57 58
42
59-60
stage
All five toes distinct Five toes elongating: femur, cubitus, and crus distinct Cloaca1 tail piece disappeared Metamorphic
st,ages
climax
Forelegs broken through --
412
HANAOKA
stage 33, the thyroid became easily recognizable under a dissecting microscope (Fig. l), and the gland, together with a small piece of the hyoid cartilage, was dissected without damage. Similar tissue samples were also taken from Xempus. These tissue samples were homogenized and used for biochemical studies. In some experiments, hypophysectomized animals were employed. Hypophysectomy was carried out by extirpation of the hypophysial anlage at the tail bud stage, after Allen (1916) and Smith (1916). Successfully hypophysectomizrd animals were distinguished by their light body color, caused by the lack of thus pars intermedia as t,he source of melanocytc-stimulating hormone. In these animals, the metamorphic process delayed and was arrested at, stage 40, caused by the lack of t,he pars distalis as the POUIW of thyroid-stimulating hormone,. ThitAayer chromntog~aphy of iodoamino acids. The thyroid-containing tissue samples, taken from individual animals at t,lre sxne stage. were pooled and homogenized with 0.3 ml of 0.1 111 phosphate buffer (pH 8.0) containing 0.2 mg Pronase E and 0.05 mg aminopeptidase M. The homogenate was incubatctl at 37°C for 48 hr to secure almost completr hydrolysis of iodinated proteins. The hydrolyzatcx was crntrifupctl at 3000 rpm for 15 min. and 0.1 ml of the supernatant, after adding authentic, MIT, DlT, 3.5.3’triiodothyroninc (T1) a.nd Ta. was applied to the plate for thin-layer chromatography (TLC) with 0.25 mm thick silica g(ll G. The proccdurcs of TLC were essentially the same as desrribed by Faircloth et crl. (1965) and Sofianidrs et al. (1966). as detailed by Soya (1968). TLC was run first with ethyl a,cetate: methanol:4N NH,OH (7:2:1. v/v) for 1 hr and successively with PI-butanol: acetic acid: water (78:5: 17, v/v) for 3 hr. Bftrr chromatography. the plate was dried and authentic iodoamino acids were made visible by spraying with ninhydrin. Silica gel at the visualized spots corresponding to each compound was scraped off, and the radioactivity was counted wit,h a liquid scintillation spectrometer, Beckman Model LS 2OOB, using 0.5% PPO and 10% naphthalene in dioxane as a scintillation mixture. After TLC of another 0.1 ml of the supernatant, radioautography was carried out by exposing the plate, usually for 10 days, to X-ray films (Fuji Film Co.). s ucrose density gradient centrijugation of iodoproteins. The thyroid-containing tissue samples were homogenized with 0.3 ml of 0.1 M phosphate buffer (pH 7.5). The homogenate was centrifuged at 10.000 rpm for 10 min, and 0.2 ml
ET
AL.
