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On dense bodies and droplets in the follicular cells of the guinea pig thyroid
© 1966 by Academic Press Inc. J. ULTRASTRUCTURE RESEARCH
16, 71-82 (1966)
71
On Dense Bodies and Droplets in the Follicular Cells of the Guinea Pi 9 Thyroid ~ R. EKHOLM AND S. SMEDS
Department of Anatomy, University of Gothenburg, Gothenburg, Sweden Received July 21, 1965 A large number of dense bodies are found in the follicular cells of nonstimulated as well as TSH-stimulated guinea pig thyroids. Administration of TSH induces the formation of numerous intracellular droplets of various appearances. The dense bodies are often topographically intimately related to the droplets. In tissues incubated for acid phosphatase activity, reaction products are found in the dense bodies and in some droplets. When 11~5 is administered several hours and TSH 1 or 2 hours before autopsy autoradiographic reaction is observed over the luminal colloid and over most, but not all, of the droplets. When TSH is given 1 or 2 hours and radioiodide 15 minutes before sacrifice, the luminal colloid is labeled but only an occasional intracellular droplet. The observations seem to corroborate the conclusions reached by previous authors that thyroglobulin is resorbed from the luminal colloid in the form of intracellular droplets and that the thyroglobulin in the droplets is hydrolyzed by enzymes derived from the dense bodies inside the cell. The heterogeneity of the intracellular droplets both with respect to morphology and labeling seem to indicate, however, that some of them represent other processes than thyroglobulin resorption and breakup. The nature of the so-called colloid droplets in the thyroid follicular cells has been a matter of dispute for m a n y years. Recently, however, a n u m b e r of observations have been presented (8, 11, 14) which favor the idea that the droplets are batches of thyroglobulin derived f r o m the luminal colloid. The droplets should then represent a step in the release process of the thyroid hormones. Since at least most of the h o r m o n e s are liberated f r o m the thyroglobulin before entering the circulation, hydrolysis of the colloid droplet content must take place within the cell. The discovery of the lysosomes has uncovered a very plausible source of the enzymes responsible for the hydrolysis of thyroglobulin. In fact, results strongly indicating this role of the thyroid lysosomes have been presented by Wollman et al. in a light microscopical study (14). z This study was supported in part by the Swedish Medical Research Council and in part by the National Institutes of Health, U.S. Public Health Service (grant AM-05254-03).
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The results of a u t o r a d i o g r a p h i c a n d histochemical studies in the electron m i c r o scope on the guinea pig t h y r o i d going on in our l a b o r a t o r y are in m o s t respects in g o o d a c c o r d a n c e with the conclusions d r a w n b y W o l l m a n et al. (14). H o w e v e r , certain observations i n d i c a t i n g a heterogeneous n a t u r e of the intracellular d r o p l e t s seem to justify a brief c o m m e n t .
MATERIAL AND METHODS The experimental animals were male guinea pigs, weighing 200-250 g and kept under normal laboratory conditions. TSH (Actyron, Ferring AB, Sweden) was given intraperitoneally in a dose of 0.5 I U 1 or 2 hours before sacrifice. 1125 (The Radiochemical Centre, Amersham, England) in the form of sodium iodide was administered by intracardial injection 15 minutes or 6 or 20 hours before autopsy. The dose varied between 500 ~C and 1.5 mC. The fixation was performed by infusion through the aorta on the anesthesized animal of a solution containing 3 To glutaraldehyde, 3 To Macrodex, and 3 % glucose and buffered to p H 7.4 with 0.075 M cacodylate (1). Postfixation was performed in 1 To OsO4 for 2 hours. F o r the demonstration of acid phosphatase activity, 50-/z sections were cut on a freezing microtome from the glands fixed only by the infusion of glutaraldehyde. The sections were rinsed in buffer and incubated for 10-20 minutes in a fresh Gomori fi-glycerophosphate medium (5) at p H 5.0 at 37°C. After fixation the sections were rinsed in buffer and postfixed in OsO4. Controls were run by omitting the substrate from the incubation medium. The specimens were embedded in Epon 812 (6), cut on an LKB Ultrotome, and stained with uranyl acetate (12) or lead (10). The sections were examined in a Siemens Elmiskop I. F o r the autoradiographic studies the Ilford L-4 emulsion was prepared according to Caro's method (2). The exposure time was 2-4 weeks. The autoradiograms were developed in Microdol for 5 minutes at 20°C, fixed in rapid fixer, and washed in distilled water.
