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The role of intracellular lumina in thyroid cells for follicle morphogenesis in vitro
JOURNAL OF IJLTRASTRUCTURE RESEARCH
61,
243-253 (1977)
The Role of Intracellular Lumina in Thyroid Cells for Follicle Morphogenesis in Vitro LIONEL REMY, MARC MICHEL-BECHET, CLAUDE CATALDO, J A N I N E BOTTINI, SONIA HOVSEPIAN,* AND GUY FAYET* Laboratoires d'Histologie I e t de *Biochimie M~dicale, Facult~ de Mddecine, 27, boulevard Jean-Moulin, 13385 Marseille Cedex 4, France Received March 22,1977, and in revised form, August 2, 1977 In a previous study, the existence of intracellular cavities was demonstrated in porcine thyroid follicle cells in culture. Utilization of the polysaccharide characterization technique with silver proteinate, and the histoautoradiographic technique with 125I, led to the conclusion that these intracellular structures contain an iodinated glycoprotein, probably thyroglobulin. The functional aspect and morphology of the intracellular lumen suggest that it undoubtedly plays a part in follicular morphogenesis. Research workers are divided as to whether the follicle lumen originates from the intercellular space or from an intracellular cavity. In this study, it is suggested that the follicle lumen results from the coalescence of several intracytoplasmic cavities with the participation of a very special intercellular space, the zonula adherens.
In a previous study (Remy et al., 1977), we demonstrated the existence, in porcine thyroid cells cultures, of an intracellular lumen containing an osmiophilic substance. This cavity seems to occur simultaneously with the appearance of numerous finger-like protrusions, which probably give rise to the future microvilli of the intracellular lumen. This led to the hypothesis that during the cellular reassociation the cells can pool their cavities to form the follicle lumen. In the first part of this work the nature of the substance inside the cavity was determined, and subsequently an attempt was made to elucidate the follicular morphogenesis mechanism. MATERIALS AND METHODS
Cell Culture Primoculture. Adult porcine thyroid glands were collected aseptically at the abbatoir and transported to the laboratory at 0°C. Muscular, fatty tissues and the thyroid capsule were removed. The glands were rinsed with Earle's saline solution, then minced with scissors, rinsed once more, and placed in a flask containing prewarmed trypsin at 35°C (0.25% proteolytic enzymes, pH 7.0, from the Institut Pasteur). The thyroid cells Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
were dissociated by means of the continuous trypsinization method derived from the methods described by Tong et al. (1962) and Fayet et al. (19701 and recently modified (Fayet and Hovsepian, 1977). The cells were placed in Falcon plastic dishes (Falcon 3024) filled with Eagle's medium (Eagle, 1955, 1959) supplemented with 20% calf serum, 200 U of penicillin G/ml, 50 t~g of streptomycin/ml, and N ~2'-O-dibutyryl cyclic AMP (DBC) at a final concentration of 0.4 mM and incubated at 35°C in a 95% air-5% CO2 atmosphere. After 36 and 48 hr, some cultures were fixed for electron microscopy and the remainder were subcultured. Subcultures. After 48 hr, follicle-reassociated cells were dissociated once more by treatment wit:h 3 mM ethylene glycol bis (fl-aminoethyl ether-N#V"tetraacetic acid (EGTA) in divalent ion-free Earle's saline solution for 14 hr at 35°C, as previously described (Fayet and Lissitzky, 1970; Fayet and Hovsepian, 1977). The isolated cells were postcu][tured in Falcon plastic dishes (Falcon 3001) wit:h crude bovine thyroid-stimulating hormone (TSH) (40 mU/ml) and fixed at 0.2, 4, 6, 8, 10, 12, and 24 hr of culture in a serum-free Eagle's medium. Control cultures without TSH were grown simultaneously.
Morphology The cells were washed three times with Earlds saline and the cell layers were fixed in situ. The first solution used was composed of 47.5 ml of 0.2 M sodium cacodylate buffer at pH 7.4, 47.5 ml of 0.4% ruthenium red, and 5 ml of 50% glutaraldehyde 243 ISSN 0022-5320
244
REMY
(Fayet et al., 1971); the cultures were fixed for 2 hr. After overnight washing with 0.1 M sodium cacodylate buffer at pH 7.4 containing 0.05 M sucrose, the cultures were postfixed for 1.5 hr in a solution of 4% osmium tetroxide, 4% ruthenium red, and 0.2 M sodium cacodylate buffer at pH 7.4 (1:1:2, v/v/v). All these procedures were carried out at 4°C. The cultures were dehydrated with successive ethanol baths (50, 70, 80, 90, and 100%) and embedded in Epon. After polymerization, ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 101 electron microscope.
