The uptake of I131 by embryonic chick thyroid glands in vivo and in vitro

THE UPTAKE O F BY EMBRYONIC CHICK THYROID GLANIIS I N VIVO AND

I N VITRO

ESTHER CARPEXTER,' J. BEATTIE AND R. D. CHAMBER8 Strangeways Researech Laboratory, Cambridge, England

SIXTEEN FIGURES

INTRODUCTION

Histological differentiation of the thyroid gland of the chick embryo generally begins on the 10th day of incubation with the appearance of small droplets of colloid near the nuclei of the epithelial cells (Hopkins, '35 ; Martindale, '41 ; Carpenter, '42; and Venzke, '49). The gland also becomes functional at about this time as indicated by the work of Sun ( '32)' Hopkins ( ' 3 5 ) , and Trunnell and Brayer ( ' 5 3 ) , using respectively, the presence of iodine in the gland, the more rapid metamorphosis of tadpoles fed with thyroids from 10-day chick embryos, and the selective concentration of radioactive iodine by the thyroid glands of embryos toward the end of the 10th day of incubation. Evidence of still earlier physiological activity will be discussed later in coiinection with the results of this investigation.

When thyroid glands are explanted whole from 8-day chick embryos and grou7n in vitro by the coverslip method, they differentiate histologically in a manner closely resembling that in vivo and at a similar rate (Carpenter, '42, and also unpublished results). The most striking difference is that the gland developing in vitr:, shows little if any increase in size; this is due in part to the removal of most of the out- growth at each transfer to fresh medium. Blitotic figures are

' Zoology Department, Smith College, Sorthanipton, Massachusetts.

249

250 E. CARPENTER, J. BEATTIE AND R. D. CHAMBERS

numerous but are apparently insufficient to compensate for the loss at each transfer.

The present investigation was undertaken to determine whether and to what extent thyroid glands developing in vitro were physiologically active as tested by their capacity to concentrate selectively the radioactive isotope, Some indication of such activity has been obtained by Gaillard ( ’53) , on photographic plates exposed to sections of thyroid glands explanted at approximately the same age as Car- penter’s and grown two or more days in vitro. We used a 24-hour exposure to the radioactive isotope and tested activity by two methods: (a) counts on known weights of thyroid tissue and (b) the stripping film technique for auto- radiography (Doniach and Pelc, ’SO), a method by which the sites of accumulation of radioactive material can be more precisely determined than by contact with a photographic plate and comparable in results to the “coated autograph” technique of Leblond and Gross (’48). Both methods clearly demonstrated the capacity of thyroid glands developing in vitro to concentrate Il3l in a way quantitatively and quali- tatively similar to that of glands of corresponding age developing in vivo.

ACKNOWLEDGMESTS

The authors thank Dr. Honor B. Fell, Director of The Strangeways Laboratory, and Dr. W. Jacobson for encourage- ment and help of various kinds during this investigation. We are grateful to Dr. S. R. Pelc and Dr. A. Glucksmann for suggestions in regard to the autoradiographic technique and to Miss June Rawlinson for drawing figure 1. We wish to express our thanks also to Dr. J. Boursnell of the Bio- chemistry Department, Cambridge University, for his as- sistaiice in making the counts of radioactivity, and to the Directors of Smith and Nephew Research, Ltd., for the loan of equipment and a grant in aid of expenses and technical assistance to one of us (J.B.).

UPTAKE O F 1"' BY EiMBRYONIC THYROIDS 251

MATERIALS AND METHODS

A. Tissue culture. The thyroid glands were obtained from chick embryos of 8 days' incubation. One drop of extract of an 11-12-day chick embryo from which the thyroids had been removed was mixed on a large coverslip with 4 drops of fowl plasma and allowed to clot. Two glands were deposited on the surface of the coagulum and the prepara- tion was then inverted and sealed over a large Maximow depression slide. The explants were transferred to fresh medium at two-day intervals and were grown for 8 days.