OBSERVATIONS I n the c y t o p l a s m of the follicular cells of n o n s t i m u l a t e d t h y r o i d s one finds n u m e r ous e l e c t r o n - o p a q u e bodies (Fig. 1). A l t h o u g h the a p p e a r a n c e of these dense b o d i e s varies considerably, they have some m o r p h o l o g i c a l p r o p e r t i e s suggesting t h a t t h e y belong to the l y s o s o m e class. The size of the bodies varies f r o m a b o u t 0.1 # to a b o u t 1.0 #. Their shape is generally r o u n d or ovoid. C o m m o n to t h e m all is a c o m p l e t e a n d distinct b o r d e r i n g m e m b r a n e . T h e y c o n t a i n a finely granular, dense, a n d evenly d i s t r i b u t e d m a t e r i a l which is often s e p a r a t e d f r o m the surface m e m b r a n e b y a less dense zone. In a d d i t i o n to this h o m o g e n e o u s m a t e r i a l s o m e bodies c o n t a i n small dense particles, small vesicles or l a m e l l a t e d myelin-like figures. The dense bodies are
FIo. 1. Numerous dense bodies of different types in the apical and middle parts of a follicular cell from a nonstimulated thyroid. Note the small dense bodies in the Golgi area (arrows). × 47,000.
R. EKHOLM AND S. SMEDS
F~G. 2. T w o large intracellular droplets in a follicular cell f r o m a thyroid s t i m u l a t e d with T S H l h o u r before autopsy. O n e of t h e droplets is enclosed by t h e s a m e m e m b r a n e as a dense b o d y × 38,000.
FIG. 3. T h e apical zone of a follicular cell f r o m a t h y r o i d stimulated with T S H 1 h o u r before fixation. Several droplets of varying size, density, a n d granularity are seen. T h e a r r o w m a r k s a f o r m a t i o n w h i c h could be a droplet or a dense body. x 28,000. FIG. 4. A n intracellular droplet containing s m a l l vesicles a n d particles in addition to a g r a n u l a r substance. T S H - s t i m u l a t e d thyroid, x 47,000. FIG. 5. A n intracellular droplet with h o m o g e n e o u s c o n t e n t in close relation to dense bodies. T S H s t i m u l a t e d thyroid. × 39,000.
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found in all parts of the cytoplasm, but the smallest ones are generally restricted to the Golgi zone. In follicular cells from the TSH-stimulated glands a similar population of dense bodies are found. TSH-stimulation induces drastic alterations of the follicular cell morphology. The most conspicuous change is the formation of large numbers of droplets (Figs. 2-5). These are rounded, membrane-bounded structures, ranging in size from a few tenths of a micron to about 2 #. On the basis of their content one may divide them into two categories. Droplets of the first category contain a finely granular, evenly distributed substance rendering them homogeneous at low magnification (Figs. 2, 3, and 5). However, their general density varies considerably. The most opaque droplets are almost as dense as the dense bodies described above. In fact, it is sometimes impossible to decide whether a certain formation should be designated as a droplet or as a dense body. The least dense droplets have a density much below that of the luminal colloid. Between these extremes all kinds of intermediates are encountered. Droplets of the second category have varying densities caused by a coarse and uneven granularity (Figs. 3 and 4). They also contain small dense vesicles and particles of various sizes. Their diameter seldom exceeds 1 #. All kinds of droplets are located in the apical half of the cell. Some of the homogeneous type are found in cytoplasmic tabs projecting from the apical cell surface. The droplets, particularly those of the first category, are often intimately related to lysosome-like bodies and, occasionally, such a body and a droplet are seen within the same limiting membrane (Fig. 2). Droplets are also observed rather closely related to the Golgi zone. Acid phosphatase activity is found in the dense bodies. Probably this activity is present in all the different types of dense bodies, although this is not definitely established, since the reaction products often conceal the interior of the bodies and