Ultrastructure of the Thyroid in Dwarf Mice ANDRd C. CORDIER,' JEAN-FRANCOIS DENEF AND STANISLAS M. HAUMONT Department of Histology, University of Louvain, Tour Vdsale U C L 5229, Avenue E . Mounier, 52, B-1200 Bruxelles, Belgium
ABSTRACT The thyroid gland of Snell's dwarf mice consists of small follicles with flattened epithelium, partly differentiated cords and undifferentiated masses. Many adipocytes are found. The ultimobranchial cysts are well developed. Parafollicular cells are normal. In the partly organized cords, microfollicular cells and some small follicles limited by two or three cells are seen. The presence of these structures led us to think that they represent the first stages of normal folliculogenesis, described as the fusion of two adjacent unicellular microfollicles. Their further growth is the result of the coalescing of small adjacent follicles or of cellular multiplication. The presence of undifferentiated masses and partly differentiated cords, in dwarf mice, seems due to a developmental arrest rather than to aberrant development. This disorder of organogenesis is ascribed mainly to a lack of pituitary growth hormone.
The recessive mutant dwarf mice, dw/ dw, were described by Snell in 1929. The growth of homozygotes practically stops on the fourteenth postnatal day. The primary lesion caused by the mutation lies in the pituitary gland (Smith and MacDowell, '31; Carsner and Rennels, '60). Acidophilic cells are missing (Ortman, '56; Elftman and Wegelius, '59; Bartke, '64). Neither STH (Lewis, '67) nor prolactin (Cheever et al., '69) are found. The TSHcells are very sparse (Ortman, '56; Elftman and Wegelius, '59; Bartke, '64). Dwarf mice are sterile even though gonadotropins are secreted (Smith and MacDowell, '3 1) ; gonads are poorly developed. In the testis spermatogenesis takes place (Smith and MacDowell, '31) but the Leydig cells present some enzymatic defects (Cavallero et al., '63). Ovaries have no corpora lutea and antral follicles are rare (Smith and MacDowell, '31). There is no deficiency of ACTH and the adrenals are normal (Bartels, '41; Shire and Hambly, '73). The PBI level is low (Wegelius, '59). The altered thyroid structure has been described for paraffin sections (Snell, '30; Smith and MacDowell, '30; Kemp and Marx, '37; Bartke, '64) but not yet at the ultrastructural level. Yet electron microscopy allows a more accurate description of the thyroid structure in dwarf mice. It also bears on important arguments in the conAM. J. ANAT.,146: 339-358.
troversy surrounding the histogenesis of thyroid follicles. MATERIALS AND METHODS
Snell's dwarf mice were obtained by mating heterozygote parents (dw/+ ). Fifteen 1- to 6-month-old dwarf mice of either sex were killed by cervical dislocation. Phenotypically normal mice were used as controls. The trachea and thyroid gland were removed at once and the two thyroid lobes were separated after immersion in fixative and fixed for two hours in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.02% CaClz. The osmolality of the buffer was adjusted with sucrose to 270 mOsm/l; the total osmolality of the fixative was 540 mOsm/l. After fixation, the fragments were rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide in a buffer of the same composition, dehydrated in graded ethanol and embedded in Epon 812 (Luft, '61). One-pm-thick sections were cut with a Reichert OMU-3 microtome, and stained with 1% toluidine blue. Thin sections were mounted on uncoated copper grids, stained with uranyl acetate (Watson, '58) and lead citrate (Reynolds, '63) and examined with Accepted April 13, '76. IResearch Fellow of the Fonds National de la Recherche Scientifique.
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periphery brought to mind the successive stages of folliculogenesis. The inner part of the cord contained undifferentiated cells RESULTS and cells with a large vacuole (fig. 8). The The thyroid of our control mice (fig. 1) wall of these vacuoles was covered with consisted of round follicles with cuboidal small microvilli, 0.2 to 0.3 pm high (fig. epithelium, separated by blood vessels and 9 ) ; their size and presence showed that a few adipocytes. It differed little from the these vacuoles were not abnormal colloid droplets but, rather, intracellular equivaglands described in the literature. In the thyroid gland of dwarf mice (fig. lents of follicular cavities. I n the middle part of the cellular cord, 2 ) , the follicles were small and separated from each other by adipose tissue. Numer- two adjacent microfollicles were joined ous partly organized epithelial cords and and their colloid vacuoles were separated undifferentiated masses were scattered be- by only a 0.2-,m-thick band of cytoplasm tween these small follicles. The ultimo- (fig. 10). Still farther out, unicellular microfolbranchial cysts, easily recognized by the presence of ciliated cells and the absence licles had fused (fig. 11) and formed small of colloid, were more numerous and more follicles limited by two or three cells (fig. developed than in the normal mouse. No 12). The contents of their cavities increased differences were noticed with regard to in size, but the limiting cells did not increase in number or volume and thus beage or sex. came flattened. Their cytoplasm was re( a ) Follicular structures duced to a thin strip not exceeding 0.2 ,m The follicles were limited by squamous in thickness, with a length of 10 or 12 ,m or cuboidal cells (fig. 3 ) . The nucleo-cyto- (fig. 13). plasmic ratio was high. The nucleus was ( c ) Undifferentiated masses ovoid or flattened, and its major axis was Among follicles and cords were undifferparallel to the base of the cell. Heterochromatin was abundant. Round to elongate entiated masses surrounded by a basement profiles of mitochondria were scattered membrane (fig. 14). The cells had heterothroughout the cytoplasm. Rough endo- chromatin-rich nuclei, R.E.R.