For purposes of control, the esophagus was removed from some of the 8-day embryos in series 11, I11 and IV, cut in pieces 1 to 2mm long, and grown on the surface of similar clots. I n series 11, the explants of esophagus were grown on separate clots throughout the 8-day period, but in series I11 and IV, after 4 days on a separate clot, each esophagus fragment was transplanted to the same clot as two thyroid cultures.

Normal thyroids from 8- and 16-day embryos were removed and fixed for comparison with the cultures in all series.

B. Applicatiom of the radioactive iodine. 1131 as NaI and substantially carrier-free was diluted with Tyrode's solution to the required concentration and either applied to the surface of the culture or injected into the yolk sac of the embryo 24 hours before the termination of the experiment. Cultures were opened on the 7th day in vitro and 0.01ml of solution containing 0.01 microcurie of 1131 was spread over the surface of the clot with a glass rod. The cultures were then resealed. Eggs which had been incubated for 7 and 15 days were injected at the yolk sac end with 0.5ml of solution containing 10 pc of The hole made by llie needle was sealed with paraffin.

C. Counting technique. In each series, 16 thyroid glands explanted from 8-day embryos and grown for 8 days in vitro were removed from the clots and placed in weighed and covered glass tubes. Bits of each clot were collected in a


similar tube. Pieces of esophagus from some of the same embryos, grown for the same length of time were likewise collected in all but series I. In series 11, fragments of clot from the esophagus cultures were collected for comparison, but in series I11 and IT; only one tube containing clot from each culture was necessary since thyroids and fragments of esophagus had been grown on the same clots during the last 4 days. All cultures had been exposed to a solution containing 0.01 pc of in 0.01 ml of fluid spread over the surface of the clot 24 hours previously.

Embryos, into the yolk sac of which 0.5ml of Tyrode's solution containing 10 pc of had been injected on the 7th and 15th days of incubation, were removed from the shell 24 hours later. From each embryo 0.2ml of amniotic fluid was removed and that for each age pooled. This was used for comparison with the thyroids since Wollman and Zwilling ( '53 ) found that the amniotic Auid in their embryos con- tained approximately the same concentration of as the carcass. From the pooled fluid 0.2ml of that of each age was used for testing. The thyroid glands were dissected out and those of each age were placed in weighing tubes and treated in the same manner as the cultures.

The wet weight of material in each tube was determined and the volume made up to 0.2ml by the addition of 10% NaOH. Except in series I, the amniotic fluid was undiluted with NaOH. All tubes were then heated over a hot-water bath until the solid material had dissolved. Aliquot parts of each solution (0.1 ml) were placed on nickel planchettes, a few drops of polyvinyl alcohol added, and the planchettes placed on a hot plate to dry. At least two counts were made on each sample by means of a Geiger-Muller end window counter and appropriate scaling unit. Counts were made within 24 hours of the termination of the experiment. Counts per gram per minute were then calculated for each of the materials tested and the iodine concentrating capacity of thyroid tissue as compared with that of the background sub- stance was determined.

CPTAKE OF BY EMBRYONIC THYROIDS 253

In connection with each series on which counts were made, the following thyroid glands were fixed for histological ob- servation and for autoradiographs: at least two from 8- and 1Bday embryos without and with exposure to Il3I, and at least one culture without and with such exposure. Susa’s fixative was used followed by Heidenhain’s modification of Mallory ’s connective tissue stain.

D. Autoradiographic techwique. The following material was preserved for autoradiographs in conjunction with each of the series on which counts were made: thyroids from 8- and 16-day embryos and at least two thyroids explanted from 8-day embryos and grown for 8 days in vitro. Esopha- gus cultures were fixed also. Two additional series of thyroid cultures provided more material for experiments with the stripping film technique. All cultures and embryos had been exposed to for 24 hours. Sections were cut at 6 p and mounted in the usual way.

