Cell Tiss. Res. 166, 49-63 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
New Ultrastructural Features on the Cream Hamster Thyroid with Special Reference to the Second Kind of Follicle* P. K e t e l b a n t - B a l a s s e a n d P. N6ve** Laboratories of Electron Microscopy, Pathology and Experimental Medicine, Universit6 Libre de Bruxelles, Brussels, Belgium
Summary. T h e t h y r o i d cells o f the c r e a m h a m s t e r , c h a r a c t e r i z e d b y a b u n d a n c e o f m i c r o t u b u l e s a n d s t r a t i f i c a t i o n o f the organelles, u n d e r g o a p a r t i c u lar e v o l u t i o n when the a n i m a l s g r o w older. These c h a n g e s are c h a r a c t e r i z e d by a n increase o f the n u m b e r o f l y s o s o m e s which in e x t r e m e cases b e c o m e so p r o m i n e n t t h a t they o c c u p y the w h o l e c y t o p l a s m o f the cell w h i c h thus loses its o r g a n e l l e stratification. A s in o t h e r species, c r e a m h a m s t e r t h y r o i d c o n t a i n s so-called u l t i m o b r a n chial follicles m a d e u p o f at least six cell t y p e s : fibrillar d a r k a n d light cells, p a r a f o l l i c u l a r cells, ciliated cells, vesicular cells, a n d cells with m y e l i n i c inclusions. T h e u l t r a s t r u c t u r e o f these follicles in the c r e a m h a m s t e r represents a m i x t u r e o f the u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s o f the s a m e follicles e n c o u n t e r e d in the rat a n d the m o u s e t h y r o i d . H e r e also m i x e d follicles are seen. N e v e r t h e l e s s vesicular cells p r e s e n t such a b u n d a n t " s e c r e t i o n g r a n u l e s " t h a t the q u e s t i o n arises as to w h e t h e r these follicles p r o d u c e a special secretion a n d p e r h a p s a new h o r m o n e . I n c u b a t i o n o f c r e a m h a m s t e r t h y r o i d s in the presence o f vincristine induces v a n i s h i n g o f m i c r o t u b u l e s , f o r m a t i o n o f p a r a c r y s t a l l i n e structures, a n d loss o f s t r a t i f i c a t i o n o f the organelles. A l t h o u g h these last effects m i g h t be due to s o m e aspecific toxic effect o f the drug, it is suggested t h a t the d i s a p p e a r i n g o f the o r g a n e l l e s t r a t i f i c a t i o n m i g h t result f r o m a specific vinc r i s t i n e - i n d u c e d d i s a g g r e g a t i o n o f the m i c r o t u b u l e s a c t i n g as a c y t o s k e l e ton. K e y words: C r e a m h a m s t e r t h y r o i d - S t r a t i f i c a t i o n o f o r g a n e l l e s s o m e s - M i c r o t u b u l e s -- U l t i m o b r a n c h i a l follicles - Vincristine.
Lyso-
S e n d offprint requests to : Dr. P. Ketelbant-Balasse, Laboratories of Electron Microscopy, Pathology and Experimental Medicine, Rue aux Laines 97, B-1000 Brussels, Belgium.
* This work was realized thanks to contracts 20013 and 3.9001.75 of the Fonds de la Recherche Scientifique M~dicale and with the help of the Caisse G~n6rale d'Epargne et de Retraite in the framework of the Association Euratom, University of Brussels and University of Pisa. ** Acknowledgments. The authors are indebted to Dr. F. Rodesch who performed the incubation experiments, to Prof. P. Dustin, Jr. for critical reading of the manuscript, to Mrs. J. SchzepesMichel, Mr. G. Vienne, and J.L. Conreur for their technical assistance, and to Mrs. B. No61-Vincke for typing the manuscript.
50
P. Ketelbant-Balasse and P. N6ve
Introduction T h e t h y r o i d o f m a m m a l s has two e m b r y o l o g i c origins (Rogers, 1927; S u g i y a m a , 1940; M c A l p i n e , 1955; Boyd, 1964): m o s t o f the follicles derive f r o m a ventral p h a r y n g e a l hernia. T h e 4th a n d 5th lateral p o u c h e s o f the b r a n c h i a l arches m a y also c o n t r i b u t e to the f o r m a t i o n o f a small p a r t o f follicles, s o m e t i m e s called u l t i m o b r a n c h i a l follicles ( D u n n , 1944; V a n D y k e , 1944, 1945; G o r b m a n , 1947; A x e l r a d a n d L e b l o n d , 1955). F o r several years, different p a p e r s have been d e v o t e d to the u l t r a s t r u c t u r a l d e s c r i p t i o n o f this s e c o n d k i n d o f follicle in different species. E a c h o f these studies has p o i n t e d out different m o r p h o l o g i c a s p e c t s c h a r a c t e r i s t i c o f each species (Wetzel a n d W o l l m a n , 1969; N6ve a n d W o l l m a n , 1971 a; W o l l m a n a n d N6ve, 1971 a; Calvert, 1972; N6ve a n d W o l l m a n , 1972). T h e a i m o f the p r e s e n t w o r k is to describe the second type o f follicle in the c r e a m h a m s t e r t h y r o i d . N e w u l t r a s t r u c t u r a l features related to the usual k i n d o f follicles will also be m e n t i o n e d . F i n a l l y , e x p e r i m e n t a l evidence will b e p r o v i d e d suggesting t h a t m i c r o t u b u l e s c o n s t i t u t e a c y t o s k e l e t o n responsible for the o r g a n e l l e s t r a t i f i c a t i o n in the u s u a l t h y r o i d cells.