Polysaccharide Cytochemistry:Periodic acid-Thiocarbohydrazide--Silver Proteinate Method (Thiery, 1967) The reaction was performed on 0.1-tLm-deep sections, collected by capillarity in small plastic rings. These rings were placed on the surface of the various solutions used in the reaction. Culture sections were oxidized with 1% periodic acid for 25 min at room temperature. The sections were washed meticulously in distilled water, twice for 15 min and once overnight (10-12 hr). The sections were then treated at room temperature with a solution composed of 0.2% thiocarbohydrazide in 20% acetic acid, washed for 20 min with 20, 10, 5, 2, and 1% acetic acid, and rinsed three times, 20 min each time, in distilled water. Subsequently, the sections were placed successively for 30 min on a 1% silver proteinate solution in the dark at room temperature and washed three times, 20 min each time, with distilled water. They were placed on grids and examined without contrast in an electron microscope. Reference sections were oxidized with 5% hydrogen peroxide instead of periodic acid. Sections that had not been oxidized were also examined.
ET AL. room, dried, and stored with P205 in opaque, airtight, plastic boxes, for 30 days. The emulsions were developed over 4 min in Microdol X at 18°C. The autoradiographs were rinsed 10 sec in distilled water, fixed in 30% thiosulfate for 5 min then washed three times for 3 min in distilled water and kept in a humid atmosphere. The celloidin films bearing the sections were removed from the slides, grids were placed on the sections, and the film was lifted off with filter paper. Film thickness was reduced by treatment with isoamyl acetate for 2 to 3 min. The sections were then examined with the electron microscope. RESULTS
Polysaccharide Cytochemistry I n a 12-hr s u b c u l t u r e , t h e i n t r a c e l l u l a r l u m i n a present silver proteinate deposits against microvilli membranes and inside t h e c a v i t y (Figs. 1 a n d 2). T h e i n t e r c e l l u l a r spaces a n d f i n g e r - l i k e p r o t r u s i o n s do n o t e x h i b i t a n y deposits. In the intracellular cavity the silver proteinate deposits forms small clumps. A c c u m u l a t i o n s of d e p o s i t s a r e o b s e r v e d running along the cavity against the microvilli, w h o s e m e m b r a n e s a r e also outl i n e d b y d e p o s i t s (Fig. 2). No s i l v e r prot e i n a t e d e p o s i t is s e e n i n t h e c o n t r o l sections.
Histoautoradiography with ~25I
I n i s o l a t e d cells, t h e i n t r a c e l l u l a r cavities p r e s e n t c o n s i d e r a b l e s i l v e r d e p o s i t s (Fig. 3). H o w e v e r , a n a l o g o u s s t r u c t u r e s i n c o n t r o l cells, t r e a t e d w i t h T a p a z o l e w h i c h Histoautoradiography with 125I i n h i b i t s i o d i n e o r g a n i f i c a t i o n , do n o t exTwo-day-old DBC-stimulated cells were dissociated by EGTA in the presence of 10 t~Ci/ml of 12~I h i b i t a n y s i l v e r deposit. with or without Tapazole (2 mM final concentration). The histoautoradiographic technique was Morphology then used (Larra and Droz, 1970). Glass slides were D u r i n g s u b c u l t u r e , t h e f i r s t c o n t a c t s beimmersed in a solution of 2% celloidin in isoamyl t w e e n ceils occur a f t e r 2 or 4 h r . A n acetate and dried. Ultrathin sections were then placed on the slides so as to stick to the celloidin a d h e s i o n o v e r a s m a l l s u r f a c e c a n b e obfilm. The slides were immersed in a solution of s e r v e d b e t w e e n opposite sectors of t h e cell 2.5% uranyl acetate in 50% ethanol, stained in the m e m b r a n e s (Fig. 4). T h e s e c o n t a c t s s p r e a d dark for 15 to 30 min, and then rinsed in three 50% o u t o n b o t h sides f r o m t h e i n i t i a l p o i n t of ethanol baths for 5 min each time. After drying, contact and entail membrane coupling the slides were plunged into a lead citrate solution for 1 to 3 min, washed with distilled water, dried, a l o n g a m o r e or less e x t e n s i v e surface. A f t e r t h e f o r m a t i o n of a follicle l u m e n and overlaid with a thin carbon layer. The slides were then soaked in Ilford L 4 emulsion in a dark- b e t w e e n t h e t w o cells, t h e j u n c t i o n a l zone
