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Thyroid follicular morphogenesis mechanism: Organ culture of the fetal gland as an experimental approach
J O U R N A L OF U L T R A S T R U C T U R E RESEARCH
82, 283-295 (1983)
Thyroid Follicular Morphogenesis Mechanism: Organ Culture of the Fetal Gland as an Experimental Approach 1 L. REMY, 2. B. VERRIER, 2,* M. MICHEL-BECHET, E. MAZZELLA, AND A. M. ATHOUEL-HAON Laboratoire d'Histologie, FacultO de M~decine/Secteur Nord, Boulevard Pierre Dramard, F-13326 Marseille Cedex 3, and *INSERM - U 38, Facultd de MOdecine/Timone, Boulevard Jean-Moulin, F-Marseille 13385, Marseille, Cedex 4, France Received September 29, 1981, and in revised form February 8, 1982~June 30, 1982 The morphological and physiological changes induced by organ culture and thyroid-stimulating hormone (TSH) stimulation in the rat fetal thyroid gland were studied. Organ culture increased Golgi activity which was further enhanced by TSH, subsequently facilitating the formation of intracellular lumina. TSH also raised the intracellular cAMP level. The intracellular lumina observed during follicular morphogenesis are structurally comparable to typical intracellular cavities formed in adult thyroid cells, which are considered as being the result of increased Golgi activity. The intracellular lumen, therefore, is probably not a physiologically significant step in thyroid morphogenesis.
Many authors have studied the morphogenesis of the thyroid follicle in various species by photon and electron microscopy, and an exhaustive bibliography has been previously published (Cau et al., 1976). The mechanism of follicle formation is, however, still controversial and two hypotheses remain. According to the first hypothesis the lumen appears in the intercellular space; the follicular cavity bounded by cellular junctions then expands by exocytosis of Golgi vesicles from the follicular cells. Several observations, namely in the thyroid of the A m m o c o e t e (Clements-Merlini, 1960), chick embryo (Hilfer, 1964; Fujita and Tanizawa, 1966; Hilfer et al., 1967, 1968), the rat fetus (Feldman et al., 1961; Ishikawa, 1965), and in adult pig thyroid cells cultivated in vitro (Michel-Bechet et al., 1973; Cau et al., 1976), support this hypothesis. ~This work was supported by a grant from the INSERM (CRL 79 154 44). 2 Research associates of the Institut National de la Sant6 et de la Recherche M6dicale (INSERM).
In the second theory, intracellular cavities in adjacent cells lead to the formation of the follicular lumen by fusion: this has been reported in the human fetus in vivo (Shepard et al., 1964; Shepard, 1968) and in vitro (Shepard, 1967), in the rat fetus (Calvert, 1973, 1975; Calvert and Pusterla, 1973), and in the dogfish (Alluchon-Gerard, 1979); it has been demonstrated in thyroid cells of adult dogs cultivated in vitro (Neve and Dumont, 1970). In adult pig thyroid cells stimulated in vitro by TSH or cAMP and under different tissue culture conditions, the prominent role in follicular formation seems to be played either by the intercellular space (MichelBechet et al., 1973; Cau et al., 1976) or by intracytoplasmic cavities ( R e m y et al., 1977a,b). An earlier study (Remy et aL, 1980) of the morphogenesis of the thyroid follicle in the rat did not demonstrate intracellular cavities similar to those found in porcine thyroid cell cultures (Remy et al., 1977a). In order to explain these conflicting results concerning the mechanism of thyroid fol283 0022-5320/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
REMY ET AL.
284
l i c u l a r m o r p h o g e n e s i s a n d to a v o i d the p h y s i o l o g i c a l p e r t u r b a t i o n s w h i c h m a y be i n d u c e d b y long t e r m c u l t u r e o f a d u l t cells, we s t i m u l a t e d o r g a n c u l t u r e d fetal t h y r o i d s w i t h T S H , a n d s u b s e q u e n t l y m e a s u r e d the i n t r a c e l l u l a r level o f c A M P . MATERIALS AND METHODS
Culture. Six female rats were caged with one male rat (Wistar) between 0800 and 1100 hr, which was considered as time 0 of gestation. Fetal rat thyroids, 14, 15, and 16 days old, were secured at about 1000 hr and placed in Coming plastic dishes filled with 2 ml of Eagle's medium (Eagle, 1955) supplemented with 2 mM glutamine, 200 U of penicillin G/ml, 50 #g of streptomycin/mt, and with or without crude bovine thyroid-stimulating hormone (TSH; TS-10, Sigma; 0.2, 20, and 40 mU/ml). The glands were incubated for various times at 37°C in a 95% air-5% CO2 atmospherel Morphology. The previously incubated thyroids and nonincubated 14-, 15-, 16-, and 17-day-old fetal glands were fixed with 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 hr, washed for 1 hr with 0. i M sodium cacodylate, postfixed for 1 hr in 1% OsO4 in 0.1 M sodium cacodylate, dehydrated in acetone, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and investigated with an Elmiskop 101 Siemens electron microscope. cAMP determination. The cAMP content of five thyroid lobes of 16-day-old fetuses was determined by placing the lobes on Millipore filters (HA; 0.45 #m) and incubatingthem described under Culture. In some instances, the medium was supplementedwith 0.6 mM isobutyl-methylxanthine (IBMX) to inhibit endogenous cAMP degradation by phosphodiesterase. At the end of the incubation the Millipore filters with the thyroid lobes were removed from the culture medium, dried on blotting paper, and placed in 200 #1 1 N perchloric acid and then quick frozen at -20°C. After thawing, 180 #1of this medium, which contains all the intracellular cAMP, was withdrawn to measure cAMP concentration by radioimmunoassay (Cailla et aL, 1973); propanol was used to separate free from bound antigen. Measures were performed in duplicate on two dilutions of the assay medium.