of the supernatant was layered on 4.7 ml of a 5 to 20% linear density gradient sucrose solution, prepared with the same buffer used for homogenization. Centrifugation was performed at 38,000 rpm for 4-5 hr at 5°C. After the cent,rifugation, the centrifuge tube was punctured with a needle at the bottom, and 5-drop fractions were taken in test tubts. Each fraction was applied to a paper strip and chromatographed with butanol: rthanol:2AV ammonia (5:1:2, v/v) for 2448 hr, in order to cause nonprotein iodocompounds to migrate away from the origin. After drying the paper, the origin arca was cut, and the radioactivity of ‘“‘I-labeled proteins was counted with a Nuclear Chicago well-type scintillation spectrometer. In the presc>nt study, since only the radioactivity of labeled iodoamino acids and iodoproteins was measured, the results do not show absolute amounts of any- iodocompounds, though the radioactivity can rrpresent the amounts of newly formed iodo~om~rounda. Carrier-frrr Nn’“‘I \vas purchased from Japan Radioisotope Association. Tokyo. Human chorionic gonadotrollin was obtained from TeikokuZoki Co., Tokyo. Pronase E and aminopeptidase M were obtained from K&n Co., Tokyo and R&m & Haas (“o., Germany (distributed by Tanpakushitsu Krnkyu Shoreikai, Osaka), respectively. Silica gel G was purchased from Merck Co., (Germany. All othrr chemicals were of commc,rcial source (reagent grade). RESULTS
Morphogenesis of the Thyoid Bufo bufo jnponicus
in
The thyroid first appears as a single median thickening of the pharyngeal epithelium, which then evaginates from the floor of the pharynx between the base of the second pair of the visceral arches at stage 26. The cvagination becomes flask-shaped at stage 27, and gradually elongates and moves away from the pharynx at stages 28 and 29 (Fig. 1, A). A split appears at the caudal end of the structure at’ stage 30 (Fig. 1, B), and the rudiment gradually becomes divided into two lobes at stages 31 and 32 (Fig. 1, C and D). Finally, at stage 33, the two lobes are completely separated and take their final position on each side of the hyoid cartilage (Fig. 1, E). At this stage, follicular structure is not observed by light
*
’
*
L
THYROID
FIG. 1. Configuration and 3X (F). The arkqe H, heart: G, gill. )(#I.
MATURATION
IN
ANURAS
LARVAE
413
of the t,hyroid of Bzrfo hlcfo japonicus at, stage 29 (.4), 30 (B), 31 (C), 32 (II), 33 (E), of the thyroid shown ill -4, B. alld C is sllrmlmded by white dashed lines. T, thyroid;
microscopy, and thyroid cells are laden with yolk. At stage 38 (Fig. 1, F), typical thyroid follicles are observable under a light microscope. Formation and Distribution 1311-Amino Acids
of
Table 2 shows the results of a series of experiments utilizing Bufo between stages 26 and 37. Considerable formation of labeled iodoamino acids, especially MIT and T,, was commenced very early (stage
26-32). After stage 33, formation of iodoamino acids, especially DIT and T,, increased rapidly with advancing stage. The values for incorporation of Ia11 into all iodoamino acids were highest at stage 35 and decreased at stage 37. This decrease might reflect an increased utilization of iodotyrosines for iodothyronine synthesis, and an increased release of synthesized iodothyronines. The ratios of labeled MIT: DIT and T, :T, (Table 3) were generally high before &age 32 and markedly low
414
HASAOKA
TABLE I)ISTRIBUTION
OF ACCUMULATED COMPOUNDS IN
AL.
ET
I~DIOIODINE Bufo TIIDPOLE:S
2 AMONG DUHIXG
Cpm Developmental stages 26 27 SO 32 33 34 35 37
No. of animals 40 40 40 20 36 23 20 10
O~b~cThyroid-containing cartilage (T), respectively.
Organic iodocompounds
Tissue samples
Cn c C c-v*
424 407 246 680 486 1022 22184 6303
TC T T T cranial
body
part
(C),
FIG. 2. Radioautographs 27 and stage 35. Solid-line iodoamino acids.