In the earlier experiments, Susa’s fixative was employed and autoradiographs made by placing sections in contact with a process plate f o r various periods of time. In all later experiments the stripping film technique was adopted since by this method it is possible to visualize more precisely the sites of accumulation of within the gland. During the mounting of the stripping film over the sections in the dark- room a dark green light was used. The slides were dried, wrapped in black paper, and kept in the refrigerator during the period of exposure of the film. The low temperature seemed to reduce greatly the blackening of the background. After development, the slides were again thoroughly dried, stained lightly with Mayer’s hematoxylin, dried from tap water, and finally mounted in Depex.

In later experiments the thyroids were fixed in forrnol- saline, acetic alcohol followed by formol-saline, or in Carnoy. Best results were obtained vhen the tissue was fixed in Carnoy, the film exposed fo r 4 days, and brought to room temperature before development. The film may become loosened if changes of temperature are too sudden or if the solutions are too warm.


E. Periodic acid Schif reactiolz. Since Wollman and Zwil- ling ( ’53 ) found numerous PAS positive droplets in the thyroid gland of the 9-day embryonic chick and a few even in the 7-day embryo, several glands from embryos of 7 and 8 days’ incubation were fixed and stained by this method.

RESULTS

Eight-day embryo& thyroids. After 8 days’ incubation, the thyroid glands of chick embryos consist of loosely arranged cords of cells separated by irregular, sinusoidal spaces (figs. 2 and 3 ) . Nuclei may be scattered through the cords or arranged at the periphery. Mitotic figures are frequent (figs. 4 and 5 ) . No colloid droplets have been ob- served in glands fixed in Susa and stained by Heidenhain’s modification of Mallory ’s connective tissue stain, but when such glands are stained by the periodic acid Schiff method it is possible to find in the epithelial cells a few small magenta stained droplets that may represent pre-colloid.

is injected into the yolk sac on the 7th day of incubation, more is found in the thyroid glands 24 hours later than in the amniotic fluid. This was true in all series although the ratio of the concentrations varied. On the other hand, sections of thyroids from %day embryos previously exposed f o r 24 hours to have not produced any localized blackening either on process plates or on stripping film. Histologically they appear to be normal and mitotic figures are present (figs. 3 and 5 ) .

Sizteewday embryolzic thyroids. After 16 days ’ incubation, follicles in all stages of formation are present in the thyroid gland. Some are outlined by a thin layer of connective tissue and contain dense, homogeneous colloid, but some of the smaller follicles are not surrounded by connective tissue and their colloid appears thin and is often non-homogeneous. There seems to be no particular pattern of distribution of follicles of different sizes. Sinusoidal spaces are still evident especially in the middle of the gland (fig. 7 ) .

As shown in table 1, when

UPTAKE OF 1131 BY EMBRYONIC THYROIDS 255

As shown in table 1, the 16-day embryonic thyroid has a greatly increased capacity to concentrate selectively Ra- dioactivity of the gland digest was so high that the solution had to be diluted 20 times in order to obtain reliable counts. Here again the comparison was made between counts on the thyroids and on the pooled amniotic fluid from the same embryos. The great increase in uptake of Il3I at this stage i s also very apparent in the autoradiographs (figs. 12, 14, and 16). I n general the film is more heavily blackened over the larger follicles. Over follicles where the deposit is less dense it is sometimes possible to see a heavier ring of black covering the periphery of the colloid and the apical parts of the follicular cells. This shows more clearly in the slides than in the photographs.

