Thyroidal Iodine Metabolism During the Development of the Chick Embryo NICOLE DAUGERAS,1 ANDR&E BRISSON, FRANgOISE LAPOINTE-BOULU, AND FRANCOIS LACHIVER Laboratoire de Physiologie Generate et Comparee du Museum et d'Endocrinologie Associe au C.N.R.S., 7, Rue Cuvier, 75005 Paris, France
studied (from day 11 to day 20). The degree of dissociation with sodium dodecyl sulfate (SDS) of the TG into 12 S subunits decreased as the degree of iodination of the TG increased. Throughout embryonic development, iodine was bound more and more to TG molecules, which were resistant to dissociation with SDS. While the average iodine content of the TG increased, no appreciable changes were found in iodotyrosine and iodothyronine percentages of TG-bound iodine: monoiodotyrosine, 26%; diiodotyrosine, 43%; thyroxine 12%; 3,5,3'triiodothyronine, 2.5%. As a consequence, a linear relationship existed for each iodoamino acid between the number of its residues per mole of TG and the iodine content of TG (127I atoms per mole)— about 30 atoms of iodine was required to form 1 mole of T4. The low efficiency of the TG of the chick embryo as a thyroidal hormone-forming protein was compensated for by its high degree of iodination. (Endocrinology 98: 1321, 1976)
ABSTRACT. Stable iodine was measured in the thyroid gland of the chick embryo from day 9 to day 20 of incubation in order to evaluate quantitatively the functional development of the gland. Total iodine content increased progressively during incubation. From day 9 to day 17 of incubation, this increase resulted from the increases of pellet-bound iodine and of soluble iodine. Afterwards, it essentially paralleled the increase of the soluble thyroglobulinbound iodine which reflected the increase in both thyroglobulin content and the degree of iodination of the thyroglobulin. The total iodine, thyroglobulinbound iodine and thyroglobulin (TG) content, increased as power functions of time during incubation, with critical times on days 11 and 15. Their concentrations also increased during the whole incubation period, while the iodide concentration remained roughly constant (25 ng/mg) from day 13 to day 19. Only one iodoprotein, 19.5 S TG, was found, and its heterogeneity of iodination was demonstrated during the whole period of incubation
R
ADIOACTIVE isotopes of iodine have been utilized by numerous workers to study the metabolism of iodine in the thyroid gland of the chick embryo (see references in 1). But, in the absence of knowledge of the specific activity of the radioactivity iodine available to the thyroid, these radioisotope studies could only give a qualitative estimate of thyroid function. There have been few studies of the metabolism of stable iodine, 127I, which is essential for a quantitative evaluation of thyroidal activity (1-4). In a previous study, we showed that there is a progressive increase in the quantity of stable iodine in the thyroid gland of the Received March 7, 1975. 1 Present address: Laboratoire Biologie-Vertebres, Universite Paris-Sud, 91 Orsay, France. Reprint request to: Laboratoire de Physiologie Generale et Comparee, Museum National d'Histoire Naturelle, 7, Rue Cuvier, 75005 Paris, France.
Comparee
chick embryo from day 7 to day 20 of incubation. However, the distribution of iodine in iodinated proteins and iodoamino acids has never been analyzed. In the present investigations, we have attempted to elucidate the functional ontogeny of the chick thyroid gland as a prelude to further studies on the hormonal control of the developing endocrine gland. Some preliminary reports of our work have already appeared.2 Materials and Methods Eggs were obtained in January, February, and March from White Leghorn fowls fed a standard diet containing 4.4 mg of 127I/kg. The eggs were incubated at a temperature of 37.8 ± 0.3 C in a 2
Daugeras, N., A. Brisson, F. Boulu, and F. Lachiver, Gen Comp Endocrinol 22: 376, 1974 (Abstract), and Lachiver, F., N. Daugeras, A. Brisson, and F. Boulu, Vth Conf European Thyroid Assoc, Jerusalem, 1973 (Abstract 62).