-poor cytoplasmic reticulum (RER) was scarce and plasm and no secretory vesicles. the poorly developed Golgi apparatus was ( d ) C cells located at the apical pole of the cell. Rare dense bodies (lysosomes) were observed, Some follicles contained C cells more but no colloid droplets. The microvilli were electron-lucent and larger than follicular scanty and very short. Some follicles con- cells. These were included in the follicusisted of very few cells with attenuated ex- lar wall, but they never came into contensions which made contact either with tact with the lumen (fig. 15a). Their cuboidal cells or with an extension froiii nucleus contained finely condensed chroanother similarly altered cell (fig. 4 ) . matin along the nuclear membrane. Mitochondria were numerous. The R.E.R. was ( b ) Partly organized cords scattered throughout the cytoplasm in the In some areas, thyroid follicles were as- form of long saccules, and the Golgi apsociated with epithelial cords. These con- paratus was well developed. Numerous tained small follicular structures and cells granules filled the entire cytoplasm, some with cytoplasm nearly filled by a large col- being very dense with a diameter of 130 to loid vesicle (fig. 5 ) . The basement mem- 150 nm, others less dense with a diameter brane which covered each follicle also sur- of 150 to 320 n m (fig. 15b). rounded any associated cord (figs. 5, 6, 7 ) . DISCUSSION However, basement membranes did not necessarily separate adjacent follicles (fig. The histogenesis of the thyroid has been 7), as in control thyroids. explained in two different ways. According The sequence of structures in one single to Norris ('18), Hilfer et al. ('68) and cord from the center of the gland to its Michel-Bechet et al. ('73), the follicles a Philips-300 electron microscope operated at 60 Kv.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE
accumulate their colloid by secreting it into an intercellular space which grows progressively and becomes a large central cavity. On the other hand, according to Bradway ('29), Waterman and Gorbman ('35) and Shepard ('67), the follicular cavity is produced by the fusion of intracellular vesicles of adjacent cells. Our observations with dwarf mouse seem to confirm this latter hypothesis. The thyroid epithelial cords, indeed, contain different structures which could represent the successive stages of folliculogenesis. I n the innermost part of the cords, the most characteristic feature is the presence of unicellular microfollicles. The following step in differentiation appears to be represented by the disappearance of the thin cytoplasmic barrier separating two neighboring unicellular microfollicles. In normal mice, folliculogenesis begins on embryonic day 16 or 17 (Shepard, '67). In dwarf mice, it seems to begin at the same time but stops quickly after starting. Although it is not possible to define the precise cause of this blockage, it is logical to suppose that it is the result of the hormonal disorder caused by the pituitary lesions. The lack of TSH or of STH could be involved in thyroid dysembryogenesis. TSH certainly influences folliculogenesis. Pituitary TSH-producing cells (Sano and Sasaki, '69) and the first thyroid follicles appear at the same time, on about day 16 or 17 of development. Furthermore. thyroid cells in culture form follicles only if their culture medium contains TSH (Fayet et al., '71, Lissitzky et al., '71). The lack of TSH could, to a certain extent, explain the presence of unorganized masses of undifferentiated cells. The structural abnormalities of the dwarf mouse thyroid cannot, however, be explained completely by this absence, because prolonged treatment with TSH causes little improvement (Bartke, '65). The lack of STH seems to be an important factor. It is known that STH has a n action on cellular multiplication (Winick and Grant, '68), and one of the characteristics of the dwarf mouse thyroid is the presence of follicles formed by a few flattened cells with extended processes. This type of follicle would result from colloid secretion accompanied by hampered divi-
34 1
sion of the cells in the follicular wall. Colloid synthesis evidently does occur in the dwarf mouse. This is compatible with the defective TSH secretion presumed to be characteristic of this mutant, because colloid production, although influenced by TSH (Tong, '67; Olin et al., '70), is not suppressed by the absence of this hormone (Bjorkman et al., '74). Normal follicular growth has been explained either by the fusion of follicles (Norris, '18; Bradway, '29) or by cellular multiplication in the follicular wall (Kull, '26; Kemp and Marx, '37). The structural defects in the thyroid of the dwarf mouse are better explained by the latter hypothesis. A block in the growth of follicles is interpreted as being due to the stretching of cells which cannot divide because of a lack of STH. Stereological analysis (to be published) has verified that the principal reason for the smaller follicular volume in the dwarf mouse is a reduced number of cells. If a defect in the histogenesis of the thyroid gland i n dwarf mice is caused by pituitary defects, it is limited to the follicles. C cells, whose structure is the same as described in the rat by Ekholm and Ericson ('68) or by Stoeckel and Porte in the chick ('69), are not disturbed. Pearse and Polak ('71) have proved by cytochemical markers the migration of these cells from the neural rhombencephalic crests to the pharyngeal pouches. Others have shown the same phenomenon in the chick (Le Douairin et al., '74; Polak et al., '74). The pituitary defects in the dwarf mouse prevent neither the migration nor the persistence of C cells in the epithelial cords. Ultimobranchial cysts are highly developed in the dwarf mouse. They are the endoderm a1 remnants of ultimobr anchi a1 bodies after the migration of C cells. Their abundance can be explained by the abnormal follicular development. The thyroid follicles normally compress these cysts and prevent their proliferation. A similar process has been described in the dysgenic thymus of the nude mouse (Cordier, '74). ACKNOWLEDGMENTS