FIG. 1. Polysaccharide localizations, 12-hr culture. Intracellular lumen (IL) with silver deposits on microvilli and inside the cavity. No silver deposits in intercellular spaces (IS). x 28 000. FIG. 2. Polysaccharide localizations, 12-hr culture. Magnification of previous figure. Details of membrane and lumen deposits (arrowheads). x 100 000. FIG. 3. Histoautoradiography. Isolated cell with deposits on the intracellular lumen, x 10 000. 245
THYROID FOLLICLE MORPHOGENESIS I N V I T R O
seems to be of zonula adherens type (Figs. 7 and 12) and may present an inconstant membrane densification (Fig. 8). The intercellular space membranes exhibit ruthenium red deposits (Fig. 7). Intracellular lumina with microvilli and osmiophilic substance are also observed. These cavities are often voluminous, and frequently two cells in contact each possess a similar cavity which is often located on both sides of the junctional zone at about the same distance from this junction (Fig. 5). Certain pictures show two cells, each containing a lumen; the cavities are separated by a cytoplasmic zone which belongs exclusively to one cell (Fig. 6). After 24 hr of culture, the follicular structures are numerous. In the same plane, they can be constituted of two or more cells (Figs. 7, 8, and 11). Under certain circumstances, cells are observed which do not seem to form a follicle and possess a strictly intracellular cavity. This is borne out by our observations of ultrathin serial sections which provide the basis for Fig. 10 (Michel-B6chet et al., 1976). DISCUSSION FUNCTIONAL ASPECT OF THE INTRACELLULAR LUMINA
Polysaccharide Localizations The presence of silver proteinate deposits in intracellular cavities indicates the existence of a polysaccharide substance. This observation agrees with that concerning the glands of Brfinner cells in human duodenum (Thiery, 1967) which show silver deposits against the microvilli and on the secretion products in the lumen. H istoautoradiograph y The presence of i25I in the intracellular
247
lumen demonstrates that iodine organification occurs in a molecule, probably iodinated thyroglobulin. Thus, the intracellular cavity seems to be a functional structure. It seems that a follicle composed of several cells is not necessary for thyroglobulin iodination. An intracellular structure appears to be sufficient for glycoprotein iodination, even in isolated cells. This indicates that, under precise conditions, isolated thyroid cells are able to synthetize and iodinate thyroglobulin (Michel-B6chet et al., 1976). Tixier-Vidal et al. (1969) observed, by histoautoradiography, silver deposits on the ergastoplasm of freshly isolated ovine thyroid cells. The existence of intracellular iodination sites is supported by the conclusions of Rousset et al. (1976). In addition, the results obtained by Van Heyningen (1961) strengthen the hypothesis of intracellular colloid formation prior to follicular lumen formation. In our cultures, morphofunctional similarity between the intracellular cavity and the follicle lumen suggests that the iodination sites are located in the membrane of the intracellular lumen as well as on the apical membrane of the cell involw~d in the formation of a follicle lumen. MORPHOGENESIS OF THE THYROID FOLLICULAR LUMEN
The cultures were fixed in a solution containing ruthenium red to facilitate the identification of intracellular structures since ruthenium red settles only on membranes which are in contact with the stain (Fayet et al., 1971). Intercellular Contacts During the first hours of culture, contacts involving only small parts of opposite
FIo. 4. Four-hour culture. Cellular aggregation. Small and continuous contacts in the center of opposite surfaces (arrows). Golgi activity, x 80 000. FIo. 5. Ten-hour culture. Two cells with intracellular l u m i n a (IL). Zonula adherens-type contacts with m e m b r a n e densification (arrowheads). × 20 000. FIo. 6. Twenty-four-hour culture. Contact between two cells, A and B. First degree of fusion. T]~e intracellular lumen of cell A is opened against the plasma m e m b r a n e of cell B (arrowheads). Opening border (arrows). × 50 000.