1321
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1322
DAUGERAS, BRISSON, LAPOINTE-BOULU AND LACHIVER
forced-air incubator. Thyroid glands were removed from embryos daily, from days 9 to 20 of incubation. At this stage, the separate lobes of the thyroid gland lie along the internal jugular veins (5) and can be readily distinguished and removed with the aid of a binocular microscope. The age of each embryo, expressed in days of incubation, was verified using the table of Hamburger and Hamilton (6). For each day of incubation, thyroid glands were weighed and pooled to obtain 10-40 mg of thyroidal tissue which was immediately frozen and stored at - 2 5 C. Up until day 13 of incubation, all studies were carried out with one pool of glands, and from day 14 to day 20, with two pools. On day 20, one of the pools was composed of thyroids from embryos which had commenced to break the shell. When, for a given day of incubation, including day 20, experiments were performed utilizing 2 different pools, the values for each parameter were always in close agreement and the mean values were consequently taken. Preparation of thyroid extracts The thawed glands were homogenized at 5 C in 0.5 ml of phosphate-buffered saline (PBS) with a composition of 0.1M NaCl-0.02M sodium phosphate, pH 7.4. Each homogenate was centrifuged at 105,000 x g, at 5 C, for 1 h. The supernatant was then immediately analyzed. Total and soluble iodine were determined on aliquots of homogenate and 105,000 x g supernatant, respectively, and pellet iodine was calculated from the difference between these two values. Thyroid extracts from iodine-deficient rats, pulse-labelled 24 h before sacrifice, were prepared in the same way as described above and used as markers in the experiments. Ninetyfive per cent of the total radioactivity was recovered in thyroglobulin (TG).3 The specificactivity of the rat thyroglobulin was sufficiently high (15,000 cpm/ng 127I) to ensure that the amount of iodine added was negligible. 3
Abbreviations used in the text: Q, total thyroidal I; TG, thyroglobulin; 1%, degree of iodination of thyroglobulin (127I /xg per 100 /xg of protein); MIT, 3 monoiodotyrosine; DIT, 3,5-diiodotyrosine; T3, 3,5,3'triiodothyronine; T4, thyroxine; BAW, n-butanol-acetic acid-water; BEA, n-butanol-ethanol-0.3N NH4OH; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid. 127
Endo • 1976 Vol 98 • No 5
Analysis of the 105,000 xg supernatant 1. Organically-bound iodine and iodide were determined using two methods: a. Paper electrophdresis in 0.3M phosphate, pH 7.4, 220 volts, for 30 min. Carrier-free 131Iiodide and 125I-labelled rat thyroid extract were added. The paper strips were cut into sections ( 1 x 3 cm), the radioactivity of each piece was counted, and the iodine was determined. b. Precipitation with 20% (vol/vol) trichloroacetic acid (TCA), followed by centrifugation (10,000 rpm for 15 min). Iodine was estimated in both the supernatant and the precipitate. 2. Iodoproteins. One hundred /xl of the 105,000 x g supernatant plus 20 /x\ of 125I-labelled rat thyroid extract (15,000 cpm) added as internal standard (7), were analyzed by centrifugation in a sucrose density gradient (5-20% in PBS) at 65,000 rpm in a Spinco ultracentrifuge (L2 65B), in a SW 65 rotor at 20 C for 105 min. In each fraction of the gradient, the radioactivity was counted, the quantity of protein and iodine was measured, and the thyroglobulin and thyroglobulin-bound iodine contents determined accordingly. Both values were then used to calculate the degree of iodination of the chick embryo TG in each fraction of the TG peak. The average degree of iodination of the TG, 1%, was then calculated. The sedimentation value of the chick embryo TG was determined by comparison with that of the 125I-labelled rat TG; a value of 19.2 S was assigned to the rat [I25I]TG in comparison with purified beef 19 S TG. From day 12 through day 20 of incubation, the dissociation of the TG into subunits was investigated by treatment with sodium dodecyl sulfate (SDS). SDS was added to an aliquot of each 105,000 x g supernatant to make a final concentration of 1.5 x 10"3M. The mixture was maintained for 1 h at 20 C and then ultracentrifuged (65,000 rpm for 210 min at 20 C) in a PBS-sucrose density gradient containing SDS (1.5 x 10" 3 M). Protein and iodine values of each fraction collected were determined. The degree of iodination of the dissociation-resistant and dissociated TG was calculated. 3. Iodoamino acids. One hundred (x\ of the 105,000 x g supernatant from each pool (except on days 10 and 11, when 300 i±\ and 200 fi\ were used, respectively) to which 20 /xl of 125 I-labelled rat thyroid extract was added, was
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127
I IN THE THYROID OF THE CHICK EMBRYO
TABLE 1. Quantitative distribution of thyroidal
1323
127
I of chick embryo from day 9 to day 20 of incubation Sol uble '"I
Age of embryos (days of incubation) 9 10 11 12 13 14 15 16 17 18 19 20
Number of thyroid glands 89.5 50.5 34 36 20 19 12
6 6 5.5 6 11
Average thyroid weight (mg) 0.28 0.44 0.81 1.20. 1.33 2.17 2.50 3.50 3.50 4.80 4.00 3.60
Iodide
Total '"I %of
Per gland (ng) 0.5 3.5 33 75 205 411 891
1,167 1,917 2,891 3,826 5,525
Per mg (ng)
Per gland (ng)
1.8 7.9 41 62 154 189 356 333 548 602 956
1,535
0.33 1.8 15 32 105 182 437 625 966
2,424 2,869 4,984
Per mg (ng)
total
1.2
67 51 45 43 51 44 49 54 50" 84 75 90
4 18 27 79 84 175 179 276
505 717
1,384
'"I
Per gland (ng) 0.31
Per mg (ng)
Organically bound %
11 26 43
1.1 3 6 9 19 20
6 28 69 66 75 77
50
20
90 93 102 125 210
26 27 21 31 58
88 86
1.3
5
90 96 96 96
Determinations were performed with a single pool of thyroids up until the 13th day, and from the 14th day onwards, with two pools, the mean values of which were calculated. All iodine assays were carried out in duplicate. See text for details.