The skillful technical assistance of Miss Liliane Vanderstraaten, of Mrs. Jacqueline Vander Perre for secretorial work and of
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Mrs. Hermine Claes for photographic work is gratefully acknowledged. LITERATURE CITED Bartels, E. 1941 Studies on hereditary dwarfism in mice. 111. Development of the adrenals i n the dwarf mice. Acta Pathol. Microbiol. Scand., 18: 20-35. Bartke, A. 1964 Histology of anterior hypophysis, thyroid and gonads of two types of dwarf mice. Anat. Rec., 149: 225-236. 1965 The response of two types of dwarf mice to growth hormone, thyrotrophin and thyroxine. Gen. Comp. Endocrinol., 5: 418-
426. Bjorkman, U., R. Ekholm, L. G. Elmqvist, L. E. Ericson, A. Melander and S. Smeds 1974 Induced unidirectional transport of Protein into the thyroid follicular lumen. EndocGinology, 95:
1506-1517. Bradway, W. 1929 The morphogenesis of the thyroid follicles of the chick. Anat. Rec., 42:
157-167. Carsner, R. L., and E. G. Rennels 1960 Primary site of gene action i n anterior pituitary dwarf mice. Science, 131: 829. Cavallero, C., M. Martinazzi, C. Baroni and U. Magrini 1963 Pituitary control of mouse testis i n hereditary dwarfism: histological and cytochemical observations. Gen. Comp. Endocrinol., 3: 636-643. Cheever, E. V., B. K. Seavey and U. J. Lewis 1969 Prolactin of normal and dwarf mice. Endocrinology, 85: 698-703. Cordier, A. C. 1974 Ultrastructure of the thymus in “nude” mice. J. Ultrastr. Res., 47: 26-40. Ekholm, R.,and L. E. Ericson 1968 The ultrastructure of the parafollicular cells i n the thyroid gland i n the rat. J. Ultrastr. Res., 23: 378-
402. Elftman, H., and 0. Wegelius 1959 Anterior Dituitarv cvtoloev of the dwarf mouse. Anat. Kec., 135: 43-15: Fayet, G., M. Michel-Bechet and. S. Lissitzky 1971 Thyrotropin-induced aggregation and reorganization into follicles of isolated porcine thyroid cells i n culture. 11. Ultrastructural studies. Eur. J. Biochem., 24: 100-111. Hilfer, S. R., L. B. Iszard and E. K. Hilfer 1968 Follicles formation i n the embryonic chick thyroid. 11. Reorganization after dissociation. Z. Zellforsch., 92: 256-269. Kemp, T., and L. Marx 1937 Beeinflussung von erblichem hypoyhysiirem Zwerguchs bei Mausen durch vershiedene Hypophysenauszuge und Thyroxin. 11. Endokrine Organe. Acta Pathol. Microbiol. Scand., 14: 197-227. Kull, H. A. 1926 The late embryonic development of the thyroid gland of the albino rat. Anat. Rec., 32: 133-150. Le Douairin, N., J. Fontaine and C. Le Lievre 1974 New studies on the neural crest origin of the avian ultimobranchial glandular cellsInterspecific combinations and cytochemical characterization of C cells based of the uptake
of biogenic amine precursors. Histochemistry,
38: 297-305. Lewis, U. J. 1967 Growth hormone of normal and dwarf mice. Mem. SOC.Endoc., 15: 179-
191. Lissitzky, S., G. Fayet, A. Giraud, B. Verrier and J. Torresani 1971 Thyrotrophin-induced aggregation and reorganization into follicles of isolated porcine thyroid cells. I. Mechanism of action of thyrotrophin and metabolic properties. Eur. J. Biochem., 24: 88-99 . Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414 Michel-Bechet, M., P. Cau and G. Fayet 1973 Etude ultrastructurale de la morphogenese du follicule thyroidien: reorganisation des cellules de porc en culture. C. R. Acad. Sc. Paris, 277: 1029-1031. Norris, E. H. 1918 The early morphogenesis of the human thyroid gland. Am. J. Anat., 24:
443-465. Olin, P., R. Ekholm and S. Almqvist 1970 Biosynthesis of thyroglobulin related to the ultrastructure of the human fetal thyroid gland. Endocrinology, 87: 1000-1014. Ortman, R. 1956 A study of some cytochemical reactions and of the hormone content of the adenohypophysis in the normal and i n genetic dwarf mice. J. Morph., 99: 417-431. Pearse, A. G. E., and J. M. Polak 1971 Cytochemical evidence for the neural crest origin of mammalian ultimobranchial C cells. Histochemie, 27: 96-102. Polak, J. M., A. G. E. Pearse, C. LeLievre, J. Fontaine and N. M. LeDouairin 1974 Immuno cytochemical confirmation of the neural crest origin of avian calcitonin-producing cells. Histochemistry, 40: 209-214. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron-opaque stain i n electron microscopy. J. Cell Biol., 17: 208-212. Sano, M., and F. Sasaki 1969 Embryonic development of the mouse anterior pituitary studied by light and electron microscopy. Z. Anat. Entwickl. Gesch., 129: 195-222. Shepard, T. H. 1967 Onset of function in the human fetal thyroid. Biochemical and radioautographic studies from organ culture. J. Clin. End. Metab., 27: 945-958. Shire, G. M. J., and E. A. Hambly 1973 The adrenal gland of mice with hereditary pituitary dwarfism. Acta Pathol. Microbiol. Scand., 81:
225-228. Smith, P. E., and E. C. MacDowell 1930 An hereditary anterior pituitary deficiency in the house mouse. Anat. Rec., 46: 249-257. 1931 The differential effect of hereditary mouse dwarfism o n the anterior-pituitary hormones. Anat. Rec., 50: 85-93. Snell, G. D. 1929 Dwarf, a new mendelian recessive character of the house mouse. Proc. Nat. Acad. Sci., 15: 733-734. 1930 Effect of injections of anterior pituitary extracts on the thyroids of mice with hereditary dwarfism. Anat. Rec., 47: 316. Stoeckel, M. E., and A. Porte 1969 Etude struc-
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE turale des corps ultimobranchiaux du poulet. I. Aspect normal et developpement embryonnaire. Z. Zellforsch., 94: 495-512. Tong, W. 1967 TSH Stimulation of I4C-amino acid incorporation i n protein by isolated bovine thyroid cells. Endocrinology, 80: 1101-1110. Waterman, A., and A. Gorbman 1935 Development of the thyroid gland of the rabbit. J. Exp. ZOO^., 132: 504-538.