Material and Methods Inbred male cream hamsters (Strain LHC/LAK) were provided by the Lakeview Hamster Colony, Newfield, N.J. The animals weighed approximately 100 g and, when used, were 6-15 months old. They were fed with Purina Laboratory Chow and received water ad libitum. After ether anesthesia and exsanguination both thyroid lobes were excised and immediately dipped for 90 min in glutaraldehyde 4.2/100 ml in 0.1 M Millonig's buffer at 20~ and at pH 7.4. After postfixation for 60 rain in 2/100 ml osmium tetroxide in phosphate buffer and after dehydration in graded concentrations of ethanol, the lobes were embedded in Epon according to Luft (1961). Serial toluidine blue-stained semithin sections were made until the second kind of follicle was recognized with the light microscope (Fig. 1). Thereafter ultrathin sections were performed with a diamond knife on a MT-2B Porter-Blum ultramicrotome. The sections were stained with uranyl-acetate and lead and observed with Siemens Elmiskop I and 101 electron microscopes. The ultrastructural aspects of the usual thyroid cells and of the second kind of follicle in 15-month-old animals were compared with those seen in 6-month-old hamsters. For experimental investigation, only 6-month-old animals were used. After rapid removal, the thyroid lobes were incubated for 4 hrs in Parker 199 culture medium at 37~ in presence or absence of vincristine sulfate (Lilly, Indianapolis) at the following concentrations: 2.10 _4 M, 6.10-4 M and 9.10 4M. At the end of incubation, fragments were fixed for electron microscope study.
Results
The Usual Thyroid Cells In the 6 - m o n t h - o l d c r e a m h a m s t e r , the usual t h y r o i d cells a p p e a r e d with the s a m e c h a r a c t e r i s t i c s as those d e s c r i b e d in a p r e v i o u s p a p e r : long microvilli, a b u n d a n c e o f m i c r o t u b u l e s a n d m i c r o f i l a m e n t s , a n d stratification o f the o r g a n e l l e s with a u n i f o r m layer o f dense r o u n d e d o r i r r e g u l a r l y s h a p e d l y s o s o m e s scattered between the a p e x a n d the nucleus (N6ve a n d W o l l m a n , 1971 b).
Ultrastructure of Cream Hamster Thyroid
51
Fig. 1. Toluidine-blue-stained semithin section in a thyroid of 15-month-oldcream hamster showing groups of so-called ultimobranchial follicles easily recognizable due to the abundance of cellular debris in the follicular lumen, x 900 In animals 15 months of age, the thyroid cells showed considerable differences in appearance. The abundance of lysosomes and the scarcity of granular reticulum was most prominent. Most of the lysosomes were large, irregularly shaped and often had a denser core. They occupied most of the space between the apex and the nucleus and between the nucleus and the lateral surfaces. In extreme cases, as shown in Fig. 2, the lysosomes occupied the whole cytoplasm with the nucleus pushed into a corner of the cell: in the remaining cytoplasm, dispersed groups of Golgi cisternae and a few rough endoplasmic cisternae were found. In these conditions stratification of the organelles was lost.
The Ultimobranchial Follicles The designation of the different cell types seen in the second kind of follicle is the same as that used in previous papers devoted to such follicles in the rat and in the mouse (Wollman and N6ve, 1971 a).
Fig. 2. Usual thyroid cell in a cream hamster 15 m o n t h s of age: most of the cytoplasm is filled with large irregular-shaped lysosomes. Golgi cisternae (go) are dispersed in several places throughout the hyaloplasm. The nucleus (N) is pushed aside in a corner of the cell. Stratification of the organelles is lost. • 10,000
Ultrastructure of Cream HamsterThyroid
53
The general properties of the ultimobranchial follicles and their cell complement were different at 6 and 15 months of age.
Six-month-old Hamster
The ultimobranchial follicles consisted of two or more layers of cells surrounding a lumen containing an amorphous material and cell debris (Fig. 3). In some follicles the multilayered U cells were connected with unilayered typical thyroid epithelium, ordinarily in contact with the lumen and indistinguishable from that in the usual follicle. When cells of the two types were in contact with each other, they were joined by desmosomes and a terminal bar at the apical end of neighboring lateral surfaces. Sections of follicles appeared surrounded by a basal membrane. The outermost or basal U cells and the more apical U cells looked somewhat different from each other and both could be distinguished from the typical thyroid epithelium. The basal U cells or fibrillar dark cells (Fig. 3) were usually flattened, with flattened nuclei, and looked darker than most apical cells. They had an abundance of free ribosomes and a relative scarcity of granular reticulum. Desmosomes and interfoldings connected these cells with each other and with the inner cells. Basal half-desmosomes and some micropinocytotic vesicles were present along the plasma membrane and bundles of fibrils were dispersed throughout the cytoplasm (Fig. 4). Bordering on the colloid lumen, several types of U cells could be recognized by their distinctive ultrastructure: fibrillar light cells, parafollicular cells, ciliated cells, vesicular cells, and cells with myelinic inclusions. The proportions between these five cell types were essentially variable from one animal to another so that it is difficult to appreciate the percentage of each cell type. The parafollicular cells, easily recognizable due to their small secretory granules, appeared as in other species and never bordered the colloid lumen although frequently separated from it by a thin layer of another cell type. The fibrillar light cells constituted the most frequent type: they differed from the basal U cells, having a low concentration of ribosomes and having fibrils largely dispersed in the apparently larger cytoplasm. Short and wide microvilli coated by an amorphous material extended into the colloid lumen. Fibrils were denser inside the microvilli. A Golgi complex with bristled coated vesicles was observed occasionally near the nucleus and mitochondria were primarily located on the sides and at the base of the nucleus (Fig. 3). The vesicular cells (Fig. 5) also had dispersed fibrils and short microvilli but were characterized along the apex by the presence of rounded vesicles of variable size, with amorphous contents. Some were in contact with the apical surface. Parallel arrays of rough endoplasmic cisternae were often seen in the cytoplasms. Cells with myelinic inclusions (Fig. 6) had the usual organelle complement but also displaced large, usually rounded inclusions with heterogeneous content and often made up of myelinic figures. In some of these inclusions tubular rods were recognizable (Figs. 7 and 8).
Fig. 3. Part of a "mixed" so-called ultimobranchial follicle in a 6-month-old hamster thyroid: unilayered usual thyroid cells (th) in the upper left of the micrograph directly continue with bilayered U cells along a single colloid lumen (Co). Dark basal and flattened U cells are easily recognizable from the upper larger fibrillar light cells with short apical microvilli, x 4,800
Ultrastructure of Cream Hamster Thyroid
55
Fig. 4. Details of a dark basal U cell: ribosomes and bundles of microfilaments are abundant. Typical hemidesmosomesexist along the basal plasma membrane. • 22,000
Ciliated cells were the rarest cell type: their ultrastructure was similar to that of ciliated cells from the second kind of thyroid follicle in C3H mouse (Fig. 9). So-called A - R cells as in mouse thyroid were also seen (Fig. 9). They were characterized by large areas of cytoplasm occupied by elongated or rounded cisternae of a granular reticulum: some of these cisternae were, however, still studded with rare ribosomes. Sometimes globular intracytoplasmic inclusions with amorphous material were found. The lumen of ultimobranchial follicles contained a cellular material, which resembled the colloid of the usual follicle and cell debris. The latter consisted principally of largely intact cell carcasses. The cytoplasm of these carcasses consisted of a relatively dense meshwork of fibers in which organelle remnants were embedded.