precipitated with 20% (vol/vol) TCA. The mixture was kept at 5 C for 1 h before centrifugation (10,000 rpm for 15 min). The precipitate was washed twice with 1 ml of ether to remove all traces of TCA. Enzymic digestion was then carried out according to the methods of Inoue and Taurog (8) and Rolland et al. (9). The precipitate was digested at 38 C in 100 /xl of 0.1M Tris-HCl, 0.05M MMI, pH 8.6, with 0.2 mg of pronase4 and a drop of toluene, in a N2 atmosphere for 48 h. Ten fxl of leucylaminopeptidase4 (70 units/ml) was then added, and the hydrolysis was continued for 24 h. At the end of the digestion period, either 20 ixl or 50 /xl (corresponding to an iodine content ranging roughly from 80 to 200 ng) from each digestion mixture was immediately applied to Whatman 1 MM chromatography paper and developed in two ascending systems, n-butanolacetic acid-water (78:5:17) (BAW) and n-butanolethanol-0.3N NH4OH (5:1:2) (BEA). The paper chromatograms were cut into sections (1 x 3 cm); their radioactivity was measured and their iodine content was estimated. Radioactivity was always counted in a welltype scintillation counter (Nuclear-ChicagoUltrascaler II). Protein determinations were performed according to the method of Lowry et al. (10) using purified beef TG as a standard. 4 Pronase, B-grade, was purchased from Calbiochem (Lucerne Switzerland), and leucylaminopeptidase from Miles Laboratories (Bridgend, England).
All the 127I assays were performed using a semi-automatic method. The samples were digested in a thermobloc (type TB4, WRW, Germany) with a mixture of concentrated sulfuric, perchloric, and nitric acids. After dilution, 127I was determined using the ceric-arsenious reaction in a Technicon autoanalyzer. The lower limit of quantitative detection was 0.5 ng. Results Evolution and distribution roidal iodine
of total
thy-
The total iodine content per gland, Q, increased steadily from day 9 to day 20 of incubation and reached a maximum of 5.5 fig (Table 1). The slope of the curve of Q against time (t) suggests a power function of the form Q = k • t a (Fig. 1). A double logarithmic transformation of Q (ng/gland) against t (days of incubation) produced three straight lines 1-2-3 (insert of Fig. 1), corresponding to, respectively: days 9-11, days 11-15, and days 15-20 of incubation. The iodine concentration (ng/mg of thyroid) increased throughout the whole period of incubation, but, when plotted doublelogarithmically against time, only two straight lines were obtained, corresponding to days 9-11 and 11-20 of incubation. The constants k and a were calculated for both iodine content and concentration using the
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Endo i 1976 Vol 98 . No 5
DAUGERAS, BRISSON, LAPOINTE-BOULU AND LACHIVER
1324
per mg, increased markedly and accounted for 90% of total iodine by day 20 (Table 1).
i/L
•=
5
d.15
Analysis of soluble thyroidal iodine Both the electrophoretic and the TCA precipitation methods gave identical results for organically-bound iodine and iodide, which are presented in Table 1. The content of soluble iodide per gland increased gradually throughout incubation. The iodide concentration increased up until day 13, then remained roughly constant at about 25 ng/mg until day 19, in spite of an increasing amount of iodine entering the thyroid gland each day. The organically-bound iodine percentage, which was only 6% of the soluble iodine on day 9, increased rapidly on days 10 and 11, and then more slowly until it accounted for up to 96% of the soluble iodine on the last days of incubation.