Eigh t -day embryonic thyro ids g r o w n in vitro for 8 days . Thyroids explanted from S-day embryos and grown for 8 days in vitro have a histological appearance similar to that of the gland of the normal 16-day embryo. While the explants are usually more compact than the normal thyroid, the loose arrangement of cords in the center of the gland in vivo is frequently seen in vitro (figs. 6 and 7). Some follicles contain dense colloid and others the thinner, non-homogeneous secretion. Mitotic figures are present, but the cultures en- large little if a t all, due in part to the removal of the out- growth at each transfer. Unlike the condition found in vivo, in the thyroid which has differentiated in vitro the larger follicles are more often at the periphery of the gland (fig. 8) or occasionally near the point of attachment of the gland to the clot. Connective tissue is also more abundant at this point and at times may be distributed through the culture unevenly giving a somewhat lobulated appearance not seen in vivo. Connective tissue at the free surface of the cnltiire may be sparse or completely absent (fig. 6).

Reference to the last column in table 1 shows that when the concentration of in the 16-day embryonic thyroid is compared with that of its amniotic fluid, and the 1131 concentration in the cultures is compared with that of the surround-


ing clot, the ratios obtained are surprisingly similar in three of the 4 series. This is shown graphically in figure 1. Pieces of esophagus from the same embryos grown on the same or similar clots in order to have another tissue for comparison with the thyroid, failed to concentrate iodine above the level in that of the surrounding clot in two out of three series.

The discordant results are all in series 11. The only explanation we can offer at present is that the embryos at this time were somewhat underdeveloped and less uniform in appearance. A. few hemorrhagic spots were noted at the time in some of the embryos. So far as the esophagus cultures were concerned, it should be noted that in series 11 in which some uptake of 1131 was recorded, the cultures were grown on separate clots throughout the 8 days in vitro. In series I11 and IV at the time the radioactive isotope was added there were two thyroids and one piece of esophagus on each clot.

Reference to the autoradiographs on plate 3 shows that blackening of the film occurs over follicles of the same size range whether they have developed in vivo or in vitro. Not all follices of a given size produce an autoradiograph, however. Where the blackening is not too dense it is possible to see over some of the follicles developing both in vivo and in vitro a ring of heavier blackening covering the periphery of the colloid and the apical parts of the folliclular cells.

CONCLUSIONS

If series I1 can be disregarded on the basis of the poor quality of some of the embryos, the results show that the capacity of the embryonic thyroid glands of the chick to concentrate reaches the same order of magnitude in the interval between the 8th ar,d 16th days & incubation, regardless of whether differentiation takes place in vivo or in vitro. This is evident from the ratios given in the last column in table 1 and from figure 1. Comparison of the autoradiographs of thyroid cultures explanted from 8-day embryos and grown in vitro for 8 days with the thyroid of the 16-day embryo

UPTAKE O F 1'"' BY EMBRYONIC THYROIDS 257

TABLE 1

Uptake of Ii3' by embryonic chick thyroids in v'ivo and in vitro

SERIES TOTAL \VET COUNT PER RATIO OF WEIGHT IN GRAM PER TISSUE TO

MILLIGRAMS MINUTB X lo3 BACKGROUND

XATBRIAL TESTED

I 8 day embryo 10 thyroids 2.80 34.8 10

Oet. 1, '53 amniotic fl. .20 ml 3.5 1

8 thyroids 11.62 73,433.8 272 amniotic fl. .16 ml 270.3 1

16 thyroids .82 5,750.0 296 clot 18.91 19.4 1

16-day embryo

%day cultures

11 8 day embryo

Oct. 28, '53 amniotic fl.

16-day embryo 6 thyroids amniotic fl.

8-day cultures 16 thyroids clot esophagus clot

8 thyroids .76 1.00 ml

6.15 .80 ml

1.03 43.85

.92 23.84

1,718.4 75 22.9 1

126,640.1 1,041 121.6 1

8,023.3 102 78.9 1

1,880.4 18 106.5 1

I11 8-day embryo 14 thyroids

Dee. 16, '53 amniotic fl.

16-day embryo 4 thyroids amniotic fl.