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Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478. Wegelius, 0. 1959 The dwarf mouse-An animal with secondary myxoedema. Proc. SOC.Exp. Biol. Med, 101: 225-227. Winick, M., and P. Grant 1968 Cellular growth in the organs of the hypopituitary dwarf mouse. Endocrinology, 83: 544-547.
PLATE 1 EXPLANATION O F FIGURES
344
1
Normal thyroid tissue in a one-month-old control mouse. Follicles are numerous and well developed. Epon section, 1 pm thick. Toluidine blue staining. x540.
2
Thyroid tissue in a one-month-old dwarf mouse. Follicles are small, sparse and lined by a flattened epithelium. They are separated by undifferentiated cellular masses ( arrows-heads) and voluminous ultimobranchial cysts ( U B ) containing ciliated cells (arrows). Epon section, 1 pm thick. Toluidine blue staining. ~ 5 4 0 .
ULTRASTRUCTURE O F T H E THYROID I N DWARF MICE A. C . Cordier, J.-F. Denef and S. M. Haumont
PLATE 1
34 5
PLATE 2 EXPLANATION O F FIGURES
346
3
Thyroid follicles of a dwarf mouse. Thyroid cells are small with a relatively large nucleus. The microvilli at their apical pole are scanty and very short. Colloid droplets are absent. ~ 4 , 8 6 0 .
4
Thyroid follicles i n a dwarf mouse. I n this follicle, the epithelium consists partly of elongated cells whose attenuated extensions are joined by desmosomes (arrows) to cuboidal cells. x 7,794.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 2
34 7
PLATE 3 EXPLANATION OF FIGURES
5
Cellular cord i n the thyroid gland of a dwarf mouse. From bottom LO top, the cord is formed by undifferentiated cells ( U C ) , by microfollicles ( M F ) and by a follicle which is lined by elongated cells (FOL). The same basement membrane (arrow-heads) covers the entire cord. x 4,275.
6 Thyroid follicles of the cord described i n figure 5. Both follicles are lined by elongated cells. They lie within the same cord and are enveloped by the same basement membrane. ~ 3 , 4 2 0 .
7 Higher power view of the area indicated by the rectangle i n figure 6. Plasmalemmas of epithelial cells belong to two adjacent follicles and make contact without interposition of any basement membrane. x 20,700.
348
ULTRASTRUCTURE O F T H E THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 3
349
PLATE 4 EXPLANATION
350
O F FIGURES
8
Epithelial cord with undifferentiated cells and unicellular microfollicle. x 6,075.
9
Unicellular microfollicle in a thyroid cord of a dwarf mouse. The nucleus is crowded toward one pole of the cell. At the opposite pole, the cytoplasm contains a colloid vesicle ( C V ) whose wall presents some microvilli (arrows). X 13,950.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 4
35 1
PLATE 5 EXPLANATION OF FIGURES
10 Unicellular microfollicles i n a thyroid cord of a dwarf mouse. Both cells contain one colloid vesicle ( C V ) . The two microfollicles are separated only by cell membranes and a narrow cytoplasmic band. X 13,140.
11
352
Epithelial cord from a dwarf mouse thyroid. Two unicellular microfollicles have fused (arrow). The other cells are undifferentiated. X 12,339.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 5
353
PLATE 6 EXPLANATION OF FIGURES
354
12
Small follicles i n a thyroid cord of a dwarf mouse. The right follicle is lined by two elongated cells. The left one consists of a large colloid droplet and three cells. The colloid masses of the two follicles are separated only by a thin cellular band. ~ 5 , 7 6 0 .
13
Thyroid follicle from a dwarf mouse. The colloid follicle is limited by some very elongated cells. They are in close contact with processes from another cell (arrows). x 5,490.
ULTRASTRUCTURE O F THE THYROID I N DWARF MICE A. C. Cordier, J.-F. Denef and S. M. H a u m o n t
PLATE 6
355
PLATE 7 EXPLANATION O F FIGURES
14 Undifferentiated cellular mass in a thyroid of a dwarf mouse. Note the presence of a basement membrane (arrows) on both sides of the mass; a polar section through a follicle is thus excluded. x 4,950.
356
x 5,040.
15a
C cell in a thyroid follicle of a dwarf mouse.
15b
Detail of a C cell. Two types of granules are recognizable.
x 16,290.