^ 3 - 2 - 1 SO o> 1.0
11
1.3
\2
lo|t [days]
Total
127
Soluble
I
1Z7
I
Pellet hound
127
I
Characterization of thyroglobulin 9
10
11
12
13
14
15
16
17
18
19
20
days of incubation
FIG. 1. Total, soluble, and pellet-bound 127I per gland plotted as a function of days of incubation. See text for details. Insert: Double-logarithmic plot of total thyroidal m I , Q, against age of embryo. See text for interpretation of 1-2-3.
method of least squares regression,5 and are listed in Table 2. From day 9 to day 17 of incubation, the amounts of pellet and soluble iodine per gland increased gradually, and each contributed approximately one-half of the total iodine content of the gland (Fig. 1). Then, from day 18 onwards, the pellet iodine per gland varied erratically (Fig. 1), and the soluble iodine, expressed both per gland and 5
None of the regressions reported in this paper differed significantly from linearity (F-test, F > 0.20), and the slopes of the various straight lines obtained for each parameter on a double logarithmic plot differed significantly (P < 0.01), as shown by analysis of covariance.
As early as day 9, iodine assays of each fraction from the sucrose gradient revealed the presence of a small amount of 127I material sedimenting near rat [125I]TG. On day 10, a quite distinct [127I]TG-peak was found, corresponding to about 28% of the TABLE 2. Values of the constants a and k in equations of the form Y = kt", which describe the changes in some parameters of iodine metabolism in chick thyroid during incubation. Period of incubation Days 9-11
Days 11-15
Q [Q] TG ITG
21 16
11
Q [Q] TG ITG
4.7 x 10"21 1.5 x 10" 15
Constants
Y
a
k
34
1.4 x 10"35
Days 15-20 6.5 6
11 12
6 8.5
2.3 x 10"10 1.9 x 10"8 5 x 10"8 1.7 x lO"6 6.5 x 10"12 3.2 x 10"12 2.9 x 10"8
Q, total I2TI (ng/gland); [Q], I27I concentration (ng/mg of thyroidal tissue); TG, soluble thyroglobulin (/itg/gland); and ITG, thyroglobulin-bound iodine (ng/gland). t = days of incubation.
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127
1325
I IN THE THYROID OF THE CHICK EMBRYO
a _ 14 t h day of incubation
t ) - 2 0 t h day of incubation
Prolrtn*
(—-•)
J] 20
400
•0 is
300
40
10
200
20
5
100
19.41
I 20.01
(.—.)!%
0.5
30
20 Fraction numb«r
10
30
20 Fraction numb*
10
FIG. 2. Sucrose gradient analyses of soluble thyroidal extracts of 14 (a) and 20 (b) day old chick embryos. In each fraction of the gradient (10 drops) a protein determination and a 127I assay were performed. The degree of iodination, 1%, (i.e., l27I ^tg per 100 /xg of protein) was then calculated for each fraction of the TG peak. Sedimentation coefficients of protein and 127I peaks of chick embryo TG were estimated by comparison with rat [I25I]TG (19.2 S) added to die gradient as internal standard (not shown). T and B are the top and bottom of the gradients, respectively.
total iodine applied to the gradient. The sedimentation coefficient of this 127I-peak was 19.5 S. From day 11 onwards, the iodinated proteins were identified both by 127 I and by protein determinations, and examples of sucrose gradient analyses are given for days 14 and 20 (Figs. 2 a and b). Iodide was identified at the top of the gradients. Thyroglobulin was found to be the only iodinated protein present in the soluble thyroid extract and had a sedimentation coefficient ranging from 19.4 S to 21 S, appreciably higher than that of the marker rat [125I]TG. No significant quantities of 12 S and 27 S iodoproteins were observed. The 19 S thyroglobulin consisted of molecules with differing degrees of iodination, increasing gradually from the
slower-sedimenting molecules to the fastersedimenting ones (Figs. 2 a and b). Evolution of thyroglobulin and thyroglobulin-bound iodine contents throughout embryonic development The amounts of thyroglobulin (TG) and TG-bound iodine (ITG), expressed per gland and per mg of thyroidal tissue, rose during the whole period of incubation (Table 3). The TG-bound iodine values were in accordance with the organically-bound iodine values obtained by analysis of the 105,000 x g supernatant by electrophoresis and TCA precipitation. Both the quantity (Figs. 3 a and b) and concentration of TG and TG-bound iodine increased as power