so make the precise identification difficult. However, bodies containing myelin-like figures are easily recognized in spite of their content of reaction precipitates (Fig. 6). Phosphatase activity can also be demonstrated in small structures, probably vesicles, in the Golgi region and in the smallest type of dense bodies found in the same area (Figs. 7 and 8). Finally, reaction products are observed in multivesiculated bodies (Fig. 8). In the cells from the TSH-stimulated glands acid phosphatase activity is found in the same loci and, in addition, in some, but not all, of the droplets (Figs. 9 and 10). The reaction intensity differs between the droplets but is generally lower in them than in the dense bodies. When iodide labeled with 112ais given at long intervals (6 hours or more) and TSH at 1 or 2 hours before autopsy an intense autoradiographic reaction is found over the luminal colloid. In addition a large number, but not all, of the intracellular droplets are labeled (Figs. 11-13). When TSH is given 1 or 2 hours and radioiodide 15 minutes
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FIa. 6. A follicular cell from a nonstimulated thyroid. The tissue was incubated for acid phosphatase activity. The reaction product is distinctly located to a few bodies, most likely lysosomes. One of the bodies contains a myelin-like structure visible through the reaction deposit. × 58,000. Fro. 7. The Golgi region in a follicular cell from a nonstimulated thyroid after incubation for acid phosphatase activity. The reaction product is found in several dense bodies, some of which are very small (arrow). The multivesicular body in the upper right part shows no reaction, x 54,000.
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R. EKHOLM AND S. SMEDS
F~G. 8. The Golgi area in a follicular cell of a TSH-stimulated thyroid after incubation for acid phosphatase activity. The reaction product is seen in small structures, probably Golgi vesicles, and in a multivesicular body. × 46,000. I~G. 9. A follicular cell from a thyroid stimulated with TSH for 1 hour. The tissue was incubated for acid phosphatase activity. The reaction product is seen in a large intracellular droplet and in a few smaller formations, probably lysosomes, x 50,000. FIG. 10. The apical zone of a follicular cell from a thyroid stimulated with TSH 1 hour before sacrifice. The tissue was incubated for acid phosphatase activity. A strong reaction is seen in dense bodies. The large droplet shows a weak but clear reaction. × 40,000.
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F~G. 11. Autoradiogram of a follicular cell 6 hours after administration of 11~5and 1 hour after TSH. Several autoradiographic grains are seen over one droplet in a cytoplasmic tab projecting from the apical cell surface. × 25,000. FIo. 12. Autoradiogram of a follicular cell 20 hours after administration of 1125and 1 hour after stimulation with TSH. Autoradiographic grains are seen over the colloid (in the upper part of the figure) and over an intracellular droplet. × 30,000.
before sacrifice, again the l u m i n a l colloid is labeled. A t this short time interval after injection of radioiodide, a u t o r a d i o g r a p h i c grains are observed only over a small n u m b e r of the intracellular droplets. DISCUSSION A t least the m a j o r i t y of the opaque bodies referred to above as dense bodies showed acid phosphatase activity. The d e m o n s t r a t i o n in these structures of only one enzyme activity, characteristic of lysosomes, does n o t prove their lysosomal nature. However, since these phosphatase-positive bodies most likely correspond to the acid phosphatase as well as esterase c o n t a i n i n g particles observed by W o l l m a n et al. (14) in the light microscope it seems legitimate to refer to them as lysosomes. Their varying m o r p h o -
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R. EKHOLM AND S. SMEDS
FIG. 13. Autoradiogram of a thyroid follicular cell 6 hours after administration of I a25 and 1 hour after TSH. Three intracellular droplets are seen, hut only two of them show autoradiographic grains. x 27,000.