RESULTS
Morphology Nonincubated glands. T h e o b s e r v a t i o n s o f fetal t h y r o i d g l a n d s o f 14, 15, 16, a n d 17 days h a v e b e e n d e s c r i b e d i n a p r e v i o u s p a p e r ( R e m y et al., 1980). T o s u m m a r i z e : -- 14-day-old thyroids showed short j u n c t i o n a l z o n e s i n the c e n t e r o f c o m p a c t cell c l u m p s (Fig. 1); - - a t 15 days, the cell j u n c t i o n s were m o r e e x t e n s i v e a n d m o r e n u m e r o u s (Fig. 2); - - a t 16 days, we o b s e r v e d large c o n t a c t s w i t h n u m e r o u s i n f o l d i n g s o f the p l a s m a m e m b r a n e (Fig. 8); - - a t 17 days, e l e m e n t a r y follicular l u m i n a a p p e a r e d b e t w e e n two cells i n the p l a n e o f the s e c t i o n (Fig. 14). A t each stage, s o m e secretion vesicles were p r e s e n t b u t we n e v e r o b s e r v e d a n y i n t r a cellular l u m e n . Organ cultured glands. T a b l e I s u m m a rizes the o b s e r v a t i o n s . I n the 14-day t h y r o i d s i n c u b a t e d for 24 h r w i t h T S H , we o b s e r v e d a n i m p o r t a n t a c c u m u l a t i o n o f clear cytoplasmic vesicles near adherens-type contacts and very extensive Golgi apparatuses (Fig. 4). I n s o m e cases, a p p a r e n t i n t r a cellular cavities bordered with microvilli were clearly v i s i b l e (Fig. 5). I n o t h e r cases, s o m e v a c u o l a r s t r u c t u r e s or m u l t i v e s i c u l a r bodies showed membrane invaginations w i t h c y t o p l a s m i c e x p a n s i o n s (Figs. 6 a n d 7). A f t e r the c o n t r o l i n c u b a t i o n , n e i t h e r m o r p h o l o g y o f this k i n d n o r a n i n c r e a s e i n G o l g i s e c r e t i o n were seen. I n the t h y r o i d s o f 1 5 - d a y fetuses i n c u b a t e d for 24 h r w i t h T S H , we o b s e r v e d extensive Golgi apparatuses with n u m e r o u s s e c r e t i o n vesicles. S o m e follicular c a v i t i e s
FIG. 1. Fourteen-day fetal thyroid. General view of several cells with membrane contact zones (arrows). Numerous plasma membrane infoldings. X 12 500. FIG. 2. Fifteen-day fetal thyroid. Membrane contact zone. Adherens-type junction with densifications (arrows). × 22 000. FIG. 3. Fourteen-day fetal thyroid incubated for 24 hr without TSH. Membrane contact zones (arrows). Numerous plasma membrane infoldings. X 12 500. FIG. 4. Fourteen-day fetal thyroid incubated for 24 hr with 20 mU TSH/ml. Membrane contact zone. Adherens-type junction. Very numerous clear vesicles. Very active Golgi apparatus (GA). × 14 400.
286
REMY ET AL. TABLE I Explantation age (days)
Incubation time (hr)
Final age (days)
GS
IL
FL
0.2 m U TSH/ml
14 15 16
24 24 24
15 16 17
++ ++ ++
-+ +
-+ +
20 m U TSH/ml
14
24
15
24
16
16
24
17
++ ++ ++
+ ++ ++
--
15
++
40 m U TSH/ml
14 15 16
24 24 24
15 16 17
++ ++ ++
+ + ++
-+ ++
Control (no TSH)
14 15 16
24 24 24
15 16 17
-+ +
-+ +
--+
Incubation medium
+
Note. GS = increase of Golgi secretion compared with that observed at the same stage in vivo, IL = intracellular lumina, FL = follicular lumina, -- = not observed, + = observed, + + = numerous.
exhibited "cytoplasmic islets" which were indented by the exocytosis of clear vesicles ( F i g s . 10 a n d l 1). C o n t r o l i n c u b a t i o n s showed an increase in Golgi secretion less m a r k e d t h a n t h a t w i t h T S H i n c u b a t i o n (Fig. 9) a n d s o m e i n t r a c e l l u l a r l u m i n a , b u t n e v e r follicular lumina. In the thyroids of 16-day-old fetuses incubated for 24 hr with TSH, two types of structures were often observed: follicular lumina and cavities which appear to be intracellu-
lar. T h e y m a y r e p r e s e n t f o l l i c u l a r l u m i n a whose cell junctions are not visible in the plane of section or they may be true intrac y t o p l a s m i c s t r u c t u r e s ( F i g . 16). A n i n t r a c e l l u l a r c a v i t y b o r d e r e d w i t h m i c r o v i l l i is s h o w n i n Fig. 13. I n t r a c e l l u l a r a n d f o l l i c u l a r lumina may exist in 16-day-old fetal thyr o i d s a f t e r c o n t r o l i n c u b a t i o n (Fig. 15), b u t they are less numerous and less developed than after incubation with TSH. cAMP
content Sixteen-day-old
fetal thy-
FIG. 5. Fourteen-day fetal thyroid incubated for 24 hours with 20 m U TSH/ml. Membrane contact zone. Very numerous clear vesicles. Cavities with microvilli which appear to be intracellular (arrows). x 20 000. FIG. 6. Fourteen-day fetal thyroid incubated for 24 hr with 20 m U TSH/ml. Golgi zone. Numerous clear vesicles and multivesicular bodies. One multivesicular body presents fingerlike cytoplasmic protrusions (arrowhead). × 20 000. FIG. 7. Fourteen-day fetal thyroid incubated for 24 hr with 20 m U TSH/ml. Follicular lumen and numerous clear vesicles. Multivesicular body with fingerlike protrusion (arrowhead). X 18 000. FIG. 8. Sixteen-day fetal thyroid. General view of several ceils with membrane contact zones (arrows). Tangential sections of infolding (arrowheads). X 11 400. Fro. 9. Fifteen-day fetal thyroid incubated for 24 hr without TSH. Membrane contact zones (arrows). Very active Golgi apparatuses (GA) and numerous clear vesicles. × 15 000. FIG. 10. Fifteen-day fetal thyroid incubated for 24 hr with 0.2 m U TSH/ml. Follicular lumen bordered with clear vesicles. In the middle of the cavity, "cytoplasmic islet" (arrow) with numerous vesicles, x 14 000. F ~ . 11. Fifteen-day fetal thyroid incubated for 24 hr with 0.2 m U TSH/ml. Magnification of previous figure. Evidence of exocytosis (arrowheads). Microvilli seem to be the result of the indentation of the cytoplasm due to the presence of exocytotic pits. Microfilamentous axis (arrows). x 50 000. FIG. 12. Fifteen-day fetal thyroid incubated for 24 hours with 0.2 m U TSH/ml. Follicular lumen with microvilli formed by microfilamentous axis (arrows). Microvillus forming a "cytoplasmic bridge" (arrowhead). × 40 000.