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1326
DAUGERAS, BRISSON, LAPOINTE-BOULU AND LACHIVER
Endo • 1976 Vol 98 • No 5
TABLE 3. Thyroglobulin and thyroglobulin-bound 127I contents in chick embryo thyroids and percentage of dissociation into 12 S subunits of the thyroglobulin after treatment with sodium dodecyl sulfate Thyroglobulin-bound
Thyroglobulin (TG)
Age of embryos (days of incubation)
I (ITG) AvpTOfyp
A
Per
gland
Per mg
(Mg)
(ng)
% dissociation into 12S
Per
gland (ng)
studied (from day 11 to day 20). The degree of dissociation with sodium dodecyl sulfate (SDS) of the TG into 12 S subunits decreased as the degree of iodination of the TG increased. Throughout embryonic development, iodine was bound more and more to TG molecules, which were resistant to dissociation with SDS. While the average iodine content of the TG increased, no appreciable changes were found in iodotyrosine and iodothyronine percentages of TG-bound iodine: monoiodotyrosine, 26%; diiodotyrosine, 43%; thyroxine 12%; 3,5,3'triiodothyronine, 2.5%. As a consequence, a linear relationship existed for each iodoamino acid between the number of its residues per mole of TG and the iodine content of TG (127I atoms per mole)— about 30 atoms of iodine was required to form 1 mole of T4. The low efficiency of the TG of the chick embryo as a thyroidal hormone-forming protein was compensated for by its high degree of iodination. (Endocrinology 98: 1321, 1976)
ABSTRACT. Stable iodine was measured in the thyroid gland of the chick embryo from day 9 to day 20 of incubation in order to evaluate quantitatively the functional development of the gland. Total iodine content increased progressively during incubation. From day 9 to day 17 of incubation, this increase resulted from the increases of pellet-bound iodine and of soluble iodine. Afterwards, it essentially paralleled the increase of the soluble thyroglobulinbound iodine which reflected the increase in both thyroglobulin content and the degree of iodination of the thyroglobulin. The total iodine, thyroglobulinbound iodine and thyroglobulin (TG) content, increased as power functions of time during incubation, with critical times on days 11 and 15. Their concentrations also increased during the whole incubation period, while the iodide concentration remained roughly constant (25 ng/mg) from day 13 to day 19. Only one iodoprotein, 19.5 S TG, was found, and its heterogeneity of iodination was demonstrated during the whole period of incubation
R
ADIOACTIVE isotopes of iodine have been utilized by numerous workers to study the metabolism of iodine in the thyroid gland of the chick embryo (see references in 1). But, in the absence of knowledge of the specific activity of the radioactivity iodine available to the thyroid, these radioisotope studies could only give a qualitative estimate of thyroid function. There have been few studies of the metabolism of stable iodine, 127I, which is essential for a quantitative evaluation of thyroidal activity (1-4). In a previous study, we showed that there is a progressive increase in the quantity of stable iodine in the thyroid gland of the Received March 7, 1975. 1 Present address: Laboratoire Biologie-Vertebres, Universite Paris-Sud, 91 Orsay, France. Reprint request to: Laboratoire de Physiologie Generale et Comparee, Museum National d'Histoire Naturelle, 7, Rue Cuvier, 75005 Paris, France.
Comparee
chick embryo from day 7 to day 20 of incubation. However, the distribution of iodine in iodinated proteins and iodoamino acids has never been analyzed. In the present investigations, we have attempted to elucidate the functional ontogeny of the chick thyroid gland as a prelude to further studies on the hormonal control of the developing endocrine gland. Some preliminary reports of our work have already appeared.2 Materials and Methods Eggs were obtained in January, February, and March from White Leghorn fowls fed a standard diet containing 4.4 mg of 127I/kg. The eggs were incubated at a temperature of 37.8 ± 0.3 C in a 2
Daugeras, N., A. Brisson, F. Boulu, and F. Lachiver, Gen Comp Endocrinol 22: 376, 1974 (Abstract), and Lachiver, F., N. Daugeras, A. Brisson, and F. Boulu, Vth Conf European Thyroid Assoc, Jerusalem, 1973 (Abstract 62).