of lalI-
OF IODO-
per animal
MIT
DIT
T3
T,
287 224 36 314 161 300 7480 1914
36 20 21 31 Yll 616 14020 3897
33 133 147 272 s 44 53 ‘2s
6X SO 42 63 11 62 631 469
cranioventral
after stage 33. On the other hand, the ratio of labeled iodotyrosine : iodothyronine increased between stages 32 and 33, indicating that the increase of iodotyrosine formation exceeds that of iodothyronine formation. Figure 2 shows representative radioautographs of chromatographed iodoamino acids. At stage 27, a dense spot of 1311MIT and a distinct spot of lS11-T3 were seen, but the spots corresponding to
V.~RIOUS ~wTIONS DEVELOPMGKT
body
part,
(C-V),
and
thyroid
wit,h
hyoid
labeled DIT and T, were faint. At stage 35, the spots of labeled DIT and T, became relatively denser and only a faint spot of labeled T, was seen. These results correspond to those listed in Tables 2 and 3, and indicate very early formation of iodoamino acids, especially of MIT and T,. Although the same concentration of 13*1was used for immersion of the animals, the absolute radioactivity counts differed remarkably among animals of different
of thin-layer chromatographed iodoamino acids obtained from circles indicate the spots corresponding to I- and ninhydrin-positive
Bufo
at st,age authentic
THYROID
TABLE RATIOS
MATURATION
IN
Bujo
Developmental stages
Iodotyrosines iodothyronines
26 27 30 32 Mean 33 34 35 37 Mean
T.DPOLES
: MIT:
DIT
3.2
8.0
1.5
11.2 1.7 10.1 7.7 0.5 0.5 0.5 0.5 0.5
0.3 1.0 1.5
33.7 6.6 31.4 11.8 21.3
Hypophysectomized 40
animals 9.0
Ta : T, 0.5 4.4 3.5 4.3 3.1 0.3 0.7 0.08 0.05 0.3
0.4
415
LARVAE
ences in absorption and excretion rates of iodide among animals at different stages. Therefore, the course of the functional development of the thyroid was clearer when the experimental results were expressed in percentage distribution of radioactivity of iodoamino acids. As shown in Fig. 3, the changes in DIT and T, were especially marked. DIT promptly increased between stages 32 and 33. T, increased from stage 26 and was high until stage 32, at stages corresponding to low DIT. T, then dropped suddenly at stage 33, corresponding to increased formation of DIT. The changes in MIT and T, were less clear. However, MIT was high at stages 26 and 27, and lou- at stages 29-31, which appeared to correspond to an increased formation of T,. With decreasing formation of T, and still low formation of DIT at stage 32, MIT increased again, thereafter decreasing gradually probably due to increased formation of DIT. The percentage of T, did not change throughout the
3
BETWEEN VARIOUS IODOCOMPOUNDS LIBELED WITH R~DIOIODINE IN DEVELOPING
ANURAN
0.5
groups (Table 2). The reasons are unknown, but this might be due to differences in lz71- content in the animal body and the immersion medium, and differ-
b all,
T’ 8 ..
60.
. a
40'
.
. .
l
i
. i
20’
0
.
. 0 -
o,
.
.
.
.
.
.
(
o
-
-
_
_
_
.
29
30
31
i1.l
.
0
0
20. l ' 0
: 0-’ 26
. 27
20. 0
0 0
; 4
28
29
30
31
32
33
_
_
_
l
34
35
36
37
Developentrl
FIG. values
3. Percentage were calculated
distribution from data
0,
; -
-
-
26
27
26
it
32
33
34
.?
35
36
31
stages
of W-labeled MIT, DIT, T3, and T1 in developing Hzljo. The plotSted shown in Table 2 and those obtained in some additional experiments.
416
HANAOKA
ET
observation period presumably due to an equilibration between formation and seof the hormone, though the cretion amount of formed 1311-Tq increased markedly between stage 34 and 35 (Table 2). Formation and Distribution of 1311-Proteins Incorporation of 1311 into proteins was evident as early as at stage 26 (Fig. 4).
From the position of the radioactivity peak, the S values of the iodinated proteins were estimated as 3-8. The SDG (sucrose density gradient) profiles of labeled proteins did not change substantially during development from stage 26 to 29, indicating no marked functional development of the thyroid with respect to iodoprotein synthesis. After stage 30, radioactive peaks appeared at a region of higher S values. At stage 32, a peak at a
4 4,
stage
02 : B
\ ‘\.
I
\ .._
-.-r-
'
10
. FRACTIONS
20
30
1
-I-,
4
0
10
FRACl TOidS 20
/../
10
20
30
0
“.
0.
\
20 FRACTIONS
31
iJ*~~~.'\i.P, .f I '.-.A.,.\.,.-.,
10
20
30
FRACTIONS
FRACTIONS
10
30
stage
1
0
30
1
7
\
"I
I
; .F s
‘r / 2'
r t
stage 26
I’!
AL.