)0
b~ O0
290
REMY ET AL.
roids are incubated with or without (basal level) 25 m U T S H / m l . T h e results (Table II) show that u n d e r the conditions used in our morphological studies, T S H induced a low but significant t i m e - d e p e n d e n t c A M P increase (2 times in 120 min). A m o r e effective stimulation was o b s e r v e d in a typical e x p e r i m e n t in the presence o f I B M X . DISCUSSION
Action of Organ Culture on Fetal Thyroid Cells Control incubation o f 14-day-old thyroids does not induce any increase o f Golgi secretion. T h e incubation o f 15- and 16d a y - o l d t h y r o i d s , h o w e v e r , i n d u c e s an increase in Golgi secretion with the appearance o f intracellular l u m i n a (15 days) and o f intracellular a n d follicular l u m i n a (16 days). We still cannot explain why organ culture in control m e d i u m induces these p h e n o m ena a n d why the increase in secretion begins after the 15th day o f pregnancy.
Action of T S H on Fetal Thyroid Cells It is well established that the anterior pituitary o f the rat fetus does not secrete T S H before the 17th day o f pregnancy (Setalo a n d N a k a n e , 1972). M o r e o v e r , m a t e r n a l T S H is unable to pass the placenta ( T h o r b u r n a n d Hopkins, 1973; Jost et al., 1974). F r o m a morphological point o f view, our observations clearly show that as early as the 14th day o f pregnancy, fetal thyroid cells are sensitive to T S H . F r o m a biochemical point o f view, the evaluation o f the c A M P level o f 16-day-old fetal thyroid cells stimulated by T S H d e m onstrates the sensitivity o f the cells to the h o r m o n e before the 17th day o f pregnancy: after 2 hr incubation, the c A M P level doubles when c o m p a r e d to the reference. We were unable to m e a s u r e the fetal thyroid c A M P level at 14 or 15 days o f preg-
nancy. F o r this experiment, it is necessary to collect only the thyroid lobes without any other tissue which is very difficult to achieve before the 16th day o f pregnancy. All these observations indicate a sensitivity o f the thyroid cells to T S H before the 17th day o f pregnancy, i.e., before the beginning o f T S H secretion by the fetal anterior pituitary. According to the results in Table I, incubation with T S H induces a m o r e i m p o r t a n t increase in secretion than incubation in the control m e d i u m . M o r e o v e r , the higher the h o r m o n e concentration, the m o r e intense is the secretion a n d the m o r e n u m e r o u s are the intracellular a n d follicular lumina. A stereological study o f the effects o f T S H on Golgi secretion has recently been carried out ( R e m y et al., in press).
Formation of the Intracellular Cavities Several cavities bordered by microvilli are visible and either represent follicular cavities which are c o m m o n to two or m o r e cells or they a p p e a r to be true intracellular lumina, as d e m o n s t r a t e d by the 22 consecutive ultrathin serial sections (Fig. 13). The existence ofintracellular cavities m a y be explained as follows: In vivo (Fig. 17A), the clear vesicles secreted by the dictyosomes migrate towards the adherens contact and are secreted by exocytosis into the intercellular space in the adherens contact region ( R e m y et al., 1980). In vitro (Fig. 17B), after T S H stimulation, the secreting activity o f the d i c t y o s o m e s is m u c h m o r e a b u n d a n t and the clear vesicles are so n u m e r o u s that they m a y coalesce before they reach the adherens contact, thus beginning the form a t i o n o f intracellular cavities. These cavities increase with the participation o f other clear vesicles a n d their progression toward the contact; then exocytosis occurs into the intercellular space near the adherens contact (see R e m y et al., 1977b).
FIr. 13. Sixteen-day fetal thyroid incubated for 30 min with 40 mU TSH/ml. Twenty-two consecutive ultrathin serial sections showing an intracellular lumen.
~ i:~~~~ ~ ~:~;~i~i,~ :~:"~~/~
16 DAY-OLD RAT FETUS THYROID CELL5 (30 MINUTES INCUBATION WITH TSH 40 mU/ml)
~:~ :~:~,-~iNTRACELLULARLUMEN 291
t~
293
RAT THYROID MORPHOGENESIS TABLE II DETERMINATION OF cAMP CONTENT
cAMP (mol × 1013)/thyroidlobe Incubation time
Without IBMX (3)~
(rain)
Basal
,'~;,
0 30 60 120
1.19 -+ 0.24b 1.14 + 0.02 1.31 _+0.02 1.0 + 0.02
With0.6 mM IBMX (1)
TSH stimulated 1.80 _+ 0.03 1.70 + 0.03 2.10 _+ 0.05
Basal
TSH stimulated
5.2 -+ 0.8 5.2 +- 0.6
39.7 +_ 3 50.7 + 2
a Number of experiments in parentheses. bMean + SD. The i m p o r t a n t role o f microfilamentous and microtubular material in the migration o f clear vesicles is shown by the fact that antiactin and antitubulin drugs inhibit the migration o f the vesicles and, consequently, folliculogenesis (Pic et al., in preparation). In conclusion, the presence or the absence o f intracellular cavities seems to be exclusively dependent on the abundance o f clear vesicles: if they are very a b u n d a n t (in vitro with TSH), they are more likely t o coalesce before reaching the contact zone than when they are less numerous (in vivo, without TSH stimulation) (Figs. 3, 8, and 14). According to our observations, although T S H enhances the p h e n o m e n o n , it is not absolutely necessary for the formation o f intracellular lumina (Denef et al., 1980). In fact, incubation without T S H m a y cause the appearance ofintracellular lumina, although this occurs less frequently.