logy is consistent with de Duve's (3) classification of the lysosomes in different functional categories. In the Golgi region acid phosphatase activity was found in structures, which probably are Golgi vesicles, and in very small bodies with dense content. This finding is consonant with the recent view (4, 7, 9) that the lysosomes are produced by the Golgi complex. When the extracellular colloid was prelabeled with I ~5, given several hours before TSH, most of the intracellular droplets showed autoradiographic reaction, Similar results have been obtained in electron microscopical autoradiographic studies on the mouse thyroid by Stein and Gross (11). These observations are also in harmony with the results of light microscopical studies presented by Nadler et al. (8) and Wollman et al. (14). The conclusions drawn by the authors referred to above that the colloid droplets have their origin in the luminal colloid seem very well founded.
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A very intimate relation was observed between lysosomes and droplets, in some instances even a fusion between them. Furthermore, some droplets showed acid phosphatase activity. These findings are in good accordance with the suggestion presented by Wollman et al. (14) that the lysosomes deliver enzymes for the hydrolysis of the thyroglobuliu contained in the droplets. However, a definite proof of this hypothesis demands the demonstration in the lysosomes not only of acid phosphatase and esterase activities but of enzymes capable of hydrolyzing thyroglobulin. Thus, the results of the present study seem to corroborate the conclusions reached by previous authors, implying that the intracellular droplets are batches of thyroglobulin resorbed from the luminal colloid. However, it seems advisable to comment upon some observations which could modify the interpretation of the nature of some of the droplets. The intracellular droplets found during the first hours after TSH stimulation are very polymorphous with respect to their size, density, and granularity. Many opaque specimens found during this period could be, judged from their morphology, either dense droplets or lysosomes. Furthermore, at long time intervals after 1125 administration most intracellular droplets, but not all, are labeled (Fig. 13). Naturally, this heterogeneity of the intracellular droplets with respect to both morphology and labeling could reflect different degrees of hydrolysis of their content. But the heterogeneity could also be explained if not all formations with droplet appearance are batches of resorbed colloid. As TSH stimulates not only the release of thyroid hormones, but also synthetic processes in the follicular cells, it appears very likely that some morphological changes observed after TSH reflect stimulated synthesis. In this connection it should be mentioned that according to Wissig (13) the formation of droplets in the follicular cells fits rather well with the picture one would expect in stimulated protein synthesis. Thus, it appears certain that thyroglobulin resorbed from the luminal colloid occurs in the follicular cells in the form of droplets. On the other hand, it seems quite possible that some intracellular droplets reflect synthetic processes rather than thyroglobulin resorption and degradation. Note added in proof." Since this paper was submitted for publication B. K. Wetzel, S. S. Spicer and S. H. Wollman have presented a very comprehensive study on the TSH effect upon the ultrastructure and acid phosphatase localization of the rat thyroid [J. Cell Biol. 25, 593 (1965)]. The conclusions reached in the present, less extensive study seem to agree well with those drawn by Wetzel et al. except for the interpretation of the heterogeneity of the intracellular droplets. They consider the heterogeneous appearance of the droplets to reflect different stages in the resorption and degradation of follicular colloid and base this interpretation on the observed correlation between droplet appearance and time after TSH administration. Our suggestion is that some of the droplets represent other processes than thyroglobulin resorption and degradation. 6 - 661833 J . Ultrastructure Re.search
82
R. EKHOLMAND S. SMEDS REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
BEHNKE,O., personal communication. (?AGO,L. G. and VAN TUnERGEN, R. P., J. Cell BioL 15, 173 (1962). DE DuvE, C., Ciba Found. Syrup. Lysosomes, p. 1. Churchill, London, 1963. GOLDFISCHER,S., ESSNER, E. and NOVIKOFF, A. B., J. Histochem. Cytochem. 12, 72 (1964). GOMORI, G., Microscopic Histochemistry. Principles and Practice, p. 189. Univ. of Chicago Press, Chicago, Illinois, 1952. L1AFT,J. H., J. Biophys. Biochem. CytoL 9, 409 (1961). MoE, H., ROSTGAARD,J. and BEHNKE, 0., J. Ultrastruct. Res. 12, 396 (1965). NADLER, N. J., SARKAR, S. K. and LEBLOND,C. P., Endocrinology 71, 120 (1962). NOVIKOFF,A. B., Ciba Found. Syrup. Lysosomes, p. 36. Churchill, London, 1963. REYNOLDS,E. S., or. Cell Biol. 17, 208 (1963). STEIN,O. and GROSS, J., Endocrinology 75, 787 (1964). WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). WISSIG,S. L., J. Cell Biol. 16, 93 (1963). WOLLMAN,S. H., SPICER, S. S. and BUgSTONE, M. S., or. Cell Biol. 21, 191 (1964).