Fifteen-month-old Hamster The ultrastructural aspect of the second kind of follicle displayed great variations from one animal to another. Similar descriptions as those made of the young hamsters could also apply to some old animals. However, a special aspect was more often observed: the follicles were larger in size with more abundant luminal contents principally made up of fibrillar cellular carcasses. The lumen
Fig. 5. Vesicular cell bordering on the colloid lumen: rounded vesicles with amorphous contents are scattered below the apex. Some of them are in close contact with the apical plasma membrane. x 22,500
Ultrastructure of Cream Hamster Thyroid
57
Fig. 6. In a mixed follicle, typical ultimobranchial cell with myelinic inclusions squeezed between a thyroid cell (th) and a fibrillar light cell. • 7,200
was bordered by bilayered flattened cells resembling the basal U cells, with m a n y free ribosomes and fibrils. However, the apical cells had m o r e cytoplasm than the basal cells and presented with some parallel arrays of rough endoplasmic cisternae and apical vesicles. Some of these cells could also be A - R cells.
Incubation Experiments The ultrastructure of the thyroid glands in vitro remained similar to that observed in vivo even after 4 hrs incubation. When incubation was performed in the presence of vincristine, at least at a concentration lower than 2. l0 -4 M, no morphologic change was noted: stratification of organelles and microtubules was maintained. On the other hand, when the glands were incubated in the presence of 6 . 1 0 - 4 M vincristine, microtubules vanished and paracrystalline structures appeared in the cytoplasm (Fig. 10). Some lysosomes were seen at the base of the cell and lateral to the nucleus although most remained located above the
Figs. 7 and 8. D e t a i l s of cells with myelinic inclusions : t u b u l a r rods are seen in some of the inclusions. x 13,500 Fig. 9. T y p i c a l A - R cell b o r d e r i n g on the colloid l u m e n and close to a ciliated cell. x 6,000
Fig. 10. Typical follicular cell of a cream hamster thyroid incubated in toto in presence of 6- 10 4 M vincristine. Paracrystalline structures (arrows) are induced by vincristine. Lysosomes (ly) and Golgi cisternae (Go) are seen at the base of the cell: stratification of organelles is not so obvious as in reference incubated glands, x 12,500
Fig. 11. Typical follicular cell in a cream hamster thyroid incubated in toto in presence of 9.10 -4 M vincristine. Apart from changes noticed in the previous figure, rounded ergastoplasmic vesicles are scattered throughout the whole cytoplasm and are no longer located at the base of the cell. x 6,500
Ultrastructure of Cream HamsterThyroid
61
nucleus. Golgi cisternae occupied less specific locations than in reference glands : they were found everywhere in the cytoplasm, even at the cellular base. The granular endoplasmic cisternae had lost their elongated shape: they appeared more or less round. The higher the concentration of vincristine in the incubation medium, the more the aforementioned changes were apparent. Rounded ergastoplasmic cisternae were found dispersed throughout the whole cytoplasm. Lysosomes were larger in size than usual. No change involved centrioles, zonula occludens, desmosomes, or mitochondria (Fig. 11).
Discussion
As previously described (N6ve and Wollman, 1971 a), the ultrastructure of the cream hamster thyroid, at least in the young animal, is characterized by special features: principally, the stratification of organelles, and the abundance of microtubules and of lysosomes. The latter are particularly large and in the young animal, constitute a layer located between the apical surface and the nucleus in each normal thyroid. When the animals grow older, these lysosomes increase in number and become more and more dispersed throughout the whole cytoplasm. In extreme cases these organelles become prominent in the cytoplasm: the lysosomes are seen everywhere whereas the other organelles are difficult to recognize. The nuclei seem to be pushed away by this abundance of lysosomes in such a way that the resultant cell type should be designed as a lysosome-rich cell. As this phenomenon of lysosomal accumulation seems to be related to age, this process might result from the exhaustion of the mechanism whereby the cell rids itself of its metabolic debris. This cell evolution is compatible with the phenomenon observed in most thyroid cells from old normal humans (N6ve, 1965; Klinck et al., 1970) and also in pathology where thyroid cells from cretinoid patients with iodoprotein defect and goiter show accumulation of lysosomes and lipofuscin residues with high cellular activities (Ketelbant-Balasse et al., 1975). The ultrastructure of the second kind of follicle or the so-called ultimobranchial follicle in rat and mouse thyroid has been reported (Wetzel and Wollman, 1969; N~ve and Wollman, 1971b, 1972; Calvert, 1972). In both species several different cell types and mixed follicles have been recognized: fibrillar cells, parafollicular cells, ciliated cells, and cell debris in the follicular lumen were common ultrastructural features. These similarities also exist in the cream hamster thyroid and allow the identification of this kind of follicle as having probably the same embryologic origin: here also, the proportion of different cell types varied with the age. Except for the parafollicular cells, the literature remains conjectural as to whether these special follicles play a physiologic role. The existence of vesicles similar to secretory granules in one of the cell types in the ultimobranchial follicles of the cream hamster suggests that these cells could have a secretory process. Can these cells give rise to a new hormone? More work is needed before answering this question. Microtubules are especially abundant in the usual thyroid cells of the cream hamster (N6ve and Woltman, 1971 b). It has been suggested that these microtubules might act as a scaffolding which holds some organelles in preferred