IO
FRACTIONS
20
proteins in developing Bufo beFIG. 4. Sucrose densit,y gradient centrifugation profiles of 1311-labeled tween &ages 26 and 32. The last fraction shown in the right, end of each pattern was obt’ained from t.he bottom 1311 expressed in cpm per animal. The arrow of the centrifuge tube. The plott,ed values are the protein-bound in the figure indicat,es 19 S position determined wit,h pig 19 S t.hyroglobulin as a reference st,andard. The number of animals used was 43, 38, 72, 40, 45, and 40, respect,ively, at each stage.
THYROID
MATURATION
IN
15-17 S region became quite clear, though slight elevations of radioactivity at a similar region were already seen at stages 30 and 31. These findings indicated that formation of new iodoproteins with higher S values was initiated after stage 30. Figure 5 shows SDG profiles of labeled proteins obtained from animals at stages between 33 and 38. At stage 33, striking differences were observed from stage 32 in
10
0
FRACTIONS
10
FRACTIOYS
ANURAN
the iodination pattern. The level of incorporated 1311 into proteins increased by a factor of 10. The radioactivity peak, seen at a 15-17 S region at stage 32 (Fig. 4), shifted to a 17-18 S region. A slight shoulder on the left slope of this peak might be a remnant of the 15-17 S peak. The peaks of radioactivity of smaller proteins lowered markedly. Based on its S value, the sharp peak at a 17-18 S region
20
10
30
"
417
LARVAE
0
10
stage
FRiCTlONS
FRACTIONS
stage
37
stage
38
20
2030
35
'-4 E 2 i 7 22
82
.I
I 0
10
‘I. ...)
FRACTIONS
20
30
FIG. 5. Sucrose density gradient centrifugation profiles of W-labeled protein in developing stages 33 and 38. Indications are the same as in Fig. 4, except that an additional arrow shows tion. The number of animals was 38, 24, 24, 30, and 4, respectively, at each &age.
Bufo between the 27 S posi-
41s
HANAOKA
ET
corresponds to “immature thyroglobulin” or newly synthesized and poorly iodinated thyroglobulin. In other words, the process of thyroglobulin formation was almost completed at stage 33. After stage 33, further maturation of the thyroid protein synthetic function took place. The iodinated small proteins practically disappeared after stage 34. The S values of the main peak increased slightly to 19 S at stage 35, indicating molecular maturation of thyroglobulin. Finally, a peak appeared at a 27 S region after stage 37, possibly indicating formation of thyroglobulin dimers. The peaks of small iodoproteins, which were high before st,age 32, were sometimes found even at later stages (Fig. 5). This suggested a possibility that the changes in the SDG profiles with advancing stage were clue to differences in the rate of aggregation of small iodoproteins. If such were the case, animals at later stages,
AL.
when immersed for a short time, would show SDG profiles similar to those observed at earlier stages. In order to examine this possibility, time course study was performed using animals at stage 41. As shown in Fig. 6, all SDG profiles obtained after immersion for l-24 hr were the same in their typical features. This result indicated that the changes in SDG profiles following development were ascribed to the changes in the species of iodoproteins, ruling out the possibility of changes in aggregative processes. The SDG analysis mentioned above was carried out only with the soluble iodoproteins. It should be noted here that less than 20% of the total labeled iodoprotein was soluble in animals before stage 32, while 60-80s was soluble after stage 33. This difference in solubility suggests another feature in the developmental changes in iodoprotein synthesis between stage 32 and 33. The properties of the insoluble
1
A
‘1 II
1
0
.i
j-i.\
lo
FRACTIONS
C
.--.-.-. 2o
30
0
__.A. 10
I
I ‘...p.
FRACTIONS
2o
30
1
10 FRACTIONS
20
FIG. 6. Time dependence of sucrose density gradient Bufo. Animals at stage 41 were immersed in ‘slI-containing 24 hr (I)), respectively.