1321
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1322
DAUGERAS, BRISSON, LAPOINTE-BOULU AND LACHIVER
forced-air incubator. Thyroid glands were removed from embryos daily, from days 9 to 20 of incubation. At this stage, the separate lobes of the thyroid gland lie along the internal jugular veins (5) and can be readily distinguished and removed with the aid of a binocular microscope. The age of each embryo, expressed in days of incubation, was verified using the table of Hamburger and Hamilton (6). For each day of incubation, thyroid glands were weighed and pooled to obtain 10-40 mg of thyroidal tissue which was immediately frozen and stored at - 2 5 C. Up until day 13 of incubation, all studies were carried out with one pool of glands, and from day 14 to day 20, with two pools. On day 20, one of the pools was composed of thyroids from embryos which had commenced to break the shell. When, for a given day of incubation, including day 20, experiments were performed utilizing 2 different pools, the values for each parameter were always in close agreement and the mean values were consequently taken. Preparation of thyroid extracts The thawed glands were homogenized at 5 C in 0.5 ml of phosphate-buffered saline (PBS) with a composition of 0.1M NaCl-0.02M sodium phosphate, pH 7.4. Each homogenate was centrifuged at 105,000 x g, at 5 C, for 1 h. The supernatant was then immediately analyzed. Total and soluble iodine were determined on aliquots of homogenate and 105,000 x g supernatant, respectively, and pellet iodine was calculated from the difference between these two values. Thyroid extracts from iodine-deficient rats, pulse-labelled 24 h before sacrifice, were prepared in the same way as described above and used as markers in the experiments. Ninetyfive per cent of the total radioactivity was recovered in thyroglobulin (TG).3 The specificactivity of the rat thyroglobulin was sufficiently high (15,000 cpm/ng 127I) to ensure that the amount of iodine added was negligible. 3
Abbreviations used in the text: Q, total thyroidal I; TG, thyroglobulin; 1%, degree of iodination of thyroglobulin (127I /xg per 100 /xg of protein); MIT, 3 monoiodotyrosine; DIT, 3,5-diiodotyrosine; T3, 3,5,3'triiodothyronine; T4, thyroxine; BAW, n-butanol-acetic acid-water; BEA, n-butanol-ethanol-0.3N NH4OH; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid. 127
Endo • 1976 Vol 98 • No 5
Analysis of the 105,000 xg supernatant 1. Organically-bound iodine and iodide were determined using two methods: a. Paper electrophdresis in 0.3M phosphate, pH 7.4, 220 volts, for 30 min. Carrier-free 131Iiodide and 125I-labelled rat thyroid extract were added. The paper strips were cut into sections ( 1 x 3 cm), the radioactivity of each piece was counted, and the iodine was determined. b. Precipitation with 20% (vol/vol) trichloroacetic acid (TCA), followed by centrifugation (10,000 rpm for 15 min). Iodine was estimated in both the supernatant and the precipitate. 2. Iodoproteins. One hundred /xl of the 105,000 x g supernatant plus 20 /x\ of 125I-labelled rat thyroid extract (15,000 cpm) added as internal standard (7), were analyzed by centrifugation in a sucrose density gradient (5-20% in PBS) at 65,000 rpm in a Spinco ultracentrifuge (L2 65B), in a SW 65 rotor at 20 C for 105 min. In each fraction of the gradient, the radioactivity was counted, the quantity of protein and iodine was measured, and the thyroglobulin and thyroglobulin-bound iodine contents determined accordingly. Both values were then used to calculate the degree of iodination of the chick embryo TG in each fraction of the TG peak. The average degree of iodination of the TG, 1%, was then calculated. The sedimentation value of the chick embryo TG was determined by comparison with that of the 125I-labelled rat TG; a value of 19.2 S was assigned to the rat [I25I]TG in comparison with purified beef 19 S TG. From day 12 through day 20 of incubation, the dissociation of the TG into subunits was investigated by treatment with sodium dodecyl sulfate (SDS). SDS was added to an aliquot of each 105,000 x g supernatant to make a final concentration of 1.5 x 10"3M. The mixture was maintained for 1 h at 20 C and then ultracentrifuged (65,000 rpm for 210 min at 20 C) in a PBS-sucrose density gradient containing SDS (1.5 x 10" 3 M). Protein and iodine values of each fraction collected were determined. The degree of iodination of the dissociation-resistant and dissociated TG was calculated. 3. Iodoamino acids. One hundred (x\ of the 105,000 x g supernatant from each pool (except on days 10 and 11, when 300 i±\ and 200 fi\ were used, respectively) to which 20 /xl of 125 I-labelled rat thyroid extract was added, was
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127
I IN THE THYROID OF THE CHICK EMBRYO
TABLE 1. Quantitative distribution of thyroidal
1323
127
I of chick embryo from day 9 to day 20 of incubation Sol uble '"I
Age of embryos (days of incubation) 9 10 11 12 13 14 15 16 17 18 19 20
Number of thyroid glands 89.5 50.5 34 36 20 19 12
6 6 5.5 6 11
Average thyroid weight (mg) 0.28 0.44 0.81 1.20. 1.33 2.17 2.50 3.50 3.50 4.80 4.00 3.60
Iodide
Total '"I %of
Per gland (ng) 0.5 3.5 33 75 205 411 891
1,167 1,917 2,891 3,826 5,525
Per mg (ng)
Per gland (ng)
1.8 7.9 41 62 154 189 356 333 548 602 956
1,535
0.33 1.8 15 32 105 182 437 625 966
2,424 2,869 4,984
Per mg (ng)
total
1.2
67 51 45 43 51 44 49 54 50" 84 75 90
4 18 27 79 84 175 179 276
505 717
1,384
'"I
Per gland (ng) 0.31
Per mg (ng)
Organically bound %
11 26 43
1.1 3 6 9 19 20
6 28 69 66 75 77
50
20
90 93 102 125 210
26 27 21 31 58
88 86
1.3
5
90 96 96 96
Determinations were performed with a single pool of thyroids up until the 13th day, and from the 14th day onwards, with two pools, the mean values of which were calculated. All iodine assays were carried out in duplicate. See text for details.