62
P. Ketelbant-Balasse and P. N6ve
p o s i t i o n s in cells a n d w o u l d a c c o u n t for the stratification o f the organelles seen on the u s u a l t h y r o i d cells o f the c r e a m hamster. M i c r o t u b u l e i n h i b i t o r s like vincristine o r colchicine d e s t r o y m i c r o t u b u l e s a n d i n h i b i t t h y r o i d secretion in vitro (N+ve et al., 1970; W i l l i a m s a n d Wolff, 1970; N~ve etal., 1972) a n d in vivo ( E k h o l m etal., 1974) after acute s t i m u l a t i o n with t h y r o t r o p i n . Vincristine also induces f o r m a t i o n o f p a r a crystalline structures. I n c u b a t i o n o f the c r e a m h a m s t e r t h y r o i d in the presence o f vincristine p r o d u c e d vanishing o f m i c r o t u b u l e s a n d f o r m a t i o n o f p a r a c r y s t a l line structures at least when the vincristine c o n c e n t r a t i o n was sufficiently h i g h : m o r e o v e r , at these c o n c e n t r a t i o n s , o t h e r m o r p h o l o g i c changes a p p e a r e d that is, r o u n d e d aspect o f the e n d o p l a s m i c cisternae a n d the loss o f organelle stratification. T h e doses o f vincristine h a d to be higher t h a n in o t h e r systems in o r d e r to d e s t r o y the m i c r o t u b u l e s . T h e higher the c o n c e n t r a t i o n s o f the drug, the m o r e intense the u l t r a s t r u c t u r a l changes. T h e latter, o f course, might be only the result o f an aspecific toxic effect o f vincristine. H o w e v e r , except for m i c r o t u bules, the intrinsic aspect o f the organelles r e m a i n e d u n c h a n g e d a n d the p l a s m a m e m b r a n e s l o o k e d n o r m a l . A n o t h e r e x p l a n a t i o n might be t h a t these m o r p h o l o g i c c h a n g e s result f r o m the absence o f m i c r o t u b u l e s : the cell in such c o n d i t i o n w o u l d lose its m i c r o t u b u l a r c y t o s k e l e t o n a n d c o n s e q u e n t l y the m a i n t e n a n c e of its organelles in preferential i n t r a c y t o p l a s m i c locations. T h e necessity to use such high doses o f vincristine for o b t a i n i n g these effects c o u l d be e x p l a i n e d by the e x c e p t i o n a l a b u n d a n c e o f m i c r o t u b u l e s in the hamster.
References Axelrad, A.A., Leblond, C.P.: Induction of thyroid tumors in rats by low iodine diet. Cancer (Philad.) 8, 339-367 (1955) Boyd, J.D.: Development of the human thyroid gland. In: The thyroid gland (Pitt-Rivers, R. and Trotter, W.R., eds.), vol. 1, p. 9-31. London: Butterworths 1964 Calvert, R.: Transitional cells in the postnatal thyroid gland of the rat. Anat. Rec. 174, 341-360 (1972) Dunn, T.B.: Ciliated cells of the thyroid of the mouse. J. nat. Cancer. Inst. 4, 555-557 (1944) Ekholm, R., Ericson, L.E., Josefsson, J.-O., Melander, A. : In vivo action of vinblastine on thyroid ultrastructure and hormone secretion. Endocrinology 94, 641-649 (1974) Gorbman, A.: Functional and morphological properties in the thyroid gland, ultimobranchial body and persisting ductus pharyngiobranchialis IV of an adult mouse. Anat. Rec. 98, 93-102 (1947) Ketelbant-Balasse, P., Glinoer, D., N+ve, P.: Ultrastructure of the thyroid gland in a case of congenital goiter with cretinism. Path. europ. 10, 155-165 (1975) Klinck, G.H., Oertel, J.E., Winship, T.: Ultrastructure of normal human thyroid. Lab. Invest. 22, 2 22 (1970) Luft, J. : Improvements in epoxy embedding methods. J. Cell Biol. 9, 409414 (1961) McAlpine, R.J.: Alkaline glycerophosphatase in the developing thyroid, parathyroid and thymus of the albino rat. Amer. J. Anat. 96, 191-223 (1955) N6ve, P.: Ultrastructure des cellules folliculaires d'une thyro'ide humaine normale. J. Microsc. 4, 811-814 (1965) N+ve, P., Ketelbant-Balasse, P., Willems, C., Dumont, J.E.: Effect of inhibitors of microtubules and microfilaments on dog thyroid slices in vitro. Exp. Cell Res. 74, 227-244 (1972)
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N~ve, P., Willems, C., Dumont, J.E.: Involvement of the microtubular-microfilament system in thyroid secretion. Exp. Cell Res. 63, 457460 (1970) N6ve, P., Wollman, S.H.: Fine structure of ultimobranchial follicles in the thyroid gland of the rat. Anat. Rec. 17L 259-272 (1971a) N6ve, P., Wollman, S.H.: Ultrastructure of the thyroid gland of the cream hamster. Anat. Rec. 168, 23~12 (1971 b) N6ve, P., Wollman, S.H.: Fine structure of a fifth type of epithelial cell in the thyroid gland of the C3H mouse. Anat. Rec. 172, 37-44 (1972) Rogers, W.M. : The fate of the ultimobranchial body in the white rat (Mus norvegicus albinus). Amer. J. Anat. 38, 349-376 (1927) Sugiyama, S.: The fate of the ultimobranchial body of the albino rat with special reference to the formation of the thyroid gland. Okajima Folia anat. jap. 19, 333-341 (1940) Van Dyke, J.H.: Behavior of ultimobranchial tissue in the postnatal thyroid gland: The origin of thyroid cystoadenomata in the rat. Anat. Rec. 88, 369-391 (1944) Van Dyke, J.H.: Behaviour of ultimobranchial tissue in the postnatal thyroid gland: epithelial cysts, their relation to thyroid parenchyma and to "new growths" in the thyroid gland of young sheep. Amer. J. Anat. 76, 201-251 (1945) Wetzel, B.K., Wollman, S.H.: Fine structure of a second kind of thyroid follicle in the C3H mouse. Endocrinology 84, 563-578 (1969) Williams, J.A., Wolff, J.: Possible role of microtubules in thyroid secretion. Proc. nat. Acad. Sci. (Wash.) 67, 1901-1908 (1970) Wollman, S.H., N6ve, P. : Ultimobranchial follicles in the thyroid glands of rats and mice. Recent Progr. Hormone Res. 27, 213-234 (1971 a) Wollman, S.H., N6ve, P.: Postnatal development and properties of ultimobranchial follicles in the rat thyroid. Anat. Rec. 171,247-258 (1971b)