ULTRASTRUCTURE OF T H E THYROID I N DWARF MICE A. C. Cordier, J.-F. Denef and S . M. H a u m o n t
PLATE 7
357
ABSTRACT The thyroid gland of Snell's dwarf mice consists of small follicles with flattened epithelium, partly differentiated cords and undifferentiated masses. Many adipocytes are found. The ultimobranchial cysts are well developed. Parafollicular cells are normal. In the partly organized cords, microfollicular cells and some small follicles limited by two or three cells are seen. The presence of these structures led us to think that they represent the first stages of normal folliculogenesis, described as the fusion of two adjacent unicellular microfollicles. Their further growth is the result of the coalescing of small adjacent follicles or of cellular multiplication. The presence of undifferentiated masses and partly differentiated cords, in dwarf mice, seems due to a developmental arrest rather than to aberrant development. This disorder of organogenesis is ascribed mainly to a lack of pituitary growth hormone.
The recessive mutant dwarf mice, dw/ dw, were described by Snell in 1929. The growth of homozygotes practically stops on the fourteenth postnatal day. The primary lesion caused by the mutation lies in the pituitary gland (Smith and MacDowell, '31; Carsner and Rennels, '60). Acidophilic cells are missing (Ortman, '56; Elftman and Wegelius, '59; Bartke, '64). Neither STH (Lewis, '67) nor prolactin (Cheever et al., '69) are found. The TSHcells are very sparse (Ortman, '56; Elftman and Wegelius, '59; Bartke, '64). Dwarf mice are sterile even though gonadotropins are secreted (Smith and MacDowell, '3 1) ; gonads are poorly developed. In the testis spermatogenesis takes place (Smith and MacDowell, '31) but the Leydig cells present some enzymatic defects (Cavallero et al., '63). Ovaries have no corpora lutea and antral follicles are rare (Smith and MacDowell, '31). There is no deficiency of ACTH and the adrenals are normal (Bartels, '41; Shire and Hambly, '73). The PBI level is low (Wegelius, '59). The altered thyroid structure has been described for paraffin sections (Snell, '30; Smith and MacDowell, '30; Kemp and Marx, '37; Bartke, '64) but not yet at the ultrastructural level. Yet electron microscopy allows a more accurate description of the thyroid structure in dwarf mice. It also bears on important arguments in the conAM. J. ANAT.,146: 339-358.
troversy surrounding the histogenesis of thyroid follicles. MATERIALS AND METHODS
Snell's dwarf mice were obtained by mating heterozygote parents (dw/+ ). Fifteen 1- to 6-month-old dwarf mice of either sex were killed by cervical dislocation. Phenotypically normal mice were used as controls. The trachea and thyroid gland were removed at once and the two thyroid lobes were separated after immersion in fixative and fixed for two hours in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.02% CaClz. The osmolality of the buffer was adjusted with sucrose to 270 mOsm/l; the total osmolality of the fixative was 540 mOsm/l. After fixation, the fragments were rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide in a buffer of the same composition, dehydrated in graded ethanol and embedded in Epon 812 (Luft, '61). One-pm-thick sections were cut with a Reichert OMU-3 microtome, and stained with 1% toluidine blue. Thin sections were mounted on uncoated copper grids, stained with uranyl acetate (Watson, '58) and lead citrate (Reynolds, '63) and examined with Accepted April 13, '76. IResearch Fellow of the Fonds National de la Recherche Scientifique.
339
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A. C. CORDIER, J.-F. D E N E F AND S . M. HAUMONT
periphery brought to mind the successive stages of folliculogenesis. The inner part of the cord contained undifferentiated cells RESULTS and cells with a large vacuole (fig. 8). The The thyroid of our control mice (fig. 1) wall of these vacuoles was covered with consisted of round follicles with cuboidal small microvilli, 0.2 to 0.3 pm high (fig. epithelium, separated by blood vessels and 9 ) ; their size and presence showed that a few adipocytes. It differed little from the these vacuoles were not abnormal colloid droplets but, rather, intracellular equivaglands described in the literature. In the thyroid gland of dwarf mice (fig. lents of follicular cavities. I n the middle part of the cellular cord, 2 ) , the follicles were small and separated from each other by adipose tissue. Numer- two adjacent microfollicles were joined ous partly organized epithelial cords and and their colloid vacuoles were separated undifferentiated masses were scattered be- by only a 0.2-,m-thick band of cytoplasm tween these small follicles. The ultimo- (fig. 10). Still farther out, unicellular microfolbranchial cysts, easily recognized by the presence of ciliated cells and the absence licles had fused (fig. 11) and formed small of colloid, were more numerous and more follicles limited by two or three cells (fig. developed than in the normal mouse. No 12). The contents of their cavities increased differences were noticed with regard to in size, but the limiting cells did not increase in number or volume and thus beage or sex. came flattened. Their cytoplasm was re( a ) Follicular structures duced to a thin strip not exceeding 0.2 ,m The follicles were limited by squamous in thickness, with a length of 10 or 12 ,m or cuboidal cells (fig. 3 ) . The nucleo-cyto- (fig. 13). plasmic ratio was high. The nucleus was ( c ) Undifferentiated masses ovoid or flattened, and its major axis was Among follicles and cords were undifferparallel to the base of the cell. Heterochromatin was abundant. Round to elongate entiated masses surrounded by a basement profiles of mitochondria were scattered membrane (fig. 14). The cells had heterothroughout the cytoplasm. Rough endo- chromatin-rich nuclei, R.E.R.