8-day cultures 16 thyroids clot esophagus same clots

.81 111.1 20 1.60 ml 5.5 1

5.03 33,614.3 246 -60 ml 136.4 1

.90 1,382.2 275 28.23 5.0 1 2.02 2.0 0.4

1 -

I V 8 day embryo 13 thyroids 2.0'7

Jan. 2 7 , '54 amniotic fl. 1.40 7.9 4.4 1.8 1

16-day embryo 6 thyroids 6.08 22,953.9 333 amniotic fl. .60 ml 68.9 1

16 thyroids .83 2,824.1 231 clot 30.54 12.2 1 esophagus 1.36 8.8 0.7 same clots 1

%day cultures


shows that the localization of 1131 within the gland is likewise similar.

The best autoradiographs were obtained when Carnoy ' s fixative was used, the stripping film exposed for 4 days, and after development o f the film, the sections were lightly stained with Mayer's hematoxylin at not more than 20°C.

a 8 day embryonic thyroid a 16 dey embryonic thyroid

0 8 day embryonic thyroid + 8 days i n v i t r o 8 day embryonic esophagus + 8 days in vitro

1040

1000 . . . . . . . . . . . . . . 340

300

200

100

0 - - -1- -JI- -=- -131-

Fig. 1 Radioiodide concentration in relation to background.

DISCUSSION

Wollnian and Zwilling ( ' 5 3 ) have demonstrated that the chick embryonic thyroid has some capacity for concentrating radioactive iodine as early as the 7th day of incubation, but in a non-precipitable form. The iodine solution was injected into the allantoic vein and material collected for testing not more than 4 hours later. Blanquet, Stoll, Maraud and Capot ( ' 5 3 ) state that the thyroid begins to take up 1131 on the 8th day of incubation when the gland consists of simple cellular cords. In all of our counting experiments the 8-day embryonic

IJPTAKE OF 1l3I BY EMBRYONIC THYROIDS 259

thyroid likewise showed some capacity to concentrate selectively. Hansborough and Kahn ( '51), however, could not demonstrate activity in the thyroids of 10-day embryos o r younger ones, and Trunnell and Brayer ('53) report a selective concentration of only toward the elid of the 10th day. Differences in methods, in the amount injected, in the sites of injection and in the time interval between injection of the isotope and the removal of the glands may account for the varying results.

We have obtained no autoradiographs from sections of 8-day embryonic thyroids exposed to for 24 hours pre- vious to removal. The normal appearance of such glands is illustrated in figure 5. From the fact that not enough I l 3 I is present in the gland to produce an autoradiograph at this age (fig. 3 ) , it is concluded that little if any organic binding of iodine has taken place. The presumption is that any inorganic iodide which may have been present would be washed out of the tissue during the usual histological manipulations. Hansborough and Khan ( '51) were unable to demonstrate before the 11th day of incubatioii when colloid-containing follicles were present, but noted a great increase in blackening of the film on the 15th day. These authors correlated the first accumulation of with the appearance of discrete follicles not only in the chick embryo, but also in the foetal hamster ('51b). Gorbman and Evans ( '43) have pointed out a similar coincidence of iodine storage and follicular differentiation in the foetal rat. However, by the newer method of filter paper radiochromatography, Gon- zales ( '54) continuing the work of Trunnell and Brayer finds mono- and diiodotyrosine and thyroxine present in the thyroid gland at the end of 8% days of incubation.

Although the usual methods of fixing and stailling shcw no colloid in the embryonic chick thyroid until the 10th day of incubation, by using the periodic acid Schiff reaction, Wollman and Zwilling ( ' 5 3 ) found many PAS-positive droplets in the 9-day embryonic gland and a few even in the 7-day one. We have confirmed their presence in the thyroid

260 E. CABPEIYTER, J. BEATTIE AND R. D. CHAMBERS

of the 8-day embryo. In the glands examined from 7-day embryos any such droplets or granules were very small and difficult to identify with certainty.