Received June 14, 1975
Cell and Tissue Research 9 by Springer-Verlag 1976
New Ultrastructural Features on the Cream Hamster Thyroid with Special Reference to the Second Kind of Follicle* P. K e t e l b a n t - B a l a s s e a n d P. N6ve** Laboratories of Electron Microscopy, Pathology and Experimental Medicine, Universit6 Libre de Bruxelles, Brussels, Belgium
Summary. T h e t h y r o i d cells o f the c r e a m h a m s t e r , c h a r a c t e r i z e d b y a b u n d a n c e o f m i c r o t u b u l e s a n d s t r a t i f i c a t i o n o f the organelles, u n d e r g o a p a r t i c u lar e v o l u t i o n when the a n i m a l s g r o w older. These c h a n g e s are c h a r a c t e r i z e d by a n increase o f the n u m b e r o f l y s o s o m e s which in e x t r e m e cases b e c o m e so p r o m i n e n t t h a t they o c c u p y the w h o l e c y t o p l a s m o f the cell w h i c h thus loses its o r g a n e l l e stratification. A s in o t h e r species, c r e a m h a m s t e r t h y r o i d c o n t a i n s so-called u l t i m o b r a n chial follicles m a d e u p o f at least six cell t y p e s : fibrillar d a r k a n d light cells, p a r a f o l l i c u l a r cells, ciliated cells, vesicular cells, a n d cells with m y e l i n i c inclusions. T h e u l t r a s t r u c t u r e o f these follicles in the c r e a m h a m s t e r represents a m i x t u r e o f the u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s o f the s a m e follicles e n c o u n t e r e d in the rat a n d the m o u s e t h y r o i d . H e r e also m i x e d follicles are seen. N e v e r t h e l e s s vesicular cells p r e s e n t such a b u n d a n t " s e c r e t i o n g r a n u l e s " t h a t the q u e s t i o n arises as to w h e t h e r these follicles p r o d u c e a special secretion a n d p e r h a p s a new h o r m o n e . I n c u b a t i o n o f c r e a m h a m s t e r t h y r o i d s in the presence o f vincristine induces v a n i s h i n g o f m i c r o t u b u l e s , f o r m a t i o n o f p a r a c r y s t a l l i n e structures, a n d loss o f s t r a t i f i c a t i o n o f the organelles. A l t h o u g h these last effects m i g h t be due to s o m e aspecific toxic effect o f the drug, it is suggested t h a t the d i s a p p e a r i n g o f the o r g a n e l l e s t r a t i f i c a t i o n m i g h t result f r o m a specific vinc r i s t i n e - i n d u c e d d i s a g g r e g a t i o n o f the m i c r o t u b u l e s a c t i n g as a c y t o s k e l e ton. K e y words: C r e a m h a m s t e r t h y r o i d - S t r a t i f i c a t i o n o f o r g a n e l l e s s o m e s - M i c r o t u b u l e s -- U l t i m o b r a n c h i a l follicles - Vincristine.
Lyso-
S e n d offprint requests to : Dr. P. Ketelbant-Balasse, Laboratories of Electron Microscopy, Pathology and Experimental Medicine, Rue aux Laines 97, B-1000 Brussels, Belgium.
* This work was realized thanks to contracts 20013 and 3.9001.75 of the Fonds de la Recherche Scientifique M~dicale and with the help of the Caisse G~n6rale d'Epargne et de Retraite in the framework of the Association Euratom, University of Brussels and University of Pisa. ** Acknowledgments. The authors are indebted to Dr. F. Rodesch who performed the incubation experiments, to Prof. P. Dustin, Jr. for critical reading of the manuscript, to Mrs. J. SchzepesMichel, Mr. G. Vienne, and J.L. Conreur for their technical assistance, and to Mrs. B. No61-Vincke for typing the manuscript.
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P. Ketelbant-Balasse and P. N6ve
Introduction T h e t h y r o i d o f m a m m a l s has two e m b r y o l o g i c origins (Rogers, 1927; S u g i y a m a , 1940; M c A l p i n e , 1955; Boyd, 1964): m o s t o f the follicles derive f r o m a ventral p h a r y n g e a l hernia. T h e 4th a n d 5th lateral p o u c h e s o f the b r a n c h i a l arches m a y also c o n t r i b u t e to the f o r m a t i o n o f a small p a r t o f follicles, s o m e t i m e s called u l t i m o b r a n c h i a l follicles ( D u n n , 1944; V a n D y k e , 1944, 1945; G o r b m a n , 1947; A x e l r a d a n d L e b l o n d , 1955). F o r several years, different p a p e r s have been d e v o t e d to the u l t r a s t r u c t u r a l d e s c r i p t i o n o f this s e c o n d k i n d o f follicle in different species. E a c h o f these studies has p o i n t e d out different m o r p h o l o g i c a s p e c t s c h a r a c t e r i s t i c o f each species (Wetzel a n d W o l l m a n , 1969; N6ve a n d W o l l m a n , 1971 a; W o l l m a n a n d N6ve, 1971 a; Calvert, 1972; N6ve a n d W o l l m a n , 1972). T h e a i m o f the p r e s e n t w o r k is to describe the second type o f follicle in the c r e a m h a m s t e r t h y r o i d . N e w u l t r a s t r u c t u r a l features related to the usual k i n d o f follicles will also be m e n t i o n e d . F i n a l l y , e x p e r i m e n t a l evidence will b e p r o v i d e d suggesting t h a t m i c r o t u b u l e s c o n s t i t u t e a c y t o s k e l e t o n responsible for the o r g a n e l l e s t r a t i f i c a t i o n in the u s u a l t h y r o i d cells.