Formation o f Microvilli The cavities so f o r m e d often contain cytoplasmic, fingerlike invaginations which m a y result from the indentation o f the cytoplasm by the clear vesicles (Figs, 5, 6, 10, 11). Part o f the cytoplasm contains microfilaments such as microvilli (Fig. 11) or future microvilli (Fig. 12).
The intracellular lumen, therefore, must not be considered as an essential structure for follicle differentiation but as the result o f an intensive secretory activity which implies increased m e m b r a n e synthesis. These observations lead to a possible explanation for the controversy surrounding thyroid follicle morphogenesis. The presence or the absence o f intracellular lumina is very likely related to the secretory activity o f the Golgi apparatus. Intracellular cavities m a y exist in vivo (Shepard et al., 1964; Heiman, 1966; Shepard, 1968); although this is rare, clear vesicles are not very n u m e r o u s and they hardly ever aggregate and coalesce. They migrate independently toward the adherens junction and fuse with the plasma m e m b r a n e . In a previous study on rat thyroid morphogenesis in vivo ( R e m y et al., 1980), no intracellular lumina were observed even when the fetal secretion has started (17 and 18 days o f pregnancy). Intracellular lumina are not only the result o f organ culture; they m a y exist in other circumstances. In C 3 H mice and during involution o f thyroid hyperplasia, M a n y et al. (1981) observed small follicular-like cavities which were d e m o n s t r a t e d to be intracellular by serial sections.
FIG. 14. Seventeen-day fetal thyroid. Follicular lumen (FL). X 14 000. FIG. 15. Sixteen-day fetal thyroid incubated for 24 hr without TSH. Intracellular lumen (arrow). X 12 000. FIG. 16. Sixteen-day fetal thyroid incubated for 24 hr with 20 mU TSH/ml. General view with several follicular or intracellular lumina at different degrees of maturation. Numerous clear vesicles adjacent to the cavities. X 8000.
294
REMY ET AL.
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FIG, 17. Interpretation of the two mechanisms of follicular morphogenesis. A. Follicular morphogenesis in vivo (without TSH stimulation). Golgi secretion with exocytosis of the clear vesicles into the intercellular space in the region of the adherens junction and infolding of the membranes. B. Follicular morphogenesis in vitro
(with TSH stimulation). IntensiveGolgi secretion of very numerous dear vesicles which fuse forming intracellular cavities before their exocytosis at the level of the zonula adherens.
I s o l a t e d a d u l t p o r c i n e t h y r o i d g l a n d s cultured with hydrocortisone frequently form intracellular lumina (unpublished, personal observation).
I n a d u l t rats t r e a t e d w i t h t h y r o x i n e , Ericson (1979) demonstrated intracellular l u m i n a i n t h y r o i d cells. H e f o u n d t h a t after 1 or 2 days o f t r e a t m e n t , these s t r u c t u r e s
RAT THYROID MORPHOGENESIS
are clearly visible, but that they disappear after 7 days of treatment: probably after exocytosis into the follicular lumen. In conclusion, intracellular lumina seem to be the result of either a nonphysiological increase of Golgi secretion due to thyroid hyperplasia or to experimental conditions such as culture or hormonal treatment. If they exist in the fetal gland in vivo and without any experimental stimulation, they would be so rare that they would not play any role in the morphogenesis of the thyroid follicle of the mammals. We are indebted to Professor H. D. Dellmann for his valuable help in the preparation of the manuscript, to Mrs. H. Cailla for the gift of labeled cAMP for radioimmunoassay, and to Mrs J. Bottini for skillful typing of the manuscript. REFERENCES ALLUCHON-GERARD,M. J. (1979) Arch. Anat. Microsc. Morphol. Exp. 68, 43-60. CAILLA, H., RACINE-WEISBUCH,M. S., AND DELAAGE, M. (1973) Anal. Biochem. 56, 394-407. CALVERT, R. (1973) Anat. Rec. 177, 359-375. CALVERT, R. (1975)Anat. Rec. 181, 561-579. CALVERT, R., AND PUSTERLA, A. (1973) Gen. Comp. Endocrinol. 20, 584-597. CAU, P., MICHEL-BECHET,M., AND FAYET, G. (1976) Advan. Anat. Embryol. Cell Biol. 52, 6-66. CLEMENTS-MERLINI,M. (1960) J. Morphol. 106, 357364. DENEF, J. F., BJORKMAN,V., AND EKHOLM, R. (1980) J. Ultrastruct. Res. 71, 185-202. EAGLE, H. (1955) J. Exp. Med. 102, 595-600. ERICSON, L. E. (1979) J. Ultrastruct. Res. 69, 297-305. FELDMAN, J. D., VAZQUEZ,J. J., AND KURTZ, S. M. (1961) J. Biophys. Biochem. Cytol. 11, 365-383.