Autoradiographs of sections of 16-day embryonic thyroids (fig. 12, 14 and 16) show localized blackening of the film in the form of circles or spheres of various sizes. These areas occur over the follicles in the underlying section and in general the blackening is denser over the larger ones. There is no particular pattern of distribution of the larger follicles and deeply blackened areas of the film, except that they are not at the periphery of the gland. In some regions where the blackening is less heavy, it can be seen that a darker ring covers the outer boundary of the colloid and the apical parts of the follicular cells (fig. 16). From an extensive study of autoradiographs of thyroids of the rat under different ex- perimental conditions and after different time intervals Le- blond and Gross ( '48) have demonstrated that thyroglobulin is formed in the apical parts of the follicular cells and rapidly secreted into the colloid. I n the developing chick thyroid, as might be expected, different stages of the process are present at the same time. The apparent lack of uniformity of distribution of II3I in the gland at this time may perhaps be accounted f o r by variations in the stage of development of the follicles and their relation to the blood supply. Whether the uniform distribution of radioiodide found by Gorbman et al. ('52) in the follicles of the thyroid of the near-term bovine foetus is attained in the embryonic chick thyroid before hatching has not yet been determined.

Thyroids explanted from 8-day embryos and grown in vitro f o r 8 days, likewise show an uneven distribution of II3I and in general more blackening of the film over the larger colloid masses. Here, also, in favorable material it is pos- sibie to see blacker rings covering the outer edge of the colloid and the apical parts of the epithelial cells. Ti'nlike the condition found in vivo, the larger follicles in the cultures are usually near the surface of the gland. This suggests that differentiation takes place more rapidly where nutritive ma-

UPTAKE O F 1"" BY EMBRYONIC THYROIUS 261

terials are more readily available. Even in such small cultures diffusion is apparently slo~v and occasionally a small, central necrotic area may be found.

Gaillard ('53 and earlier) using thyroids explanted from 84-day embryos arid a medium containing varying amounts of 11"1, was able to get autoradiographs from sections of cultures fixed two and three days after explantation. He states that although colloid droplets were found near the nnclei in cultures fixed after three days, none were seen in those fixed after two days. Carpenter ('42 and later) has found colloid appearing regularly in glands from 8-day embryos after two days of cultivation. The rate of colloid for- ination was at least as fast in vitro as in vivo and on the third day some follicular arrangement was usually present. The extract used in the medium was made from 11-day embryos, wliile that used by Gaillard mas from 9-day embryos. Gonzales ( '54) by the filter paper radiochromatography method has identified mono- and diiodotyrosine and thyroxine at the end of two days cultivation of thyroids explanted from 7-day embryos.

The counting technique reveals that the thyroid g l a d of the embryonic chick has begun to concentrate iodine selectively before any obvious histological differentiation has occurred and shows quantitatively that the concentrating capacity fo r 1131 which develops in vitro is of the same order of magnitude in relation to the background as in the gland developing undisturbed in vivo. The autoradiographic stripping film technique provides a method by vhieh the distribution of 1'"l within the gland can be visualized after the iodine is organically bound and shows that the distribution is similar whether the gland is developing in vivo or in vitro. Thinner sections and more experience may make it possible to localize the sites of accumulation of radioiodide still more precisely (Pelc and Hmvard, '52 ; and Fitzgerald, '53). Furthermore the results presented indicate that the thyroid cultivated in vitro is reliable material for experiinents to elucidate thyroid function under controlled conditions.

262 E. CARPEXTEK, J. BEATTIE AND R. D. CHAMBERS

SUMMARY

1. The thyroid gland of the 8-day embryonic chick was found to have some capacity to concentrate P1 as shown by counting niethods, confirming the work of other investi- gators. Since the autoradiographic technique failed to reveal the presence of the radioactive isotope in the gland at this age, it is assumed that little if any organic binding of iodine has occurred. Such binding may be closely correlated with the formation of colloid. Although the latter is not detectable at this age by the usual histological methods, very small PAS-positive droplets can be demonstrated in the cells by the periodic acid Schiff reaction.