Material and Methods Inbred male cream hamsters (Strain LHC/LAK) were provided by the Lakeview Hamster Colony, Newfield, N.J. The animals weighed approximately 100 g and, when used, were 6-15 months old. They were fed with Purina Laboratory Chow and received water ad libitum. After ether anesthesia and exsanguination both thyroid lobes were excised and immediately dipped for 90 min in glutaraldehyde 4.2/100 ml in 0.1 M Millonig's buffer at 20~ and at pH 7.4. After postfixation for 60 rain in 2/100 ml osmium tetroxide in phosphate buffer and after dehydration in graded concentrations of ethanol, the lobes were embedded in Epon according to Luft (1961). Serial toluidine blue-stained semithin sections were made until the second kind of follicle was recognized with the light microscope (Fig. 1). Thereafter ultrathin sections were performed with a diamond knife on a MT-2B Porter-Blum ultramicrotome. The sections were stained with uranyl-acetate and lead and observed with Siemens Elmiskop I and 101 electron microscopes. The ultrastructural aspects of the usual thyroid cells and of the second kind of follicle in 15-month-old animals were compared with those seen in 6-month-old hamsters. For experimental investigation, only 6-month-old animals were used. After rapid removal, the thyroid lobes were incubated for 4 hrs in Parker 199 culture medium at 37~ in presence or absence of vincristine sulfate (Lilly, Indianapolis) at the following concentrations: 2.10 _4 M, 6.10-4 M and 9.10 4M. At the end of incubation, fragments were fixed for electron microscope study.
Results
The Usual Thyroid Cells In the 6 - m o n t h - o l d c r e a m h a m s t e r , the usual t h y r o i d cells a p p e a r e d with the s a m e c h a r a c t e r i s t i c s as those d e s c r i b e d in a p r e v i o u s p a p e r : long microvilli, a b u n d a n c e o f m i c r o t u b u l e s a n d m i c r o f i l a m e n t s , a n d stratification o f the o r g a n e l l e s with a u n i f o r m layer o f dense r o u n d e d o r i r r e g u l a r l y s h a p e d l y s o s o m e s scattered between the a p e x a n d the nucleus (N6ve a n d W o l l m a n , 1971 b).
Ultrastructure of Cream Hamster Thyroid
51
Fig. 1. Toluidine-blue-stained semithin section in a thyroid of 15-month-oldcream hamster showing groups of so-called ultimobranchial follicles easily recognizable due to the abundance of cellular debris in the follicular lumen, x 900 In animals 15 months of age, the thyroid cells showed considerable differences in appearance. The abundance of lysosomes and the scarcity of granular reticulum was most prominent. Most of the lysosomes were large, irregularly shaped and often had a denser core. They occupied most of the space between the apex and the nucleus and between the nucleus and the lateral surfaces. In extreme cases, as shown in Fig. 2, the lysosomes occupied the whole cytoplasm with the nucleus pushed into a corner of the cell: in the remaining cytoplasm, dispersed groups of Golgi cisternae and a few rough endoplasmic cisternae were found. In these conditions stratification of the organelles was lost.
The Ultimobranchial Follicles The designation of the different cell types seen in the second kind of follicle is the same as that used in previous papers devoted to such follicles in the rat and in the mouse (Wollman and N6ve, 1971 a).
Fig. 2. Usual thyroid cell in a cream hamster 15 m o n t h s of age: most of the cytoplasm is filled with large irregular-shaped lysosomes. Golgi cisternae (go) are dispersed in several places throughout the hyaloplasm. The nucleus (N) is pushed aside in a corner of the cell. Stratification of the organelles is lost. • 10,000
Ultrastructure of Cream HamsterThyroid
53
The general properties of the ultimobranchial follicles and their cell complement were different at 6 and 15 months of age.
Six-month-old Hamster
The ultimobranchial follicles consisted of two or more layers of cells surrounding a lumen containing an amorphous material and cell debris (Fig. 3). In some follicles the multilayered U cells were connected with unilayered typical thyroid epithelium, ordinarily in contact with the lumen and indistinguishable from that in the usual follicle. When cells of the two types were in contact with each other, they were joined by desmosomes and a terminal bar at the apical end of neighboring lateral surfaces. Sections of follicles appeared surrounded by a basal membrane. The outermost or basal U cells and the more apical U cells looked somewhat different from each other and both could be distinguished from the typical thyroid epithelium. The basal U cells or fibrillar dark cells (Fig. 3) were usually flattened, with flattened nuclei, and looked darker than most apical cells. They had an abundance of free ribosomes and a relative scarcity of granular reticulum. Desmosomes and interfoldings connected these cells with each other and with the inner cells. Basal half-desmosomes and some micropinocytotic vesicles were present along the plasma membrane and bundles of fibrils were dispersed throughout the cytoplasm (Fig. 4). Bordering on the colloid lumen, several types of U cells could be recognized by their distinctive ultrastructure: fibrillar light cells, parafollicular cells, ciliated cells, vesicular cells, and cells with myelinic inclusions. The proportions between these five cell types were essentially variable from one animal to another so that it is difficult to appreciate the percentage of each cell type. The parafollicular cells, easily recognizable due to their small secretory granules, appeared as in other species and never bordered the colloid lumen although frequently separated from it by a thin layer of another cell type. The fibrillar light cells constituted the most frequent type: they differed from the basal U cells, having a low concentration of ribosomes and having fibrils largely dispersed in the apparently larger cytoplasm. Short and wide microvilli coated by an amorphous material extended into the colloid lumen. Fibrils were denser inside the microvilli. A Golgi complex with bristled coated vesicles was observed occasionally near the nucleus and mitochondria were primarily located on the sides and at the base of the nucleus (Fig. 3). The vesicular cells (Fig. 5) also had dispersed fibrils and short microvilli but were characterized along the apex by the presence of rounded vesicles of variable size, with amorphous contents. Some were in contact with the apical surface. Parallel arrays of rough endoplasmic cisternae were often seen in the cytoplasms. Cells with myelinic inclusions (Fig. 6) had the usual organelle complement but also displaced large, usually rounded inclusions with heterogeneous content and often made up of myelinic figures. In some of these inclusions tubular rods were recognizable (Figs. 7 and 8).
Fig. 3. Part of a "mixed" so-called ultimobranchial follicle in a 6-month-old hamster thyroid: unilayered usual thyroid cells (th) in the upper left of the micrograph directly continue with bilayered U cells along a single colloid lumen (Co). Dark basal and flattened U cells are easily recognizable from the upper larger fibrillar light cells with short apical microvilli, x 4,800
Ultrastructure of Cream Hamster Thyroid
55
Fig. 4. Details of a dark basal U cell: ribosomes and bundles of microfilaments are abundant. Typical hemidesmosomesexist along the basal plasma membrane. • 22,000
Ciliated cells were the rarest cell type: their ultrastructure was similar to that of ciliated cells from the second kind of thyroid follicle in C3H mouse (Fig. 9). So-called A - R cells as in mouse thyroid were also seen (Fig. 9). They were characterized by large areas of cytoplasm occupied by elongated or rounded cisternae of a granular reticulum: some of these cisternae were, however, still studded with rare ribosomes. Sometimes globular intracytoplasmic inclusions with amorphous material were found. The lumen of ultimobranchial follicles contained a cellular material, which resembled the colloid of the usual follicle and cell debris. The latter consisted principally of largely intact cell carcasses. The cytoplasm of these carcasses consisted of a relatively dense meshwork of fibers in which organelle remnants were embedded.