295
FUJITA, H., AND TANIZAWA, Y. (1966) Z. Anat. Entwicklungs gesch. 125, 132-151. HEIMANN, P. (1966) Acta Endocrinol. SuppL, 53, 1102. HILLER, S. R. (1964) J. Morphol. 115, 135-152. HILFER, S. R., HILFER, E. K., AND ISZARD,L. B. (1967) J. MorphoL 123, 199-212. HILFER, S. R., ISZARO,L. B., AND HILFER, E. K. (1968) Z. Zellforsch. 92, 256-269. ISHIKAWA, K. (1965) Okajimas Folia Anat. Jpn. 41, 295-311. JOST, A., DuPouY, J. P., AND RIEUTORT, M. (1974) in SWAAB,D. F., AND SCHAD~,J. P. (Eds), Progress in Brain Research, vol. 41 : Integrative hypothalamic activity, pp. 209-215, Elsevier, Amsterdam. MANY, M. C., DENEF, J. F., AND HAUMONT, S. (1981) Ann. Endocrinol. 42, 41 A. MICHEL-BECHET,M., CAU, P., AND FAYET, G. (1973) C. R. Acad. Sci. D. (Paris) 277, 1029-1032. NEVE, P., ANDDUMONT,J. E. (1970) Z. Zellforsch. 103, 61-74. REMY, L., MICHEL-BECHET,M., ATHOUEL-HAON,A. M., H'~VSEPIAN,S., ANO FAVET,G. (1977a) Ann. Sci. Nat. 18, 21-35. REMY, L., MICrtEL-BECHET, M., ATHOUEL-HAoN, A. M., MAGRE, S., CATALDO,C., AND JOST, A. (1980) Arch. Anat. Microsc. Morphol. Exp. 69, 91-108. REMY, L., MICHEL-BECHET,M., CATALDO,C., BOTTINI, J., HOVSEPIAN,S., AND FAYET, G. (1977b) J. Ultrastruct, Res. 61, 243-253. REMV, L., PENEL,C., RUA, A., ANDMAZZELLA,E. (1982) Biol. Cell. (abstr), in press. SETALO,G., ANDNAKANE,P. K. (1972) Anat. Rec. 172, 403--404. SHEPARD, T. H. (1967) £ Clin. Endocrinol. 27, 945958. SHEPARO, T. H. (1968) Gen. Comp. Endocrinol. 10, 174-181. SHEPARD, T. H., ANDERSEN,H., AND ANDERSEN,H. S. (1964) Anat. Rec. 149, 363-380. THORBURN,G. D., ANDHOPKINS,P. S. (1973) in Foetal and neonatal physiology, Barcroft Centenary Symposium, Cambridge, p. 448.
82, 283-295 (1983)
Thyroid Follicular Morphogenesis Mechanism: Organ Culture of the Fetal Gland as an Experimental Approach 1 L. REMY, 2. B. VERRIER, 2,* M. MICHEL-BECHET, E. MAZZELLA, AND A. M. ATHOUEL-HAON Laboratoire d'Histologie, FacultO de M~decine/Secteur Nord, Boulevard Pierre Dramard, F-13326 Marseille Cedex 3, and *INSERM - U 38, Facultd de MOdecine/Timone, Boulevard Jean-Moulin, F-Marseille 13385, Marseille, Cedex 4, France Received September 29, 1981, and in revised form February 8, 1982~June 30, 1982 The morphological and physiological changes induced by organ culture and thyroid-stimulating hormone (TSH) stimulation in the rat fetal thyroid gland were studied. Organ culture increased Golgi activity which was further enhanced by TSH, subsequently facilitating the formation of intracellular lumina. TSH also raised the intracellular cAMP level. The intracellular lumina observed during follicular morphogenesis are structurally comparable to typical intracellular cavities formed in adult thyroid cells, which are considered as being the result of increased Golgi activity. The intracellular lumen, therefore, is probably not a physiologically significant step in thyroid morphogenesis.
Many authors have studied the morphogenesis of the thyroid follicle in various species by photon and electron microscopy, and an exhaustive bibliography has been previously published (Cau et al., 1976). The mechanism of follicle formation is, however, still controversial and two hypotheses remain. According to the first hypothesis the lumen appears in the intercellular space; the follicular cavity bounded by cellular junctions then expands by exocytosis of Golgi vesicles from the follicular cells. Several observations, namely in the thyroid of the A m m o c o e t e (Clements-Merlini, 1960), chick embryo (Hilfer, 1964; Fujita and Tanizawa, 1966; Hilfer et al., 1967, 1968), the rat fetus (Feldman et al., 1961; Ishikawa, 1965), and in adult pig thyroid cells cultivated in vitro (Michel-Bechet et al., 1973; Cau et al., 1976), support this hypothesis. ~This work was supported by a grant from the INSERM (CRL 79 154 44). 2 Research associates of the Institut National de la Sant6 et de la Recherche M6dicale (INSERM).
In the second theory, intracellular cavities in adjacent cells lead to the formation of the follicular lumen by fusion: this has been reported in the human fetus in vivo (Shepard et al., 1964; Shepard, 1968) and in vitro (Shepard, 1967), in the rat fetus (Calvert, 1973, 1975; Calvert and Pusterla, 1973), and in the dogfish (Alluchon-Gerard, 1979); it has been demonstrated in thyroid cells of adult dogs cultivated in vitro (Neve and Dumont, 1970). In adult pig thyroid cells stimulated in vitro by TSH or cAMP and under different tissue culture conditions, the prominent role in follicular formation seems to be played either by the intercellular space (MichelBechet et al., 1973; Cau et al., 1976) or by intracytoplasmic cavities ( R e m y et al., 1977a,b). An earlier study (Remy et aL, 1980) of the morphogenesis of the thyroid follicle in the rat did not demonstrate intracellular cavities similar to those found in porcine thyroid cell cultures (Remy et al., 1977a). In order to explain these conflicting results concerning the mechanism of thyroid fol283 0022-5320/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
REMY ET AL.
284
l i c u l a r m o r p h o g e n e s i s a n d to a v o i d the p h y s i o l o g i c a l p e r t u r b a t i o n s w h i c h m a y be i n d u c e d b y long t e r m c u l t u r e o f a d u l t cells, we s t i m u l a t e d o r g a n c u l t u r e d fetal t h y r o i d s w i t h T S H , a n d s u b s e q u e n t l y m e a s u r e d the i n t r a c e l l u l a r level o f c A M P . MATERIALS AND METHODS
Culture. Six female rats were caged with one male rat (Wistar) between 0800 and 1100 hr, which was considered as time 0 of gestation. Fetal rat thyroids, 14, 15, and 16 days old, were secured at about 1000 hr and placed in Coming plastic dishes filled with 2 ml of Eagle's medium (Eagle, 1955) supplemented with 2 mM glutamine, 200 U of penicillin G/ml, 50 #g of streptomycin/mt, and with or without crude bovine thyroid-stimulating hormone (TSH; TS-10, Sigma; 0.2, 20, and 40 mU/ml). The glands were incubated for various times at 37°C in a 95% air-5% CO2 atmospherel Morphology. The previously incubated thyroids and nonincubated 14-, 15-, 16-, and 17-day-old fetal glands were fixed with 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 hr, washed for 1 hr with 0. i M sodium cacodylate, postfixed for 1 hr in 1% OsO4 in 0.1 M sodium cacodylate, dehydrated in acetone, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and investigated with an Elmiskop 101 Siemens electron microscope. cAMP determination. The cAMP content of five thyroid lobes of 16-day-old fetuses was determined by placing the lobes on Millipore filters (HA; 0.45 #m) and incubatingthem described under Culture. In some instances, the medium was supplementedwith 0.6 mM isobutyl-methylxanthine (IBMX) to inhibit endogenous cAMP degradation by phosphodiesterase. At the end of the incubation the Millipore filters with the thyroid lobes were removed from the culture medium, dried on blotting paper, and placed in 200 #1 1 N perchloric acid and then quick frozen at -20°C. After thawing, 180 #1of this medium, which contains all the intracellular cAMP, was withdrawn to measure cAMP concentration by radioimmunoassay (Cailla et aL, 1973); propanol was used to separate free from bound antigen. Measures were performed in duplicate on two dilutions of the assay medium.