precipitated with 20% (vol/vol) TCA. The mixture was kept at 5 C for 1 h before centrifugation (10,000 rpm for 15 min). The precipitate was washed twice with 1 ml of ether to remove all traces of TCA. Enzymic digestion was then carried out according to the methods of Inoue and Taurog (8) and Rolland et al. (9). The precipitate was digested at 38 C in 100 /xl of 0.1M Tris-HCl, 0.05M MMI, pH 8.6, with 0.2 mg of pronase4 and a drop of toluene, in a N2 atmosphere for 48 h. Ten fxl of leucylaminopeptidase4 (70 units/ml) was then added, and the hydrolysis was continued for 24 h. At the end of the digestion period, either 20 ixl or 50 /xl (corresponding to an iodine content ranging roughly from 80 to 200 ng) from each digestion mixture was immediately applied to Whatman 1 MM chromatography paper and developed in two ascending systems, n-butanolacetic acid-water (78:5:17) (BAW) and n-butanolethanol-0.3N NH4OH (5:1:2) (BEA). The paper chromatograms were cut into sections (1 x 3 cm); their radioactivity was measured and their iodine content was estimated. Radioactivity was always counted in a welltype scintillation counter (Nuclear-ChicagoUltrascaler II). Protein determinations were performed according to the method of Lowry et al. (10) using purified beef TG as a standard. 4 Pronase, B-grade, was purchased from Calbiochem (Lucerne Switzerland), and leucylaminopeptidase from Miles Laboratories (Bridgend, England).
All the 127I assays were performed using a semi-automatic method. The samples were digested in a thermobloc (type TB4, WRW, Germany) with a mixture of concentrated sulfuric, perchloric, and nitric acids. After dilution, 127I was determined using the ceric-arsenious reaction in a Technicon autoanalyzer. The lower limit of quantitative detection was 0.5 ng. Results Evolution and distribution roidal iodine
of total
thy-
The total iodine content per gland, Q, increased steadily from day 9 to day 20 of incubation and reached a maximum of 5.5 fig (Table 1). The slope of the curve of Q against time (t) suggests a power function of the form Q = k • t a (Fig. 1). A double logarithmic transformation of Q (ng/gland) against t (days of incubation) produced three straight lines 1-2-3 (insert of Fig. 1), corresponding to, respectively: days 9-11, days 11-15, and days 15-20 of incubation. The iodine concentration (ng/mg of thyroid) increased throughout the whole period of incubation, but, when plotted doublelogarithmically against time, only two straight lines were obtained, corresponding to days 9-11 and 11-20 of incubation. The constants k and a were calculated for both iodine content and concentration using the
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Endo i 1976 Vol 98 . No 5
DAUGERAS, BRISSON, LAPOINTE-BOULU AND LACHIVER
1324
per mg, increased markedly and accounted for 90% of total iodine by day 20 (Table 1).
i/L
•=
5
d.15
Analysis of soluble thyroidal iodine Both the electrophoretic and the TCA precipitation methods gave identical results for organically-bound iodine and iodide, which are presented in Table 1. The content of soluble iodide per gland increased gradually throughout incubation. The iodide concentration increased up until day 13, then remained roughly constant at about 25 ng/mg until day 19, in spite of an increasing amount of iodine entering the thyroid gland each day. The organically-bound iodine percentage, which was only 6% of the soluble iodine on day 9, increased rapidly on days 10 and 11, and then more slowly until it accounted for up to 96% of the soluble iodine on the last days of incubation.