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THYROID FOLLICLE MORPHOGENESIS I N V I T R O
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F[o. 9. Hypothetical mechanism of follicular lumen morphogenesis. (A) Two cells each with a n intracellular lumen. 1-2: E s t a b l i s h m e n t of a zonula adherens-like contact. Growth of the intracellular cavity by Golgi secretions. 1'-2': Opening of the 1' cell cavity against the 2' cell surface. M e m b r a n e continuity between p l a s m a l e m m a and cavity membrane of 1' cell. l"-g': Opening of g' cell cavity by the same mechanism as before; follicular l u m e n constitution by fusion. 1" - 2 " . Growth and extension of the follicular lumen. (B) Only one cell with a n intracellular lumen. 1-2: E s t a b l i s h m e n t of a zonula adherens-like contact. Growth of the 1 cell cavity by Golgi secretions in 2 cell, in front of the contact. 1'-2': Opening of the 1' cell cavity against the 2' cell surface. M e m b r a n e continuity between plasmalemma and cavity m e m b r a n e of 1' cell. Differentiation of microvilli on the 2' cell plasmalemma; formation of the follicular lumen. 1"-~': Growth of the follicular lumen, particularly in ~' cell, by intensive Golgi secretions. 1 " - 2 " : Growth and extension of the follicular lumen.
membranes of two cells are observed. These contacts then spread out and resemble zonulae adherentes. After 24 hr of culture, which is the most advanced stage we observed, the membrane contacts have the appearance ofzonulae adherentes with or without membrane densification. This latter may be characteristic of evolution.
249
The Role of the Intracellular L u m i n a
Our observations showed that 40% of the cells present intracytoplasmic cavities (Remy et al., 1977). However, this phenomenon was not universal. Some cells never presented such structures from the time they were isolated to the time they reconstituted a follicular structure. So, it may be envisaged that the way in which the follicle lumen is constituted is different for each of these cell types. The two cells present one intracellular l u m e n each. After coupling along, or almost along, their whole opposite faces and then forming one zonula adherens-type contact, the cytoplasmic zone located between the lumen and the plasma membrane of each cell is greatly reduced. At a certain moment the cavity membrane is directly in contact with the plasmalemma. A membrane adherence then occurs which allows the cavity to open outside the cell, like an exocytosis vesicle (Fig. 6). The neighboring cell behaves more or less simultaneously in a similar fashion and the two cavities become connected and give rise to an early follicular structure comprising only two cells (Figs. 5-7, and 9A). It can be noted that the zonula adherens limits the extension of the ruthenium red deposits (Fig. 7). Only one cell presents an intracellular l u m e n . The process of follicle lumen constitution is different from the previous case as one of the cells has no intracellu.lar c a v i t y . Membrane adhesion of zonula adherens type also occurs between the two cells, and the cell with a cavity behaves like each of the cells in the previous instance, i.e., its cavity opens out. On the opposite area of the other cell's surface, microvilli are built up by membrane foldings similar to those observed in Golgi stacks (Remy et al., 1977).
FIG. 7. Twenty-four-hour culture. Follicular l u m e n (FL), result of the fusion of two intracellular cavities. Zonulae adherentes (arrows). Intercellular space (IS). R u t h e n i u m red (RR). x 40 000. FIG. 8. Twelve-hour culture. Follicular l u m e n (FL) resulting from the intracellular lumen opening (cell A) against the surface of cell B, which shows microvilli (MV). Hypothetical follicular formation using only one intracellular l u m e n per two cells. Zonulae adherentes with m e m b r a n e densification (arrows). × 30 000.