RESULTS
Morphology Nonincubated glands. T h e o b s e r v a t i o n s o f fetal t h y r o i d g l a n d s o f 14, 15, 16, a n d 17 days h a v e b e e n d e s c r i b e d i n a p r e v i o u s p a p e r ( R e m y et al., 1980). T o s u m m a r i z e : -- 14-day-old thyroids showed short j u n c t i o n a l z o n e s i n the c e n t e r o f c o m p a c t cell c l u m p s (Fig. 1); - - a t 15 days, the cell j u n c t i o n s were m o r e e x t e n s i v e a n d m o r e n u m e r o u s (Fig. 2); - - a t 16 days, we o b s e r v e d large c o n t a c t s w i t h n u m e r o u s i n f o l d i n g s o f the p l a s m a m e m b r a n e (Fig. 8); - - a t 17 days, e l e m e n t a r y follicular l u m i n a a p p e a r e d b e t w e e n two cells i n the p l a n e o f the s e c t i o n (Fig. 14). A t each stage, s o m e secretion vesicles were p r e s e n t b u t we n e v e r o b s e r v e d a n y i n t r a cellular l u m e n . Organ cultured glands. T a b l e I s u m m a rizes the o b s e r v a t i o n s . I n the 14-day t h y r o i d s i n c u b a t e d for 24 h r w i t h T S H , we o b s e r v e d a n i m p o r t a n t a c c u m u l a t i o n o f clear cytoplasmic vesicles near adherens-type contacts and very extensive Golgi apparatuses (Fig. 4). I n s o m e cases, a p p a r e n t i n t r a cellular cavities bordered with microvilli were clearly v i s i b l e (Fig. 5). I n o t h e r cases, s o m e v a c u o l a r s t r u c t u r e s or m u l t i v e s i c u l a r bodies showed membrane invaginations w i t h c y t o p l a s m i c e x p a n s i o n s (Figs. 6 a n d 7). A f t e r the c o n t r o l i n c u b a t i o n , n e i t h e r m o r p h o l o g y o f this k i n d n o r a n i n c r e a s e i n G o l g i s e c r e t i o n were seen. I n the t h y r o i d s o f 1 5 - d a y fetuses i n c u b a t e d for 24 h r w i t h T S H , we o b s e r v e d extensive Golgi apparatuses with n u m e r o u s s e c r e t i o n vesicles. S o m e follicular c a v i t i e s
FIG. 1. Fourteen-day fetal thyroid. General view of several cells with membrane contact zones (arrows). Numerous plasma membrane infoldings. X 12 500. FIG. 2. Fifteen-day fetal thyroid. Membrane contact zone. Adherens-type junction with densifications (arrows). × 22 000. FIG. 3. Fourteen-day fetal thyroid incubated for 24 hr without TSH. Membrane contact zones (arrows). Numerous plasma membrane infoldings. X 12 500. FIG. 4. Fourteen-day fetal thyroid incubated for 24 hr with 20 mU TSH/ml. Membrane contact zone. Adherens-type junction. Very numerous clear vesicles. Very active Golgi apparatus (GA). × 14 400.
286
REMY ET AL. TABLE I Explantation age (days)
Incubation time (hr)
Final age (days)
GS
IL
FL
0.2 m U TSH/ml
14 15 16
24 24 24
15 16 17
++ ++ ++
-+ +
-+ +
20 m U TSH/ml
14
24
15
24
16
16
24
17
++ ++ ++
+ ++ ++
--
15
++
40 m U TSH/ml
14 15 16
24 24 24
15 16 17
++ ++ ++
+ + ++
-+ ++
Control (no TSH)
14 15 16
24 24 24
15 16 17
-+ +
-+ +
--+
Incubation medium
+
Note. GS = increase of Golgi secretion compared with that observed at the same stage in vivo, IL = intracellular lumina, FL = follicular lumina, -- = not observed, + = observed, + + = numerous.