10
centrifugation medium
for
FRACTIONS
20
profile of 1311-labeled proteins in 1 hr (A), 3 hr (B), 6 hr (C), and
THYROID
MATURATION
iodoproteins are unknown at present and further detailed studies are in progress. Effects of Hypophysectomy Functional Development
on the of the Thyroid
The results mentioned in the previous sections show that distinct functional changes during the morphological development of the thyroid. However, the relation of these changes to the pituitary is unsettled since animals can develop up to stage 40 in the absence of the pituitary. When measured in hypophysectomized animals at stage 40, the labeled MIT:DIT ratio was 0.4 and labeled T, :T, ratio 0.5. Similar ratios were obtained in normal animals after stage 33 (Table 3). The ratio of labeled iodotyrosine : iodothyronine (9.0) was much higher in hypophysectomized animals than normal animals before stage 32, and close to that of normal animals after stage 33 (Table 3).
0
10
20
30
FRACTIONS
FIG. 7. Effect of protein profiles of as in Fig. 4. (A) stage 40; (B) normal ber of animals w&s
hypophysectomy on ‘3’1-labeled Bufo. Indications are the same Hypophysectomized animals at animals at stage 41. The num20 and 8, respectively.
IN
ANURAN
419
LARVAE
The SDG profile of the 1311-labeled proteins obtained from hypophysectomized animals was the same as that of normal animals, though the total amount of the labeled proteins was only one fortieth of that of the normal animals (Fig. 7). These similarities and differences indicated that, even in the absence of the pituitary, essentially normal functional development of the thyroid took place until stage 40 in the qualitative sense t.hough the iodination efficiency dropped markedly. Maturation of Thyroid Developing Xenopus
Function
in
The question whether the results obtained with Bufo are also observable in other species of anura, was examined employing Xenopus larvae. The morphological development of the Xenopus between stages 40 and 50 was approximately the same as that of Bufo between stages 30 and 35 (Table 1). The tendency of the functional changes in Xenopus was found similar to those seen in Bufo as follows: In developing Xenopus, the percentage distribution of 13’1-labeled DIT increased, that of labeled T, decreased and those of labeled MIT and T, did not, change notably (Fig. 8). The ratios between newly formed iodoamino acids shown in Table 4 generally decreased with advancing stage. The reversal of the values for MIT:DIT and for T,: T, took place between stages 40 and 46 (corresponding to stages 30 and 34 in Bufo). These changes were qualitatively the same as in Bufo. However, a critical period of rapid and extensive changes was not apparent in this study. Figure 9 shows the changes in SDG profiles of 1311-labeled iodoproteins in Xenopus. At stage 46, a distinct peak of labeled 17-18 S immature thyroglobulin appeared, together with peaks of iodinated smaller proteins. With advancing stages, labeled thyroglobulin formation increased and the proportion of smaller iodoproteins decreased. At stage 53, a stage corresponding to stage 38 in Bufo, labeled 19 S thyroglobulin was clearly demonstrated.
420
HANAOKA
RATIOS
BETWEEN
Developmental stages
No.
a,b Same c Thyroid
of animals
Tissue
10 10 10 20 20 10 5 5 5 2.5
symbols only.
as in Table
AL.
TABLE 4 IODOCOMPOUNDS LABELED ?ienopus TADPOLES
VARIOUS
40 45 -46 46 48 48 N 49 49 N 50 49 - 51 52 - 54 53 - 55 60
ET
WITH
Iodotyrosines iodothyronines
samples co C-V* F T T T T T T T
RADIOIODINE
IN
DEVELOPING
: MIT:
0.4 0.5 1.2 0.9 0 3 0 s 0.5 0. :3 0 ,5 0.3
DIT
Ts:Tr
3.9 1.1 0.7 0.4 0.7 0 .4 0.6 0 6 0.1 0.3
2.4 2.3 0. 3 0 7 0.4 0.7 0 3 0 3 0.6 0.04
2.