-poor cytoplasmic reticulum (RER) was scarce and plasm and no secretory vesicles. the poorly developed Golgi apparatus was ( d ) C cells located at the apical pole of the cell. Rare dense bodies (lysosomes) were observed, Some follicles contained C cells more but no colloid droplets. The microvilli were electron-lucent and larger than follicular scanty and very short. Some follicles con- cells. These were included in the follicusisted of very few cells with attenuated ex- lar wall, but they never came into contensions which made contact either with tact with the lumen (fig. 15a). Their cuboidal cells or with an extension froiii nucleus contained finely condensed chroanother similarly altered cell (fig. 4 ) . matin along the nuclear membrane. Mitochondria were numerous. The R.E.R. was ( b ) Partly organized cords scattered throughout the cytoplasm in the In some areas, thyroid follicles were as- form of long saccules, and the Golgi apsociated with epithelial cords. These con- paratus was well developed. Numerous tained small follicular structures and cells granules filled the entire cytoplasm, some with cytoplasm nearly filled by a large col- being very dense with a diameter of 130 to loid vesicle (fig. 5 ) . The basement mem- 150 nm, others less dense with a diameter brane which covered each follicle also sur- of 150 to 320 n m (fig. 15b). rounded any associated cord (figs. 5, 6, 7 ) . DISCUSSION However, basement membranes did not necessarily separate adjacent follicles (fig. The histogenesis of the thyroid has been 7), as in control thyroids. explained in two different ways. According The sequence of structures in one single to Norris ('18), Hilfer et al. ('68) and cord from the center of the gland to its Michel-Bechet et al. ('73), the follicles a Philips-300 electron microscope operated at 60 Kv.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE
accumulate their colloid by secreting it into an intercellular space which grows progressively and becomes a large central cavity. On the other hand, according to Bradway ('29), Waterman and Gorbman ('35) and Shepard ('67), the follicular cavity is produced by the fusion of intracellular vesicles of adjacent cells. Our observations with dwarf mouse seem to confirm this latter hypothesis. The thyroid epithelial cords, indeed, contain different structures which could represent the successive stages of folliculogenesis. I n the innermost part of the cords, the most characteristic feature is the presence of unicellular microfollicles. The following step in differentiation appears to be represented by the disappearance of the thin cytoplasmic barrier separating two neighboring unicellular microfollicles. In normal mice, folliculogenesis begins on embryonic day 16 or 17 (Shepard, '67). In dwarf mice, it seems to begin at the same time but stops quickly after starting. Although it is not possible to define the precise cause of this blockage, it is logical to suppose that it is the result of the hormonal disorder caused by the pituitary lesions. The lack of TSH or of STH could be involved in thyroid dysembryogenesis. TSH certainly influences folliculogenesis. Pituitary TSH-producing cells (Sano and Sasaki, '69) and the first thyroid follicles appear at the same time, on about day 16 or 17 of development. Furthermore. thyroid cells in culture form follicles only if their culture medium contains TSH (Fayet et al., '71, Lissitzky et al., '71). The lack of TSH could, to a certain extent, explain the presence of unorganized masses of undifferentiated cells. The structural abnormalities of the dwarf mouse thyroid cannot, however, be explained completely by this absence, because prolonged treatment with TSH causes little improvement (Bartke, '65). The lack of STH seems to be an important factor. It is known that STH has a n action on cellular multiplication (Winick and Grant, '68), and one of the characteristics of the dwarf mouse thyroid is the presence of follicles formed by a few flattened cells with extended processes. This type of follicle would result from colloid secretion accompanied by hampered divi-
34 1
sion of the cells in the follicular wall. Colloid synthesis evidently does occur in the dwarf mouse. This is compatible with the defective TSH secretion presumed to be characteristic of this mutant, because colloid production, although influenced by TSH (Tong, '67; Olin et al., '70), is not suppressed by the absence of this hormone (Bjorkman et al., '74). Normal follicular growth has been explained either by the fusion of follicles (Norris, '18; Bradway, '29) or by cellular multiplication in the follicular wall (Kull, '26; Kemp and Marx, '37). The structural defects in the thyroid of the dwarf mouse are better explained by the latter hypothesis. A block in the growth of follicles is interpreted as being due to the stretching of cells which cannot divide because of a lack of STH. Stereological analysis (to be published) has verified that the principal reason for the smaller follicular volume in the dwarf mouse is a reduced number of cells. If a defect in the histogenesis of the thyroid gland i n dwarf mice is caused by pituitary defects, it is limited to the follicles. C cells, whose structure is the same as described in the rat by Ekholm and Ericson ('68) or by Stoeckel and Porte in the chick ('69), are not disturbed. Pearse and Polak ('71) have proved by cytochemical markers the migration of these cells from the neural rhombencephalic crests to the pharyngeal pouches. Others have shown the same phenomenon in the chick (Le Douairin et al., '74; Polak et al., '74). The pituitary defects in the dwarf mouse prevent neither the migration nor the persistence of C cells in the epithelial cords. Ultimobranchial cysts are highly developed in the dwarf mouse. They are the endoderm a1 remnants of ultimobr anchi a1 bodies after the migration of C cells. Their abundance can be explained by the abnormal follicular development. The thyroid follicles normally compress these cysts and prevent their proliferation. A similar process has been described in the dysgenic thymus of the nude mouse (Cordier, '74). ACKNOWLEDGMENTS