exhibited "cytoplasmic islets" which were indented by the exocytosis of clear vesicles ( F i g s . 10 a n d l 1). C o n t r o l i n c u b a t i o n s showed an increase in Golgi secretion less m a r k e d t h a n t h a t w i t h T S H i n c u b a t i o n (Fig. 9) a n d s o m e i n t r a c e l l u l a r l u m i n a , b u t n e v e r follicular lumina. In the thyroids of 16-day-old fetuses incubated for 24 hr with TSH, two types of structures were often observed: follicular lumina and cavities which appear to be intracellu-
lar. T h e y m a y r e p r e s e n t f o l l i c u l a r l u m i n a whose cell junctions are not visible in the plane of section or they may be true intrac y t o p l a s m i c s t r u c t u r e s ( F i g . 16). A n i n t r a c e l l u l a r c a v i t y b o r d e r e d w i t h m i c r o v i l l i is s h o w n i n Fig. 13. I n t r a c e l l u l a r a n d f o l l i c u l a r lumina may exist in 16-day-old fetal thyr o i d s a f t e r c o n t r o l i n c u b a t i o n (Fig. 15), b u t they are less numerous and less developed than after incubation with TSH. cAMP
content Sixteen-day-old
fetal thy-
FIG. 5. Fourteen-day fetal thyroid incubated for 24 hours with 20 m U TSH/ml. Membrane contact zone. Very numerous clear vesicles. Cavities with microvilli which appear to be intracellular (arrows). x 20 000. FIG. 6. Fourteen-day fetal thyroid incubated for 24 hr with 20 m U TSH/ml. Golgi zone. Numerous clear vesicles and multivesicular bodies. One multivesicular body presents fingerlike cytoplasmic protrusions (arrowhead). × 20 000. FIG. 7. Fourteen-day fetal thyroid incubated for 24 hr with 20 m U TSH/ml. Follicular lumen and numerous clear vesicles. Multivesicular body with fingerlike protrusion (arrowhead). X 18 000. FIG. 8. Sixteen-day fetal thyroid. General view of several ceils with membrane contact zones (arrows). Tangential sections of infolding (arrowheads). X 11 400. Fro. 9. Fifteen-day fetal thyroid incubated for 24 hr without TSH. Membrane contact zones (arrows). Very active Golgi apparatuses (GA) and numerous clear vesicles. × 15 000. FIG. 10. Fifteen-day fetal thyroid incubated for 24 hr with 0.2 m U TSH/ml. Follicular lumen bordered with clear vesicles. In the middle of the cavity, "cytoplasmic islet" (arrow) with numerous vesicles, x 14 000. F ~ . 11. Fifteen-day fetal thyroid incubated for 24 hr with 0.2 m U TSH/ml. Magnification of previous figure. Evidence of exocytosis (arrowheads). Microvilli seem to be the result of the indentation of the cytoplasm due to the presence of exocytotic pits. Microfilamentous axis (arrows). x 50 000. FIG. 12. Fifteen-day fetal thyroid incubated for 24 hours with 0.2 m U TSH/ml. Follicular lumen with microvilli formed by microfilamentous axis (arrows). Microvillus forming a "cytoplasmic bridge" (arrowhead). × 40 000.
)0
b~ O0
290
REMY ET AL.
roids are incubated with or without (basal level) 25 m U T S H / m l . T h e results (Table II) show that u n d e r the conditions used in our morphological studies, T S H induced a low but significant t i m e - d e p e n d e n t c A M P increase (2 times in 120 min). A m o r e effective stimulation was o b s e r v e d in a typical e x p e r i m e n t in the presence o f I B M X . DISCUSSION
Action of Organ Culture on Fetal Thyroid Cells Control incubation o f 14-day-old thyroids does not induce any increase o f Golgi secretion. T h e incubation o f 15- and 16d a y - o l d t h y r o i d s , h o w e v e r , i n d u c e s an increase in Golgi secretion with the appearance o f intracellular l u m i n a (15 days) and o f intracellular a n d follicular l u m i n a (16 days). We still cannot explain why organ culture in control m e d i u m induces these p h e n o m ena a n d why the increase in secretion begins after the 15th day o f pregnancy.
Action of T S H on Fetal Thyroid Cells It is well established that the anterior pituitary o f the rat fetus does not secrete T S H before the 17th day o f pregnancy (Setalo a n d N a k a n e , 1972). M o r e o v e r , m a t e r n a l T S H is unable to pass the placenta ( T h o r b u r n a n d Hopkins, 1973; Jost et al., 1974). F r o m a morphological point o f view, our observations clearly show that as early as the 14th day o f pregnancy, fetal thyroid cells are sensitive to T S H . F r o m a biochemical point o f view, the evaluation o f the c A M P level o f 16-day-old fetal thyroid cells stimulated by T S H d e m onstrates the sensitivity o f the cells to the h o r m o n e before the 17th day o f pregnancy: after 2 hr incubation, the c A M P level doubles when c o m p a r e d to the reference. We were unable to m e a s u r e the fetal thyroid c A M P level at 14 or 15 days o f preg-
nancy. F o r this experiment, it is necessary to collect only the thyroid lobes without any other tissue which is very difficult to achieve before the 16th day o f pregnancy. All these observations indicate a sensitivity o f the thyroid cells to T S H before the 17th day o f pregnancy, i.e., before the beginning o f T S H secretion by the fetal anterior pituitary. According to the results in Table I, incubation with T S H induces a m o r e i m p o r t a n t increase in secretion than incubation in the control m e d i u m . M o r e o v e r , the higher the h o r m o n e concentration, the m o r e intense is the secretion a n d the m o r e n u m e r o u s are the intracellular a n d follicular lumina. A stereological study o f the effects o f T S H on Golgi secretion has recently been carried out ( R e m y et al., in press).
Formation of the Intracellular Cavities Several cavities bordered by microvilli are visible and either represent follicular cavities which are c o m m o n to two or m o r e cells or they a p p e a r to be true intracellular lumina, as d e m o n s t r a t e d by the 22 consecutive ultrathin serial sections (Fig. 13). The existence ofintracellular cavities m a y be explained as follows: In vivo (Fig. 17A), the clear vesicles secreted by the dictyosomes migrate towards the adherens contact and are secreted by exocytosis into the intercellular space in the adherens contact region ( R e m y et al., 1980). In vitro (Fig. 17B), after T S H stimulation, the secreting activity o f the d i c t y o s o m e s is m u c h m o r e a b u n d a n t and the clear vesicles are so n u m e r o u s that they m a y coalesce before they reach the adherens contact, thus beginning the form a t i o n o f intracellular cavities. These cavities increase with the participation o f other clear vesicles a n d their progression toward the contact; then exocytosis occurs into the intercellular space near the adherens contact (see R e m y et al., 1977b).
FIr. 13. Sixteen-day fetal thyroid incubated for 30 min with 40 mU TSH/ml. Twenty-two consecutive ultrathin serial sections showing an intracellular lumen.