16, 71-82 (1966)
71
On Dense Bodies and Droplets in the Follicular Cells of the Guinea Pi 9 Thyroid ~ R. EKHOLM AND S. SMEDS
Department of Anatomy, University of Gothenburg, Gothenburg, Sweden Received July 21, 1965 A large number of dense bodies are found in the follicular cells of nonstimulated as well as TSH-stimulated guinea pig thyroids. Administration of TSH induces the formation of numerous intracellular droplets of various appearances. The dense bodies are often topographically intimately related to the droplets. In tissues incubated for acid phosphatase activity, reaction products are found in the dense bodies and in some droplets. When 11~5 is administered several hours and TSH 1 or 2 hours before autopsy autoradiographic reaction is observed over the luminal colloid and over most, but not all, of the droplets. When TSH is given 1 or 2 hours and radioiodide 15 minutes before sacrifice, the luminal colloid is labeled but only an occasional intracellular droplet. The observations seem to corroborate the conclusions reached by previous authors that thyroglobulin is resorbed from the luminal colloid in the form of intracellular droplets and that the thyroglobulin in the droplets is hydrolyzed by enzymes derived from the dense bodies inside the cell. The heterogeneity of the intracellular droplets both with respect to morphology and labeling seem to indicate, however, that some of them represent other processes than thyroglobulin resorption and breakup. The nature of the so-called colloid droplets in the thyroid follicular cells has been a matter of dispute for m a n y years. Recently, however, a n u m b e r of observations have been presented (8, 11, 14) which favor the idea that the droplets are batches of thyroglobulin derived f r o m the luminal colloid. The droplets should then represent a step in the release process of the thyroid hormones. Since at least most of the h o r m o n e s are liberated f r o m the thyroglobulin before entering the circulation, hydrolysis of the colloid droplet content must take place within the cell. The discovery of the lysosomes has uncovered a very plausible source of the enzymes responsible for the hydrolysis of thyroglobulin. In fact, results strongly indicating this role of the thyroid lysosomes have been presented by Wollman et al. in a light microscopical study (14). z This study was supported in part by the Swedish Medical Research Council and in part by the National Institutes of Health, U.S. Public Health Service (grant AM-05254-03).
72
R. EKHOLM AND S. SMEDS
The results of a u t o r a d i o g r a p h i c a n d histochemical studies in the electron m i c r o scope on the guinea pig t h y r o i d going on in our l a b o r a t o r y are in m o s t respects in g o o d a c c o r d a n c e with the conclusions d r a w n b y W o l l m a n et al. (14). H o w e v e r , certain observations i n d i c a t i n g a heterogeneous n a t u r e of the intracellular d r o p l e t s seem to justify a brief c o m m e n t .