^ 3 - 2 - 1 SO o> 1.0
11
1.3
\2
lo|t [days]
Total
127
Soluble
I
1Z7
I
Pellet hound
127
I
Characterization of thyroglobulin 9
10
11
12
13
14
15
16
17
18
19
20
days of incubation
FIG. 1. Total, soluble, and pellet-bound 127I per gland plotted as a function of days of incubation. See text for details. Insert: Double-logarithmic plot of total thyroidal m I , Q, against age of embryo. See text for interpretation of 1-2-3.
method of least squares regression,5 and are listed in Table 2. From day 9 to day 17 of incubation, the amounts of pellet and soluble iodine per gland increased gradually, and each contributed approximately one-half of the total iodine content of the gland (Fig. 1). Then, from day 18 onwards, the pellet iodine per gland varied erratically (Fig. 1), and the soluble iodine, expressed both per gland and 5
None of the regressions reported in this paper differed significantly from linearity (F-test, F > 0.20), and the slopes of the various straight lines obtained for each parameter on a double logarithmic plot differed significantly (P < 0.01), as shown by analysis of covariance.
As early as day 9, iodine assays of each fraction from the sucrose gradient revealed the presence of a small amount of 127I material sedimenting near rat [125I]TG. On day 10, a quite distinct [127I]TG-peak was found, corresponding to about 28% of the TABLE 2. Values of the constants a and k in equations of the form Y = kt", which describe the changes in some parameters of iodine metabolism in chick thyroid during incubation. Period of incubation Days 9-11
Days 11-15
Q [Q] TG ITG
21 16
11
Q [Q] TG ITG
4.7 x 10"21 1.5 x 10" 15
Constants
Y
a
k
34
1.4 x 10"35
Days 15-20 6.5 6
11 12
6 8.5
2.3 x 10"10 1.9 x 10"8 5 x 10"8 1.7 x lO"6 6.5 x 10"12 3.2 x 10"12 2.9 x 10"8
Q, total I2TI (ng/gland); [Q], I27I concentration (ng/mg of thyroidal tissue); TG, soluble thyroglobulin (/itg/gland); and ITG, thyroglobulin-bound iodine (ng/gland). t = days of incubation.
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127
1325
I IN THE THYROID OF THE CHICK EMBRYO
a _ 14 t h day of incubation
t ) - 2 0 t h day of incubation
Prolrtn*
(—-•)
J] 20
400
•0 is
300
40
10
200
20
5
100
19.41
I 20.01
(.—.)!%
0.5
30
20 Fraction numb«r
10
30
20 Fraction numb*
10
FIG. 2. Sucrose gradient analyses of soluble thyroidal extracts of 14 (a) and 20 (b) day old chick embryos. In each fraction of the gradient (10 drops) a protein determination and a 127I assay were performed. The degree of iodination, 1%, (i.e., l27I ^tg per 100 /xg of protein) was then calculated for each fraction of the TG peak. Sedimentation coefficients of protein and 127I peaks of chick embryo TG were estimated by comparison with rat [I25I]TG (19.2 S) added to die gradient as internal standard (not shown). T and B are the top and bottom of the gradients, respectively.
total iodine applied to the gradient. The sedimentation coefficient of this 127I-peak was 19.5 S. From day 11 onwards, the iodinated proteins were identified both by 127 I and by protein determinations, and examples of sucrose gradient analyses are given for days 14 and 20 (Figs. 2 a and b). Iodide was identified at the top of the gradients. Thyroglobulin was found to be the only iodinated protein present in the soluble thyroid extract and had a sedimentation coefficient ranging from 19.4 S to 21 S, appreciably higher than that of the marker rat [125I]TG. No significant quantities of 12 S and 27 S iodoproteins were observed. The 19 S thyroglobulin consisted of molecules with differing degrees of iodination, increasing gradually from the
slower-sedimenting molecules to the fastersedimenting ones (Figs. 2 a and b). Evolution of thyroglobulin and thyroglobulin-bound iodine contents throughout embryonic development The amounts of thyroglobulin (TG) and TG-bound iodine (ITG), expressed per gland and per mg of thyroidal tissue, rose during the whole period of incubation (Table 3). The TG-bound iodine values were in accordance with the organically-bound iodine values obtained by analysis of the 105,000 x g supernatant by electrophoresis and TCA precipitation. Both the quantity (Figs. 3 a and b) and concentration of TG and TG-bound iodine increased as power
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1326
DAUGERAS, BRISSON, LAPOINTE-BOULU AND LACHIVER
Endo • 1976 Vol 98 • No 5
TABLE 3. Thyroglobulin and thyroglobulin-bound 127I contents in chick embryo thyroids and percentage of dissociation into 12 S subunits of the thyroglobulin after treatment with sodium dodecyl sulfate Thyroglobulin-bound
Thyroglobulin (TG)
Age of embryos (days of incubation)
I (ITG) AvpTOfyp
A
Per
gland
Per mg
(Mg)
(ng)
% dissociation into 12S
Per
gland (ng)