~ i:~~~~ ~ ~:~;~i~i,~ :~:"~~/~
16 DAY-OLD RAT FETUS THYROID CELL5 (30 MINUTES INCUBATION WITH TSH 40 mU/ml)
~:~ :~:~,-~iNTRACELLULARLUMEN 291
t~
293
RAT THYROID MORPHOGENESIS TABLE II DETERMINATION OF cAMP CONTENT
cAMP (mol × 1013)/thyroidlobe Incubation time
Without IBMX (3)~
(rain)
Basal
,'~;,
0 30 60 120
1.19 -+ 0.24b 1.14 + 0.02 1.31 _+0.02 1.0 + 0.02
With0.6 mM IBMX (1)
TSH stimulated 1.80 _+ 0.03 1.70 + 0.03 2.10 _+ 0.05
Basal
TSH stimulated
5.2 -+ 0.8 5.2 +- 0.6
39.7 +_ 3 50.7 + 2
a Number of experiments in parentheses. bMean + SD. The i m p o r t a n t role o f microfilamentous and microtubular material in the migration o f clear vesicles is shown by the fact that antiactin and antitubulin drugs inhibit the migration o f the vesicles and, consequently, folliculogenesis (Pic et al., in preparation). In conclusion, the presence or the absence o f intracellular cavities seems to be exclusively dependent on the abundance o f clear vesicles: if they are very a b u n d a n t (in vitro with TSH), they are more likely t o coalesce before reaching the contact zone than when they are less numerous (in vivo, without TSH stimulation) (Figs. 3, 8, and 14). According to our observations, although T S H enhances the p h e n o m e n o n , it is not absolutely necessary for the formation o f intracellular lumina (Denef et al., 1980). In fact, incubation without T S H m a y cause the appearance ofintracellular lumina, although this occurs less frequently.
Formation o f Microvilli The cavities so f o r m e d often contain cytoplasmic, fingerlike invaginations which m a y result from the indentation o f the cytoplasm by the clear vesicles (Figs, 5, 6, 10, 11). Part o f the cytoplasm contains microfilaments such as microvilli (Fig. 11) or future microvilli (Fig. 12).
The intracellular lumen, therefore, must not be considered as an essential structure for follicle differentiation but as the result o f an intensive secretory activity which implies increased m e m b r a n e synthesis. These observations lead to a possible explanation for the controversy surrounding thyroid follicle morphogenesis. The presence or the absence o f intracellular lumina is very likely related to the secretory activity o f the Golgi apparatus. Intracellular cavities m a y exist in vivo (Shepard et al., 1964; Heiman, 1966; Shepard, 1968); although this is rare, clear vesicles are not very n u m e r o u s and they hardly ever aggregate and coalesce. They migrate independently toward the adherens junction and fuse with the plasma m e m b r a n e . In a previous study on rat thyroid morphogenesis in vivo ( R e m y et al., 1980), no intracellular lumina were observed even when the fetal secretion has started (17 and 18 days o f pregnancy). Intracellular lumina are not only the result o f organ culture; they m a y exist in other circumstances. In C 3 H mice and during involution o f thyroid hyperplasia, M a n y et al. (1981) observed small follicular-like cavities which were d e m o n s t r a t e d to be intracellular by serial sections.
FIG. 14. Seventeen-day fetal thyroid. Follicular lumen (FL). X 14 000. FIG. 15. Sixteen-day fetal thyroid incubated for 24 hr without TSH. Intracellular lumen (arrow). X 12 000. FIG. 16. Sixteen-day fetal thyroid incubated for 24 hr with 20 mU TSH/ml. General view with several follicular or intracellular lumina at different degrees of maturation. Numerous clear vesicles adjacent to the cavities. X 8000.
294
REMY ET AL.
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FIG, 17. Interpretation of the two mechanisms of follicular morphogenesis. A. Follicular morphogenesis in vivo (without TSH stimulation). Golgi secretion with exocytosis of the clear vesicles into the intercellular space in the region of the adherens junction and infolding of the membranes. B. Follicular morphogenesis in vitro
(with TSH stimulation). IntensiveGolgi secretion of very numerous dear vesicles which fuse forming intracellular cavities before their exocytosis at the level of the zonula adherens.
I s o l a t e d a d u l t p o r c i n e t h y r o i d g l a n d s cultured with hydrocortisone frequently form intracellular lumina (unpublished, personal observation).
I n a d u l t rats t r e a t e d w i t h t h y r o x i n e , Ericson (1979) demonstrated intracellular l u m i n a i n t h y r o i d cells. H e f o u n d t h a t after 1 or 2 days o f t r e a t m e n t , these s t r u c t u r e s
RAT THYROID MORPHOGENESIS
are clearly visible, but that they disappear after 7 days of treatment: probably after exocytosis into the follicular lumen. In conclusion, intracellular lumina seem to be the result of either a nonphysiological increase of Golgi secretion due to thyroid hyperplasia or to experimental conditions such as culture or hormonal treatment. If they exist in the fetal gland in vivo and without any experimental stimulation, they would be so rare that they would not play any role in the morphogenesis of the thyroid follicle of the mammals. We are indebted to Professor H. D. Dellmann for his valuable help in the preparation of the manuscript, to Mrs. H. Cailla for the gift of labeled cAMP for radioimmunoassay, and to Mrs J. Bottini for skillful typing of the manuscript. REFERENCES ALLUCHON-GERARD,M. J. (1979) Arch. Anat. Microsc. Morphol. Exp. 68, 43-60. CAILLA, H., RACINE-WEISBUCH,M. S., AND DELAAGE, M. (1973) Anal. Biochem. 56, 394-407. CALVERT, R. (1973) Anat. Rec. 177, 359-375. CALVERT, R. (1975)Anat. Rec. 181, 561-579. CALVERT, R., AND PUSTERLA, A. (1973) Gen. Comp. Endocrinol. 20, 584-597. CAU, P., MICHEL-BECHET,M., AND FAYET, G. (1976) Advan. Anat. Embryol. Cell Biol. 52, 6-66. CLEMENTS-MERLINI,M. (1960) J. Morphol. 106, 357364. DENEF, J. F., BJORKMAN,V., AND EKHOLM, R. (1980) J. Ultrastruct. Res. 71, 185-202. EAGLE, H. (1955) J. Exp. Med. 102, 595-600. ERICSON, L. E. (1979) J. Ultrastruct. Res. 69, 297-305. FELDMAN, J. D., VAZQUEZ,J. J., AND KURTZ, S. M. (1961) J. Biophys. Biochem. Cytol. 11, 365-383.
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