MATERIAL AND METHODS The experimental animals were male guinea pigs, weighing 200-250 g and kept under normal laboratory conditions. TSH (Actyron, Ferring AB, Sweden) was given intraperitoneally in a dose of 0.5 I U 1 or 2 hours before sacrifice. 1125 (The Radiochemical Centre, Amersham, England) in the form of sodium iodide was administered by intracardial injection 15 minutes or 6 or 20 hours before autopsy. The dose varied between 500 ~C and 1.5 mC. The fixation was performed by infusion through the aorta on the anesthesized animal of a solution containing 3 To glutaraldehyde, 3 To Macrodex, and 3 % glucose and buffered to p H 7.4 with 0.075 M cacodylate (1). Postfixation was performed in 1 To OsO4 for 2 hours. F o r the demonstration of acid phosphatase activity, 50-/z sections were cut on a freezing microtome from the glands fixed only by the infusion of glutaraldehyde. The sections were rinsed in buffer and incubated for 10-20 minutes in a fresh Gomori fi-glycerophosphate medium (5) at p H 5.0 at 37°C. After fixation the sections were rinsed in buffer and postfixed in OsO4. Controls were run by omitting the substrate from the incubation medium. The specimens were embedded in Epon 812 (6), cut on an LKB Ultrotome, and stained with uranyl acetate (12) or lead (10). The sections were examined in a Siemens Elmiskop I. F o r the autoradiographic studies the Ilford L-4 emulsion was prepared according to Caro's method (2). The exposure time was 2-4 weeks. The autoradiograms were developed in Microdol for 5 minutes at 20°C, fixed in rapid fixer, and washed in distilled water.
OBSERVATIONS I n the c y t o p l a s m of the follicular cells of n o n s t i m u l a t e d t h y r o i d s one finds n u m e r ous e l e c t r o n - o p a q u e bodies (Fig. 1). A l t h o u g h the a p p e a r a n c e of these dense b o d i e s varies considerably, they have some m o r p h o l o g i c a l p r o p e r t i e s suggesting t h a t t h e y belong to the l y s o s o m e class. The size of the bodies varies f r o m a b o u t 0.1 # to a b o u t 1.0 #. Their shape is generally r o u n d or ovoid. C o m m o n to t h e m all is a c o m p l e t e a n d distinct b o r d e r i n g m e m b r a n e . T h e y c o n t a i n a finely granular, dense, a n d evenly d i s t r i b u t e d m a t e r i a l which is often s e p a r a t e d f r o m the surface m e m b r a n e b y a less dense zone. In a d d i t i o n to this h o m o g e n e o u s m a t e r i a l s o m e bodies c o n t a i n small dense particles, small vesicles or l a m e l l a t e d myelin-like figures. The dense bodies are
FIo. 1. Numerous dense bodies of different types in the apical and middle parts of a follicular cell from a nonstimulated thyroid. Note the small dense bodies in the Golgi area (arrows). × 47,000.
R. EKHOLM AND S. SMEDS
F~G. 2. T w o large intracellular droplets in a follicular cell f r o m a thyroid s t i m u l a t e d with T S H l h o u r before autopsy. O n e of t h e droplets is enclosed by t h e s a m e m e m b r a n e as a dense b o d y × 38,000.
FIG. 3. T h e apical zone of a follicular cell f r o m a t h y r o i d stimulated with T S H 1 h o u r before fixation. Several droplets of varying size, density, a n d granularity are seen. T h e a r r o w m a r k s a f o r m a t i o n w h i c h could be a droplet or a dense body. x 28,000. FIG. 4. A n intracellular droplet containing s m a l l vesicles a n d particles in addition to a g r a n u l a r substance. T S H - s t i m u l a t e d thyroid, x 47,000. FIG. 5. A n intracellular droplet with h o m o g e n e o u s c o n t e n t in close relation to dense bodies. T S H s t i m u l a t e d thyroid. × 39,000.