These results indicated the same tendency of the developmental changes as in Bufo.
results, other investigators reported that the synthesis of thyroid hormones (T, and T,) was detectable after formation of the thyroid follicles in later developmental stages (Table 1). Shellabarger and Brown (1959)) studying on Xelzopus Zaevis, were able to detect T, in the thyroid at the beginning of the prometamorphic stage. Flickinger (1964) showed that T, is detectable in premetamorphic larvae (16
DISCUSSION
The present results have provided clear evidence for very early formation of thyroid hormones, especially of T,, in Bufo bufo japonicus at stage 26, when the thyroid appears only as a thickening of the pharyngeal epithelium. In contrast to our
l
20t
0
l
40
l
l
. .
l 0
8
b
l 0
40
45
50
55
60 0eve10pmenta1
FIG.
8. Percentage
distribution
of 1311-labeled
MIT,
stages
DIT,
T,,
and Tb in developing
Xempus.
THYROID
MATURATION
IN
ANURAN
421
LARVAE
C
0 10 -
lo-
20
FRACTIklNS
30
0
10
FRACTIONS
20
B
30
0 2. ":
Y ;
2 2 e
5'
5
1'
0
FIG.
Indications
10
FRACTIONS
20
9. Sucrose density gradient are the same as in Fig.
mm) of the
30
00
10
20
30
FRACTIOWS
centrifugation profiles of W-labeled prot,eins in developing Xenopus. 4. A, stage 46; B, stages 47 and 48; C, &ages 49-51; D, stage 53.
frog (presumably Rana pipiens, though he did not show the species). In these cases, at the time of thyroid hormone formation, the thyroid was found to be filled with the follicles containing colloid. However, a recent report on the chick by Shain et al. (1972) is in agreement with our finding. They clearly demonstrated that T, and T, are formed at a very early stage of thyroid development, just when the thickening of the pharyngeal epithelium occurs. The discrepancy in the time of appearance of thyroid hormones may be resulted from different analytical procedures. Concerning the successive appearance of iodoamino acids, some investigators support the view that MIT, DIT, T,, and T, are formed in this sequence in rabbit (Waterman and Gorbman, 1953), chick (Trunnell and Wade, 1955)) Xenopus (Shellabarger and Brown, 1959) and probably in Ranu (Flickinger, 1964). Our data indicate that these iodoamino acids do not appear in this sequence, since all iodo-
amino acids are formed early at stage 26 in Bufo. However, the relative proportions of these compounds at different developmental stages indicates that the production increases in the order of MIT, T,, DIT, and T,. The early relative increase of labeled T, production can be accounted for by the facts that MIT is the first iodotyrosine amply formed, and the efficiency of coupling of iodotyrosines is unexpectedly high, as shown by a low ratio of iodotyrosines : iodothyronines, at early developmental stages. Another interesting finding is the presence of a critical period of rapid functional changes and morphological transformation of the thyroid between stages 32 and 33. While formation of labeled MIT exceeds that of DIT and formation of labeled T, exceeds that of T, before stage 32, labeled DIT is formed more than MIT and T, more than T, after stage 33. The pattern of labeled iodoprotein formation also strikingly changes between stages 32 and 33. While 3-8 protein(s) and
422
HANAOKA
then 15-17 S protein(s) are mainly labeled with 1311 before stage 32, 17-18 S 1311-labeled protein is amply formed for the first time at stage 33 and simultaneously smaller iodoproteins practically disof appear. At the same time, solubility 1311-labeled protein increases strikingly. The protein synthetic functional maturation of the thyroid is thus almost completed at stage 33. It is surprising that all these changes occur rapidly in only 20 hr during development from stage 32 to 33. Along with these functional changes, the thyroid first appears in its typical form of two separated lobes and takes the final anatomical position at both sides of the hyoid cartilage at stage 33. Colloid-containing intercellular spaces are first observed at stage 33 in our preliminary electron microscopy study. Olin et nl. (1970) and Lissitzky et al. (1971)) studying the thyroid of the human fetus and the rabbit fetus, respectively, have shown that 17-18 S iodoprotein appears at the period of formation of the intercellular colloid spaces. Since the 17-18 S iodoprotein is assumed to be immature thyroglobulin, it appears that the formation of immature thyroglobulin occurs synchronously with the formation of the thyroid follicles. On the other hand, however, such synchronization is not observed in the ammocoetes endostyle (Suzuki and Kondo, 1973). This tissue produces labeled thyroglobulin before intercellular spaces are formed. In such lower vertebrates, thyroglobulin may be synthesized without follicle formation. In Bufo, large molecules of 15-17 S 1311-protein(s) are formed during the early stages of 30-32, just before the appearance of 17-18 S immature thyroglobulin. The 15-17 S molecules do not seem to be immature thyroglobulin based on their lower S values than those of immature thyroglobulin ever reported, though the relationship between these two iodoproteins remains to be investigated. In relation to the finding of 15-17 S iodoprotein(s), it is noted that the branchial sac of tunicates, which can accumulate iodide and store organic-bound iodine, produces similar sized iodoprotein (16 S)
ET
AL.