Fifteen-month-old Hamster The ultrastructural aspect of the second kind of follicle displayed great variations from one animal to another. Similar descriptions as those made of the young hamsters could also apply to some old animals. However, a special aspect was more often observed: the follicles were larger in size with more abundant luminal contents principally made up of fibrillar cellular carcasses. The lumen
Fig. 5. Vesicular cell bordering on the colloid lumen: rounded vesicles with amorphous contents are scattered below the apex. Some of them are in close contact with the apical plasma membrane. x 22,500
Ultrastructure of Cream Hamster Thyroid
57
Fig. 6. In a mixed follicle, typical ultimobranchial cell with myelinic inclusions squeezed between a thyroid cell (th) and a fibrillar light cell. • 7,200
was bordered by bilayered flattened cells resembling the basal U cells, with m a n y free ribosomes and fibrils. However, the apical cells had m o r e cytoplasm than the basal cells and presented with some parallel arrays of rough endoplasmic cisternae and apical vesicles. Some of these cells could also be A - R cells.
Incubation Experiments The ultrastructure of the thyroid glands in vitro remained similar to that observed in vivo even after 4 hrs incubation. When incubation was performed in the presence of vincristine, at least at a concentration lower than 2. l0 -4 M, no morphologic change was noted: stratification of organelles and microtubules was maintained. On the other hand, when the glands were incubated in the presence of 6 . 1 0 - 4 M vincristine, microtubules vanished and paracrystalline structures appeared in the cytoplasm (Fig. 10). Some lysosomes were seen at the base of the cell and lateral to the nucleus although most remained located above the
Figs. 7 and 8. D e t a i l s of cells with myelinic inclusions : t u b u l a r rods are seen in some of the inclusions. x 13,500 Fig. 9. T y p i c a l A - R cell b o r d e r i n g on the colloid l u m e n and close to a ciliated cell. x 6,000
Fig. 10. Typical follicular cell of a cream hamster thyroid incubated in toto in presence of 6- 10 4 M vincristine. Paracrystalline structures (arrows) are induced by vincristine. Lysosomes (ly) and Golgi cisternae (Go) are seen at the base of the cell: stratification of organelles is not so obvious as in reference incubated glands, x 12,500
Fig. 11. Typical follicular cell in a cream hamster thyroid incubated in toto in presence of 9.10 -4 M vincristine. Apart from changes noticed in the previous figure, rounded ergastoplasmic vesicles are scattered throughout the whole cytoplasm and are no longer located at the base of the cell. x 6,500
Ultrastructure of Cream HamsterThyroid
61
nucleus. Golgi cisternae occupied less specific locations than in reference glands : they were found everywhere in the cytoplasm, even at the cellular base. The granular endoplasmic cisternae had lost their elongated shape: they appeared more or less round. The higher the concentration of vincristine in the incubation medium, the more the aforementioned changes were apparent. Rounded ergastoplasmic cisternae were found dispersed throughout the whole cytoplasm. Lysosomes were larger in size than usual. No change involved centrioles, zonula occludens, desmosomes, or mitochondria (Fig. 11).
Discussion
As previously described (N6ve and Wollman, 1971 a), the ultrastructure of the cream hamster thyroid, at least in the young animal, is characterized by special features: principally, the stratification of organelles, and the abundance of microtubules and of lysosomes. The latter are particularly large and in the young animal, constitute a layer located between the apical surface and the nucleus in each normal thyroid. When the animals grow older, these lysosomes increase in number and become more and more dispersed throughout the whole cytoplasm. In extreme cases these organelles become prominent in the cytoplasm: the lysosomes are seen everywhere whereas the other organelles are difficult to recognize. The nuclei seem to be pushed away by this abundance of lysosomes in such a way that the resultant cell type should be designed as a lysosome-rich cell. As this phenomenon of lysosomal accumulation seems to be related to age, this process might result from the exhaustion of the mechanism whereby the cell rids itself of its metabolic debris. This cell evolution is compatible with the phenomenon observed in most thyroid cells from old normal humans (N6ve, 1965; Klinck et al., 1970) and also in pathology where thyroid cells from cretinoid patients with iodoprotein defect and goiter show accumulation of lysosomes and lipofuscin residues with high cellular activities (Ketelbant-Balasse et al., 1975). The ultrastructure of the second kind of follicle or the so-called ultimobranchial follicle in rat and mouse thyroid has been reported (Wetzel and Wollman, 1969; N~ve and Wollman, 1971b, 1972; Calvert, 1972). In both species several different cell types and mixed follicles have been recognized: fibrillar cells, parafollicular cells, ciliated cells, and cell debris in the follicular lumen were common ultrastructural features. These similarities also exist in the cream hamster thyroid and allow the identification of this kind of follicle as having probably the same embryologic origin: here also, the proportion of different cell types varied with the age. Except for the parafollicular cells, the literature remains conjectural as to whether these special follicles play a physiologic role. The existence of vesicles similar to secretory granules in one of the cell types in the ultimobranchial follicles of the cream hamster suggests that these cells could have a secretory process. Can these cells give rise to a new hormone? More work is needed before answering this question. Microtubules are especially abundant in the usual thyroid cells of the cream hamster (N6ve and Woltman, 1971 b). It has been suggested that these microtubules might act as a scaffolding which holds some organelles in preferred