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found in all parts of the cytoplasm, but the smallest ones are generally restricted to the Golgi zone. In follicular cells from the TSH-stimulated glands a similar population of dense bodies are found. TSH-stimulation induces drastic alterations of the follicular cell morphology. The most conspicuous change is the formation of large numbers of droplets (Figs. 2-5). These are rounded, membrane-bounded structures, ranging in size from a few tenths of a micron to about 2 #. On the basis of their content one may divide them into two categories. Droplets of the first category contain a finely granular, evenly distributed substance rendering them homogeneous at low magnification (Figs. 2, 3, and 5). However, their general density varies considerably. The most opaque droplets are almost as dense as the dense bodies described above. In fact, it is sometimes impossible to decide whether a certain formation should be designated as a droplet or as a dense body. The least dense droplets have a density much below that of the luminal colloid. Between these extremes all kinds of intermediates are encountered. Droplets of the second category have varying densities caused by a coarse and uneven granularity (Figs. 3 and 4). They also contain small dense vesicles and particles of various sizes. Their diameter seldom exceeds 1 #. All kinds of droplets are located in the apical half of the cell. Some of the homogeneous type are found in cytoplasmic tabs projecting from the apical cell surface. The droplets, particularly those of the first category, are often intimately related to lysosome-like bodies and, occasionally, such a body and a droplet are seen within the same limiting membrane (Fig. 2). Droplets are also observed rather closely related to the Golgi zone. Acid phosphatase activity is found in the dense bodies. Probably this activity is present in all the different types of dense bodies, although this is not definitely established, since the reaction products often conceal the interior of the bodies and so make the precise identification difficult. However, bodies containing myelin-like figures are easily recognized in spite of their content of reaction precipitates (Fig. 6). Phosphatase activity can also be demonstrated in small structures, probably vesicles, in the Golgi region and in the smallest type of dense bodies found in the same area (Figs. 7 and 8). Finally, reaction products are observed in multivesiculated bodies (Fig. 8). In the cells from the TSH-stimulated glands acid phosphatase activity is found in the same loci and, in addition, in some, but not all, of the droplets (Figs. 9 and 10). The reaction intensity differs between the droplets but is generally lower in them than in the dense bodies. When iodide labeled with 112ais given at long intervals (6 hours or more) and TSH at 1 or 2 hours before autopsy an intense autoradiographic reaction is found over the luminal colloid. In addition a large number, but not all, of the intracellular droplets are labeled (Figs. 11-13). When TSH is given 1 or 2 hours and radioiodide 15 minutes
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FIa. 6. A follicular cell from a nonstimulated thyroid. The tissue was incubated for acid phosphatase activity. The reaction product is distinctly located to a few bodies, most likely lysosomes. One of the bodies contains a myelin-like structure visible through the reaction deposit. × 58,000. Fro. 7. The Golgi region in a follicular cell from a nonstimulated thyroid after incubation for acid phosphatase activity. The reaction product is found in several dense bodies, some of which are very small (arrow). The multivesicular body in the upper right part shows no reaction, x 54,000.
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F~G. 8. The Golgi area in a follicular cell of a TSH-stimulated thyroid after incubation for acid phosphatase activity. The reaction product is seen in small structures, probably Golgi vesicles, and in a multivesicular body. × 46,000. I~G. 9. A follicular cell from a thyroid stimulated with TSH for 1 hour. The tissue was incubated for acid phosphatase activity. The reaction product is seen in a large intracellular droplet and in a few smaller formations, probably lysosomes, x 50,000. FIG. 10. The apical zone of a follicular cell from a thyroid stimulated with TSH 1 hour before sacrifice. The tissue was incubated for acid phosphatase activity. A strong reaction is seen in dense bodies. The large droplet shows a weak but clear reaction. × 40,000.
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F~G. 11. Autoradiogram of a follicular cell 6 hours after administration of 11~5and 1 hour after TSH. Several autoradiographic grains are seen over one droplet in a cytoplasmic tab projecting from the apical cell surface. × 25,000. FIo. 12. Autoradiogram of a follicular cell 20 hours after administration of 1125and 1 hour after stimulation with TSH. Autoradiographic grains are seen over the colloid (in the upper part of the figure) and over an intracellular droplet. × 30,000.