in addition to smaller iodoproteins (Suzuki and Kondo, 1971). The formation of these various sized iodoproteins is a possibly significant fact for understanding evolution of t,hyroid functions. Before stage 29, 3-8 S iodoprotein(s) is the main component among labeled iodoproteins. Protein(s) of this size is not labeled with Ia11 in the well-developed normal thyroid of higher vertebrates but is iodinated in some thyroid tumors (Salabe and Robbins, 1970) and in the ammocoetes endostyle (Suzuki and Kondo, 1973). At present, the properties of this small iodoprotein(s) and its relationship to noniodinated 3-8 S precursors of thyroglobulin arc obscure. The possibility of extrathyroidal origin cannot be excluded, since our preliminary studies indicated the formation of similar iodoprotein(s) in nonthyroidal tissues of Bzhfo larvae. Detailed studies are in progress. The role of the anterior pituitary in thyroid morphogenesis and functional maturation of the thyroid is not clear at present. In external appearance, Bufo larvae develop up to stage 40 and thyroid functions mature even in the absence of the pituitary. The thyroid of hypophysectomized animals at stage 40 forms-labeled S 19 thyroglobulin, though in a very small amount, and the main iodotyrosine and iodothyronine are DIT and T, as in normal animals. Moreover, previous investigators have reported that the thyroid forms typical follicles in hypophysectomized anura (Allen, 1929; Etkin, 1964; Hanaoka, 1967). These observations appear to indicate that the pituitary is not necessary for the morphological and functional maturation of the thyroid during early development of anuran larvae. However, this may not necessarily exclude the possibility that the pituitary becomes involved at early developmental stages in normal animals to accelerate the observed rapid changes between stages 32 and 33, or to enhance quantitatively the functions of the mature thyroid after stage 33. The precise evidence will be obtainable when TSH assay becomes applicable to anuran larvae. In Rana pipiens, Kaye (1961) has
THYROID
IN
RIATURATION
reported that the first evidence of cir;ulating TSH is found at stage III, at which stage the thyroid contains follicles filled with colloid. REFEREXCES ALLEN, B. M. (1916). Extirpation experiments in 1 Rana pipiens larvae. Science 44, 755-757. ALLEN, B. M. (1929). The influence of the thyroid gland and the hypophysis upon growth and development of amphibian larvae. Quart. Rev, ETKIN,
Biol.
4, 32&352.
W. (1964). Metamorphosis. In “Physiology of Amphibia” (J. A. Moore, ed.), pp. 427-468. Academic Press, New York. FAIRCLOTH, M. A., WILLIAMS, A. D., AND FLORSHEIM, W. H. (1965). A thin-layer chromatographic method for the analysis of thyroidal ’ iodoamino acids. Anal. Biochem. 12, 437443. FLICKINGER, R. A. (1964). Sequential appearance of monoiodotyrosine, diiodotyrosine, and thyroxine in the developing frog embryo. Gen. Comp. HANAOKA,
Endocrinol.
4, 285-289.
Y. (1966). Uptake of 1311by the thyroid gland during metamorphosis in Xenopus laevis. J. Fat. Sci. Hokkaido Univ. 16, 106-112. BANAOKA, Y. (1967). The effects of posterior hypothalectomy upon the growth and metamorphosis of the tadpole of Rana pipiens. Gen. Comp. Endocrinol. 8, 417-431. <
Comp.