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P. Ketelbant-Balasse and P. N6ve
p o s i t i o n s in cells a n d w o u l d a c c o u n t for the stratification o f the organelles seen on the u s u a l t h y r o i d cells o f the c r e a m hamster. M i c r o t u b u l e i n h i b i t o r s like vincristine o r colchicine d e s t r o y m i c r o t u b u l e s a n d i n h i b i t t h y r o i d secretion in vitro (N+ve et al., 1970; W i l l i a m s a n d Wolff, 1970; N~ve etal., 1972) a n d in vivo ( E k h o l m etal., 1974) after acute s t i m u l a t i o n with t h y r o t r o p i n . Vincristine also induces f o r m a t i o n o f p a r a crystalline structures. I n c u b a t i o n o f the c r e a m h a m s t e r t h y r o i d in the presence o f vincristine p r o d u c e d vanishing o f m i c r o t u b u l e s a n d f o r m a t i o n o f p a r a c r y s t a l line structures at least when the vincristine c o n c e n t r a t i o n was sufficiently h i g h : m o r e o v e r , at these c o n c e n t r a t i o n s , o t h e r m o r p h o l o g i c changes a p p e a r e d that is, r o u n d e d aspect o f the e n d o p l a s m i c cisternae a n d the loss o f organelle stratification. T h e doses o f vincristine h a d to be higher t h a n in o t h e r systems in o r d e r to d e s t r o y the m i c r o t u b u l e s . T h e higher the c o n c e n t r a t i o n s o f the drug, the m o r e intense the u l t r a s t r u c t u r a l changes. T h e latter, o f course, might be only the result o f an aspecific toxic effect o f vincristine. H o w e v e r , except for m i c r o t u bules, the intrinsic aspect o f the organelles r e m a i n e d u n c h a n g e d a n d the p l a s m a m e m b r a n e s l o o k e d n o r m a l . A n o t h e r e x p l a n a t i o n might be t h a t these m o r p h o l o g i c c h a n g e s result f r o m the absence o f m i c r o t u b u l e s : the cell in such c o n d i t i o n w o u l d lose its m i c r o t u b u l a r c y t o s k e l e t o n a n d c o n s e q u e n t l y the m a i n t e n a n c e of its organelles in preferential i n t r a c y t o p l a s m i c locations. T h e necessity to use such high doses o f vincristine for o b t a i n i n g these effects c o u l d be e x p l a i n e d by the e x c e p t i o n a l a b u n d a n c e o f m i c r o t u b u l e s in the hamster.
References Axelrad, A.A., Leblond, C.P.: Induction of thyroid tumors in rats by low iodine diet. Cancer (Philad.) 8, 339-367 (1955) Boyd, J.D.: Development of the human thyroid gland. In: The thyroid gland (Pitt-Rivers, R. and Trotter, W.R., eds.), vol. 1, p. 9-31. London: Butterworths 1964 Calvert, R.: Transitional cells in the postnatal thyroid gland of the rat. Anat. Rec. 174, 341-360 (1972) Dunn, T.B.: Ciliated cells of the thyroid of the mouse. J. nat. Cancer. Inst. 4, 555-557 (1944) Ekholm, R., Ericson, L.E., Josefsson, J.-O., Melander, A. : In vivo action of vinblastine on thyroid ultrastructure and hormone secretion. Endocrinology 94, 641-649 (1974) Gorbman, A.: Functional and morphological properties in the thyroid gland, ultimobranchial body and persisting ductus pharyngiobranchialis IV of an adult mouse. Anat. Rec. 98, 93-102 (1947) Ketelbant-Balasse, P., Glinoer, D., N+ve, P.: Ultrastructure of the thyroid gland in a case of congenital goiter with cretinism. Path. europ. 10, 155-165 (1975) Klinck, G.H., Oertel, J.E., Winship, T.: Ultrastructure of normal human thyroid. Lab. Invest. 22, 2 22 (1970) Luft, J. : Improvements in epoxy embedding methods. J. Cell Biol. 9, 409414 (1961) McAlpine, R.J.: Alkaline glycerophosphatase in the developing thyroid, parathyroid and thymus of the albino rat. Amer. J. Anat. 96, 191-223 (1955) N6ve, P.: Ultrastructure des cellules folliculaires d'une thyro'ide humaine normale. J. Microsc. 4, 811-814 (1965) N+ve, P., Ketelbant-Balasse, P., Willems, C., Dumont, J.E.: Effect of inhibitors of microtubules and microfilaments on dog thyroid slices in vitro. Exp. Cell Res. 74, 227-244 (1972)
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N~ve, P., Willems, C., Dumont, J.E.: Involvement of the microtubular-microfilament system in thyroid secretion. Exp. Cell Res. 63, 457460 (1970) N6ve, P., Wollman, S.H.: Fine structure of ultimobranchial follicles in the thyroid gland of the rat. Anat. Rec. 17L 259-272 (1971a) N6ve, P., Wollman, S.H.: Ultrastructure of the thyroid gland of the cream hamster. Anat. Rec. 168, 23~12 (1971 b) N6ve, P., Wollman, S.H.: Fine structure of a fifth type of epithelial cell in the thyroid gland of the C3H mouse. Anat. Rec. 172, 37-44 (1972) Rogers, W.M. : The fate of the ultimobranchial body in the white rat (Mus norvegicus albinus). Amer. J. Anat. 38, 349-376 (1927) Sugiyama, S.: The fate of the ultimobranchial body of the albino rat with special reference to the formation of the thyroid gland. Okajima Folia anat. jap. 19, 333-341 (1940) Van Dyke, J.H.: Behavior of ultimobranchial tissue in the postnatal thyroid gland: The origin of thyroid cystoadenomata in the rat. Anat. Rec. 88, 369-391 (1944) Van Dyke, J.H.: Behaviour of ultimobranchial tissue in the postnatal thyroid gland: epithelial cysts, their relation to thyroid parenchyma and to "new growths" in the thyroid gland of young sheep. Amer. J. Anat. 76, 201-251 (1945) Wetzel, B.K., Wollman, S.H.: Fine structure of a second kind of thyroid follicle in the C3H mouse. Endocrinology 84, 563-578 (1969) Williams, J.A., Wolff, J.: Possible role of microtubules in thyroid secretion. Proc. nat. Acad. Sci. (Wash.) 67, 1901-1908 (1970) Wollman, S.H., N6ve, P. : Ultimobranchial follicles in the thyroid glands of rats and mice. Recent Progr. Hormone Res. 27, 213-234 (1971 a) Wollman, S.H., N6ve, P.: Postnatal development and properties of ultimobranchial follicles in the rat thyroid. Anat. Rec. 171,247-258 (1971b)
Received June 14, 1975