before sacrifice, again the l u m i n a l colloid is labeled. A t this short time interval after injection of radioiodide, a u t o r a d i o g r a p h i c grains are observed only over a small n u m b e r of the intracellular droplets. DISCUSSION A t least the m a j o r i t y of the opaque bodies referred to above as dense bodies showed acid phosphatase activity. The d e m o n s t r a t i o n in these structures of only one enzyme activity, characteristic of lysosomes, does n o t prove their lysosomal nature. However, since these phosphatase-positive bodies most likely correspond to the acid phosphatase as well as esterase c o n t a i n i n g particles observed by W o l l m a n et al. (14) in the light microscope it seems legitimate to refer to them as lysosomes. Their varying m o r p h o -
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FIG. 13. Autoradiogram of a thyroid follicular cell 6 hours after administration of I a25 and 1 hour after TSH. Three intracellular droplets are seen, hut only two of them show autoradiographic grains. x 27,000.
logy is consistent with de Duve's (3) classification of the lysosomes in different functional categories. In the Golgi region acid phosphatase activity was found in structures, which probably are Golgi vesicles, and in very small bodies with dense content. This finding is consonant with the recent view (4, 7, 9) that the lysosomes are produced by the Golgi complex. When the extracellular colloid was prelabeled with I ~5, given several hours before TSH, most of the intracellular droplets showed autoradiographic reaction, Similar results have been obtained in electron microscopical autoradiographic studies on the mouse thyroid by Stein and Gross (11). These observations are also in harmony with the results of light microscopical studies presented by Nadler et al. (8) and Wollman et al. (14). The conclusions drawn by the authors referred to above that the colloid droplets have their origin in the luminal colloid seem very well founded.
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A very intimate relation was observed between lysosomes and droplets, in some instances even a fusion between them. Furthermore, some droplets showed acid phosphatase activity. These findings are in good accordance with the suggestion presented by Wollman et al. (14) that the lysosomes deliver enzymes for the hydrolysis of the thyroglobuliu contained in the droplets. However, a definite proof of this hypothesis demands the demonstration in the lysosomes not only of acid phosphatase and esterase activities but of enzymes capable of hydrolyzing thyroglobulin. Thus, the results of the present study seem to corroborate the conclusions reached by previous authors, implying that the intracellular droplets are batches of thyroglobulin resorbed from the luminal colloid. However, it seems advisable to comment upon some observations which could modify the interpretation of the nature of some of the droplets. The intracellular droplets found during the first hours after TSH stimulation are very polymorphous with respect to their size, density, and granularity. Many opaque specimens found during this period could be, judged from their morphology, either dense droplets or lysosomes. Furthermore, at long time intervals after 1125 administration most intracellular droplets, but not all, are labeled (Fig. 13). Naturally, this heterogeneity of the intracellular droplets with respect to both morphology and labeling could reflect different degrees of hydrolysis of their content. But the heterogeneity could also be explained if not all formations with droplet appearance are batches of resorbed colloid. As TSH stimulates not only the release of thyroid hormones, but also synthetic processes in the follicular cells, it appears very likely that some morphological changes observed after TSH reflect stimulated synthesis. In this connection it should be mentioned that according to Wissig (13) the formation of droplets in the follicular cells fits rather well with the picture one would expect in stimulated protein synthesis. Thus, it appears certain that thyroglobulin resorbed from the luminal colloid occurs in the follicular cells in the form of droplets. On the other hand, it seems quite possible that some intracellular droplets reflect synthetic processes rather than thyroglobulin resorption and degradation. Note added in proof." Since this paper was submitted for publication B. K. Wetzel, S. S. Spicer and S. H. Wollman have presented a very comprehensive study on the TSH effect upon the ultrastructure and acid phosphatase localization of the rat thyroid [J. Cell Biol. 25, 593 (1965)]. The conclusions reached in the present, less extensive study seem to agree well with those drawn by Wetzel et al. except for the interpretation of the heterogeneity of the intracellular droplets. They consider the heterogeneous appearance of the droplets to reflect different stages in the resorption and degradation of follicular colloid and base this interpretation on the observed correlation between droplet appearance and time after TSH administration. Our suggestion is that some of the droplets represent other processes than thyroglobulin resorption and degradation. 6 - 661833 J . Ultrastructure Re.search
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R. EKHOLMAND S. SMEDS REFERENCES
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