Endocrinol.
1, 1-19.
S. M. (1968). The application layer chromatography to the thyroid and analogues. Folia Endocrinol. 64%654.
LISSITZKY,
S.,
ROQUES,
M.,
of thinhormones Jap. 44,
TORRESANI,
J..
AND
A. (1971). Relationship between thyroglobulin biosynthesis, thyroid hormone formation and histogenesis of the fetal rabbit thyroid. In vivo studies. In “Further Advances in Thyroid Research” (K. Fellinger and R. Hofer, eds.), Vol. 1, pp. 189-199. Verlag der Wiener Medisinishen Akademie, Vienna. NIEUWKOOP, P. D., AND FABER, J. (1956). Normal JOST,
Table
of
Xenopus
laevk
Daudin.
North
Hol-
land Publ., Amsterdam. OLIN, P., EKHOLM. R., AND ALMQUIST, S. (1970). Biosynthesis of thyroglobulin related to the ultrastructure of the human fetal thyroid gland. Endocrinology 87, 1000-1014. REGAUD, E., AND MAUCHAMP, J. (1971). Ultra-
ANURAN
423
LARVAE
structure de la glande thyro’ide du Xknope larvaire normal et hypophysectomisC : corrClations avec la biosynthhse de la thyroglobuline. J. Ultrastruct. Res. 37, 664-678. SALABE, G. B., AND ROBBINS, J. (1970). The nature of particulate thyroid proteins in an experimental rat thyroid tumor. Biochim. Biophys. Acta 214, 198-206 SAXON, L., SAXI~N, E., TOIVONEN, S., AND SALIM~KI, K. (1957). Quantitative investigation on the anterior pituitary-thyroid mechanism during metamorphosis. Endocrinology 61, 35-44. SHAIN, W. G., HILFER, R., AND FONTE, V. G. (1972). Early organogenesis of the embryonic chick thyroid. I. Morphology and biochemistry. Develop. Biol 28, 202-218. SHELLABARGER, C. J., AND BROWN, J. R. (1959). The biosynthesis of thyroxine and 3: 5:3’triiodothyronine in larval and adult toads. J. Endocrinol. SHEPARD, T.
18, 9%101.
H. (1971). Development of the human fetal thyroid. 11%“Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.), pp. 767-780. Appleton-CenturyCrofts, New York. SMITH, P. E. (1916). Experimental ablation of the hypophysis in the frog embryo. Science 44,
28&282.
T., MELONI, C. R., ALGER, E., AND J. J. (1966). Separation and quantitative recovery of iodinated amino acids and iodide by thin-layer chromatography. Proc.
SOFIANIDES, C.\N.~RY.
Sot.
Exp.
Biol.
Med.
123,
646650.
S., ASD KONDO, Y. (1971). Demonstration of thyroglobulin-like iodinated proteins in t,he branchial sac of tunicntes. Gen. Comp.
SUZUKI,
Endocrinol.
17, 402-406.
S.. AND KONDO, Y. (1973). Thyroidal morphogenesis and biosynthesis of thyroglobulin before and after metamorphosis in the lamprey- Lampetru reissneri. Gen. Comp.
SUZUKI,
Endocrkol. TAHARA,
21,
451460.
U.. AND ICHIKAWA, M. (1965). Stages in the normal development of Bufo bufo japonicus. Zn “Embryology of Vertebrate” (M. Kume, ed.), pp. 178-195. (in Japanese.) Baifukan Publ., Tokyo. TRUNNELL, J. B., AND WADE, P. (1955). Fact.ors governing the development of the chick embryo thyroid. II. Chronology of the synthesis of iodinated compounds studied by chromatographic analysis. J. Clin. Endocrinol. Metab. 15, 107-117. WATERMAN, A. J., AND G~RBMAN, A. (1953). Onset of function of the foetal rabbit thyroid gland. Anat. Rec. 117, 612-613.