The skillful technical assistance of Miss Liliane Vanderstraaten, of Mrs. Jacqueline Vander Perre for secretorial work and of
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A. C. CORDIER, J.-F. DENEF AND S. M. HAUMONT
Mrs. Hermine Claes for photographic work is gratefully acknowledged. LITERATURE CITED Bartels, E. 1941 Studies on hereditary dwarfism in mice. 111. Development of the adrenals i n the dwarf mice. Acta Pathol. Microbiol. Scand., 18: 20-35. Bartke, A. 1964 Histology of anterior hypophysis, thyroid and gonads of two types of dwarf mice. Anat. Rec., 149: 225-236. 1965 The response of two types of dwarf mice to growth hormone, thyrotrophin and thyroxine. Gen. Comp. Endocrinol., 5: 418-
426. Bjorkman, U., R. Ekholm, L. G. Elmqvist, L. E. Ericson, A. Melander and S. Smeds 1974 Induced unidirectional transport of Protein into the thyroid follicular lumen. EndocGinology, 95:
1506-1517. Bradway, W. 1929 The morphogenesis of the thyroid follicles of the chick. Anat. Rec., 42:
157-167. Carsner, R. L., and E. G. Rennels 1960 Primary site of gene action i n anterior pituitary dwarf mice. Science, 131: 829. Cavallero, C., M. Martinazzi, C. Baroni and U. Magrini 1963 Pituitary control of mouse testis i n hereditary dwarfism: histological and cytochemical observations. Gen. Comp. Endocrinol., 3: 636-643. Cheever, E. V., B. K. Seavey and U. J. Lewis 1969 Prolactin of normal and dwarf mice. Endocrinology, 85: 698-703. Cordier, A. C. 1974 Ultrastructure of the thymus in “nude” mice. J. Ultrastr. Res., 47: 26-40. Ekholm, R.,and L. E. Ericson 1968 The ultrastructure of the parafollicular cells i n the thyroid gland i n the rat. J. Ultrastr. Res., 23: 378-
402. Elftman, H., and 0. Wegelius 1959 Anterior Dituitarv cvtoloev of the dwarf mouse. Anat. Kec., 135: 43-15: Fayet, G., M. Michel-Bechet and. S. Lissitzky 1971 Thyrotropin-induced aggregation and reorganization into follicles of isolated porcine thyroid cells i n culture. 11. Ultrastructural studies. Eur. J. Biochem., 24: 100-111. Hilfer, S. R., L. B. Iszard and E. K. Hilfer 1968 Follicles formation i n the embryonic chick thyroid. 11. Reorganization after dissociation. Z. Zellforsch., 92: 256-269. Kemp, T., and L. Marx 1937 Beeinflussung von erblichem hypoyhysiirem Zwerguchs bei Mausen durch vershiedene Hypophysenauszuge und Thyroxin. 11. Endokrine Organe. Acta Pathol. Microbiol. Scand., 14: 197-227. Kull, H. A. 1926 The late embryonic development of the thyroid gland of the albino rat. Anat. Rec., 32: 133-150. Le Douairin, N., J. Fontaine and C. Le Lievre 1974 New studies on the neural crest origin of the avian ultimobranchial glandular cellsInterspecific combinations and cytochemical characterization of C cells based of the uptake
of biogenic amine precursors. Histochemistry,
38: 297-305. Lewis, U. J. 1967 Growth hormone of normal and dwarf mice. Mem. SOC.Endoc., 15: 179-
191. Lissitzky, S., G. Fayet, A. Giraud, B. Verrier and J. Torresani 1971 Thyrotrophin-induced aggregation and reorganization into follicles of isolated porcine thyroid cells. I. Mechanism of action of thyrotrophin and metabolic properties. Eur. J. Biochem., 24: 88-99 . Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414 Michel-Bechet, M., P. Cau and G. Fayet 1973 Etude ultrastructurale de la morphogenese du follicule thyroidien: reorganisation des cellules de porc en culture. C. R. Acad. Sc. Paris, 277: 1029-1031. Norris, E. H. 1918 The early morphogenesis of the human thyroid gland. Am. J. Anat., 24:
443-465. Olin, P., R. Ekholm and S. Almqvist 1970 Biosynthesis of thyroglobulin related to the ultrastructure of the human fetal thyroid gland. Endocrinology, 87: 1000-1014. Ortman, R. 1956 A study of some cytochemical reactions and of the hormone content of the adenohypophysis in the normal and i n genetic dwarf mice. J. Morph., 99: 417-431. Pearse, A. G. E., and J. M. Polak 1971 Cytochemical evidence for the neural crest origin of mammalian ultimobranchial C cells. Histochemie, 27: 96-102. Polak, J. M., A. G. E. Pearse, C. LeLievre, J. Fontaine and N. M. LeDouairin 1974 Immuno cytochemical confirmation of the neural crest origin of avian calcitonin-producing cells. Histochemistry, 40: 209-214. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron-opaque stain i n electron microscopy. J. Cell Biol., 17: 208-212. Sano, M., and F. Sasaki 1969 Embryonic development of the mouse anterior pituitary studied by light and electron microscopy. Z. Anat. Entwickl. Gesch., 129: 195-222. Shepard, T. H. 1967 Onset of function in the human fetal thyroid. Biochemical and radioautographic studies from organ culture. J. Clin. End. Metab., 27: 945-958. Shire, G. M. J., and E. A. Hambly 1973 The adrenal gland of mice with hereditary pituitary dwarfism. Acta Pathol. Microbiol. Scand., 81:
225-228. Smith, P. E., and E. C. MacDowell 1930 An hereditary anterior pituitary deficiency in the house mouse. Anat. Rec., 46: 249-257. 1931 The differential effect of hereditary mouse dwarfism o n the anterior-pituitary hormones. Anat. Rec., 50: 85-93. Snell, G. D. 1929 Dwarf, a new mendelian recessive character of the house mouse. Proc. Nat. Acad. Sci., 15: 733-734. 1930 Effect of injections of anterior pituitary extracts on the thyroids of mice with hereditary dwarfism. Anat. Rec., 47: 316. Stoeckel, M. E., and A. Porte 1969 Etude struc-
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE turale des corps ultimobranchiaux du poulet. I. Aspect normal et developpement embryonnaire. Z. Zellforsch., 94: 495-512. Tong, W. 1967 TSH Stimulation of I4C-amino acid incorporation i n protein by isolated bovine thyroid cells. Endocrinology, 80: 1101-1110. Waterman, A., and A. Gorbman 1935 Development of the thyroid gland of the rabbit. J. Exp. ZOO^., 132: 504-538.