2. By the 16th day in vivo, the capacity of embryonic chick thyroid to concentrate 11:$' has increased greatly and is demonstrated easily by either counting or autoradiographic methods. The most successful autoradiographs were obtained when the thyroids were fixed in Carnoy and the stripping film exposed to the sections for 4 days.

3. When thyroid glands are explanted from embryos of 8 days' incubation and grown for 8 days in vitro, they display, by counting methods, a selective absorption of 11"' of the same order of magnitude as that of thyroids from 16-day embryos. When studied by the stripping film autoradiographic technique, both the explanted glands and the normal ones of corresponding age show a similar distribution of the radioactive isotope in follicles of the same size range. A darker ring over the area including the outer edge of the colloid and the apical parts of the follicular cells indicates that this is a region of high activity. Enlargement of the gland in vitro is slight under the conditions employed although differentiation proceeds at a normal rate.

4. I n conclnsion, it has heen demonstrated tha t eirihrponic chick thyroids which have differentiated in vitro resemble glands of a corresponding age which have developed in vivo not only in their histological appearance, but also in their physiological capacity to concentrate II"' from the surrounding medium. The embryonic thyroid growing in vitro thus

TJPTAICE OF 1"" BY EMBRYONIC THYROIDS 263

provides suitable inaterial for further physiological esperi- ments under the simplified conditions of organ culture.

LITERATURE CITED

BLANQUET, P., R. STOLL, R. MARAUD ~ N D L. CAPOT 1953 The building up of tliyroxirre by the thyroid of tile chick embiyo. Bull. Soc. Chim. biol., 5.5: 627-631.

I)ifferentiation of cliiclr ciriiti yo thyro ds in tissue cultiire. J. Exp. Zool., 89: 407-431.

1954 The physiological activity of chitl: embryo thyroids grown in ri tro as indicated by uptake of radioactive iodine. Anat. Rec., 11S: 446-447.

DONIACH, I., AND S. R. PELC 1950 Biitoiadiograph teclinique. Brit. J. Radial.,

FITZGERALD, P. J. 1953 Radioautograpliy in cytology. Texas Reports on Bid. aiid Med., 11: 6i1-677.

GAILLARD, P. J . 1953 Growth and differentiation of explanted tissues. 4. Cul- ture of thyroid gland froin chick embryos. Internat. Rev. of Cytol., 2: 361-367.

CARPENTER, E.

CAXPENTER, E., J. BEATTIE A N D R. I). C H ~ X B E R S

1942

23: 184-192.

GOXZ4LFS, F. 1954 Peisoiial coniinunication. GORBMAK, A., AND €I. M. F~VANS 1943

GORBMAN, A., S. LISSITZKY, 0. M I ~ I ~ E L , R. NICHEL AND J. ROCHE

Beginning of fniictioii in the thyroid of the fe ta l rat. Kndocriuologp, :?a: 113-115.

Metabo- lism of radioiodiirr by the near teiin bovine fetus. Endocrinology,

The inLtial function of the chick thyroid gland with the use of radioiodine (Ixq1). J. Exp. Zool., 116: 447-454.

___- 195111 Accumulation of radioiodine (1'") in the thyroid gland of the liaiiister embryo. Proc. Soc. Exp. Giol. Med., 78: 481-483.

HOPICINS, 31. I,. 1933 The development of the thyroid gland in the chick embryo. J. Morph., 58: 585-604.

LEBLOND, C. P. 1943 Localization of newly adniinisterrd iodine in the thyroid glaiid as indicated hy radio-active iodine. J. Anat., 7 7 : 149-152.

LEBLOND, C. P., AND J. GROSS 1948 Thyroglobulin formation in the thyroid follicle visualized by the " Coatcrl Autograph " techniqiie. Endocrin

LILLIE, R. 11. 1948 Histopatlrologic Teclruic, p. 143. Tire Blakistoii Co., Phila- delphia.