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Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478. Wegelius, 0. 1959 The dwarf mouse-An animal with secondary myxoedema. Proc. SOC.Exp. Biol. Med, 101: 225-227. Winick, M., and P. Grant 1968 Cellular growth in the organs of the hypopituitary dwarf mouse. Endocrinology, 83: 544-547.
PLATE 1 EXPLANATION O F FIGURES
344
1
Normal thyroid tissue in a one-month-old control mouse. Follicles are numerous and well developed. Epon section, 1 pm thick. Toluidine blue staining. x540.
2
Thyroid tissue in a one-month-old dwarf mouse. Follicles are small, sparse and lined by a flattened epithelium. They are separated by undifferentiated cellular masses ( arrows-heads) and voluminous ultimobranchial cysts ( U B ) containing ciliated cells (arrows). Epon section, 1 pm thick. Toluidine blue staining. ~ 5 4 0 .
ULTRASTRUCTURE O F T H E THYROID I N DWARF MICE A. C . Cordier, J.-F. Denef and S. M. Haumont
PLATE 1
34 5
PLATE 2 EXPLANATION O F FIGURES
346
3
Thyroid follicles of a dwarf mouse. Thyroid cells are small with a relatively large nucleus. The microvilli at their apical pole are scanty and very short. Colloid droplets are absent. ~ 4 , 8 6 0 .
4
Thyroid follicles i n a dwarf mouse. I n this follicle, the epithelium consists partly of elongated cells whose attenuated extensions are joined by desmosomes (arrows) to cuboidal cells. x 7,794.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 2
34 7
PLATE 3 EXPLANATION OF FIGURES
5
Cellular cord i n the thyroid gland of a dwarf mouse. From bottom LO top, the cord is formed by undifferentiated cells ( U C ) , by microfollicles ( M F ) and by a follicle which is lined by elongated cells (FOL). The same basement membrane (arrow-heads) covers the entire cord. x 4,275.
6 Thyroid follicles of the cord described i n figure 5. Both follicles are lined by elongated cells. They lie within the same cord and are enveloped by the same basement membrane. ~ 3 , 4 2 0 .
7 Higher power view of the area indicated by the rectangle i n figure 6. Plasmalemmas of epithelial cells belong to two adjacent follicles and make contact without interposition of any basement membrane. x 20,700.
348
ULTRASTRUCTURE O F T H E THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 3
349
PLATE 4 EXPLANATION
350
O F FIGURES
8
Epithelial cord with undifferentiated cells and unicellular microfollicle. x 6,075.
9
Unicellular microfollicle in a thyroid cord of a dwarf mouse. The nucleus is crowded toward one pole of the cell. At the opposite pole, the cytoplasm contains a colloid vesicle ( C V ) whose wall presents some microvilli (arrows). X 13,950.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 4
35 1
PLATE 5 EXPLANATION OF FIGURES
10 Unicellular microfollicles i n a thyroid cord of a dwarf mouse. Both cells contain one colloid vesicle ( C V ) . The two microfollicles are separated only by cell membranes and a narrow cytoplasmic band. X 13,140.
11
352
Epithelial cord from a dwarf mouse thyroid. Two unicellular microfollicles have fused (arrow). The other cells are undifferentiated. X 12,339.
ULTRASTRUCTURE OF THE THYROID IN DWARF MICE A. C. Cordier, J.-F. Denef and S. M. Haumont
PLATE 5
353
PLATE 6 EXPLANATION OF FIGURES
354
12
Small follicles i n a thyroid cord of a dwarf mouse. The right follicle is lined by two elongated cells. The left one consists of a large colloid droplet and three cells. The colloid masses of the two follicles are separated only by a thin cellular band. ~ 5 , 7 6 0 .
13
Thyroid follicle from a dwarf mouse. The colloid follicle is limited by some very elongated cells. They are in close contact with processes from another cell (arrows). x 5,490.
ULTRASTRUCTURE O F THE THYROID I N DWARF MICE A. C. Cordier, J.-F. Denef and S. M. H a u m o n t
PLATE 6
355
PLATE 7 EXPLANATION O F FIGURES
14 Undifferentiated cellular mass in a thyroid of a dwarf mouse. Note the presence of a basement membrane (arrows) on both sides of the mass; a polar section through a follicle is thus excluded. x 4,950.
356
x 5,040.
15a
C cell in a thyroid follicle of a dwarf mouse.
15b
Detail of a C cell. Two types of granules are recognizable.
x 16,290.
ULTRASTRUCTURE OF T H E THYROID I N DWARF MICE A. C. Cordier, J.-F. Denef and S . M. H a u m o n t
PLATE 7
357