MARTINDALE. F. M. Initiation aiid earlv derelopment of thyrotvopic fiinc- tion in the incubating chick. Anat. Bee., 79 : 373-385.

PELC, 8. R., AND A. HOWARD 1952 Twliniqiirs of aiitoradiography aiid the application of the stripping-film method to problems of nuclear metabolism. Brit. Med. Bull., 8: 132-135.

Histo-physiogenesis of the glands of internal secretion of tlie chicken embryo. Pligsiol. Zool., 5 : 384-396.

1952

5 2 : 546-561. RIXSBOROCGH, L. A., AND M. KHAX 1931a

o l o g ~ , 4 3 : 306-324.

1941

SUK, T. P. 1932

264 E. CABPENTEIL, J. BEATTIE AND R. D. CHAMBERS

TRUNNELL, J. R., AND F. T. BRAYER Factors governing the developmont of the chick embryo. 1. Determination of the time at which col- lection begins. J. Clin. Endocr. and Metab., 13: 88-94.

Morphogenesis of the thyroid glanils of chicken embryos. Am. J. Vet. Res., 10: 272-281.

1953 Radioiodine metabolism in the chick embryo. Endocrinology, 52: 526-535.

1953

VENZKE, IT. G.

WOLLMAN, S. IT., AND E. BVC-ILLING

1949

PLATE 1

EXPLANATION O F FIGURES

2 Normal 8-day embryonic chick thyroid. X 140.

3 Thyroid from 8-day chick embryo. X 140. Radioactive iodine, 1131, was injected into the yolk sac 24 hours before the embryo was remored from the shell. The section is covered with stripping film but no autoradiograph is present. The dark spots are either erythrocytes or mitotic figures. See figure 5.

4 Par t of thyroid of figure 2. X 430.

5 P a r t of thyroid of figure 3. X 430.

UPTAKE O F 11% BY EMBRTONIO THYROIDS I? CARPBNTER, J . BRATTIE AND R. I). CHAMBERS

PLATE 1

265

P L S T E 2

EXPLANATION OF FIGURES

Figures 6, 7 , 8 and 9 are all a t tlie same magnification, X 430.

6 P a r t of a thyroid gland explanted from an %day embryo and grown 8 days in ritro. The spatial arrangement of the cords is similar to that of the ceiitral region of a thyroid gland from a 16-day embryo.

Central region of the thyroid gland of a 16-day embryo. Compare with figure 6.

P a r t of a thyroid gland explanted from an 8-day embryo and growii 8 days in vitro. The gland is more compact than tha t shown in figure 6, and shows a greater accumulation of colloid a t the periphery of the culture. A number of mitotic figures a re present.

7

8

9 P a r t of tlie thyroid gland of a 16-day embryo. The region shown is more peripherally located than that in figure 7.

266

UPTAKE O F 1'" B Y EMBRYONIC THYROIDS F C A R P E N T E R , J. BE1TTIE A N D R. D. CHAMBERS

PJdATE 2

267

PLATE 3

EXPLANATION OF FIGURES

Figures 10, 11, 13 and 13 s h o ~ v sections of thyroid glands n4iicb Tvere explanted from M a y enibryos and groivn 8 days in r i t io . Figures 13, 14 and 16 are pfioto- graphs of sections of thvroid glands froin 16-day embryos. All glands bad been exposed to the radioactive isotope, P, for 24 hours before fixation. The scctious Tveie covered with photographic film. The more heavily blackened areas are the mtoradiograplrs produced in the overlying film after suitable exposure and derelopment. S o t e the similarity in the autoradiographs of the cultures and the glands f rom normal embryos of corresponding total age.

Figures 10, 11 and 12 are X 100; figures 13 and 14, X 170; figures 15 and 16, X 430.

UPTAKE OF 1181 BY EMBRYONIC THYROII>S E. CARPENTER, J. BEATTIE AND R D. CHAMBERS

PLATE 3

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The uptake of I131 by embryonic chick thyroid glands in vivo and in vitro