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THYROID FOLLICLE MORPHOGENESIS I N V I T R O
Thus a cavity is formed by two cells and is entirely bordered by microvilli; it subsequently enlarges to become a primitive follicular lumen and the result is similar to that in the previous case. However, this follicular lumen is initially assymetrical and extends preferentially at the expense of the cell which formerly enclosed an intracellular cavity (Michel-B~chet et al., 1976). Neither cell encloses an intracellular lumen. Observation of two cells in contact, both lacking an intracellular lumen, suggests at first sight that no follicle morphogenesis could occur between these two cells. However, formation of the follicular cavity from a very modified Golgi complex in the process of becoming a follicle-like intracellular lumen (Remy et al., 1977) can be envisaged. Under these circumstances, a follicle lumen could be constituted directly from one or two very modified Golgi complexes without previous formation of an intracellular cavity. This is an allowable hypothesis although it does not correspond with any observation in our material. Two requirements would then have to be fulfilled: the presence of one or several very modified Golgi complexes close to the cell membrane and a contact between two cells before the Golgi complexes are changed into intracellular cavities. This hypothesis is possible because the Golgi activities, and the biological properties of the apical membrane, under our culture conditions, are not necessarily the same as they would be under other culture conditions or in vivo. According to these observations the intracellular lumen really seems to be the
251
source of follicular lumen formation. This hypothesis is supported by the nature of its components, such as microvilli and osmiophilic substance. Follicle Growth
After two cells have formed a follicular lumen, follicle growth occurs by the addition of other cells to the primitive structure (Fig. 7). Contacts are established between the early follicular lumen and cells having no intracellular lumina or between several early follicular lumina. Depending on the circumstance, either a fusion of follicular structures occurs or there is adjunction of cells, without intracellular cavities, which would "benefit" from the follicular structure previously elaborated by the neighboring cells. The follicle growth could then be dependent on continuous adjunction of cells as well as glycoproteins of Golgi origin, provided by vesicles blending with the apical membrane and pouring their contents into the colloid lumen. Relationship with the previous studies. At the present time, two very different hypotheses have been postulated for thyroid follicle morphogenesis: According to the first hypothesis, the follicle lumen is of extracellular origin. These authors state that junctional complexes delimit parts of intercellular spaces resulting in closed cavities bordered with microvilli which are subsequently enlarged by Golgi secretion. These studies, on porcine thyroid cell cultures, should be compared with those on embryonic chick organ cultures (Porte and Petrovic, 1961; Petrovic and Porte, 1961). Many other authors consider that the follicle lumen orig-
Fro. 10. Twenty-four-hour culture. Follicular lumen (FL). One cell, playing no role in follicular constitution, presents a strictly intracellular lumen (IL). Micrograph derived from consecutive serial sections. Ruthenium red deposits (RR). × 15 000. FIG. 11. Twenty-four-hour culture. '~Cloverleaf" follicular lumen (FL) using five cells in the plane and resulting from true intracellular cavity fusion. × 8000. Fia. 12. Twenty-four-hour culture. Magnification of previous figure. Intercellular contact detail; zonula adherens (arrowheads). × 40 000.
252
REMY E T AL.
inates in the intercellular space. Some research workers have observed structures similar to intracellular cavities in chick (Stoll et al., 1958; Hilfer, 1964; H i l f e r a n d Hilfer, 1966) and in porcine thyroid cell cultures (Michel-B~chet et al., 1973; Cau, 1974; Cau et al., 1976), but they have always inferred that these structures were tangential sections of actual follicular lumina. According to the second hypothesis, the follicle lumen is of intracellular origin with or without participation of the intercellular space; the follicle origin is a genuine intracellular cavity. Intracellular lumina with microvilli and osmiophilic substance have been described by Shepard (1967, 1968), Garcia-Bunuel et al. (1972), and Fisher and Dussault (1974). Analogous observations have been made in the rat embryo (Calvert, 1973; Calvert and Pusterla, 1973), in the human fetus (Olin et al., 1970), in human pathological thyroids (Heimann, 1966; Michel-B~chet et al., 1968, 1969), and in thyroid cell cultures of chicks (Gaillard, 1953), and sheep (Neve et al., 1968; Neve and Dumont, 1970). Some of these authors think that the intercellular space plays a role in morphogenesis. Studies in vivo (Shepard et al., 1964) and in vitro (Shepard, 1967) suggest that in each cell the intracellular lumen is connected to a closed intercellular space by a fine canaliculus and that the follicle lumen is the result of the growth of this structure. In our experimental model the morphogenetic mechanism is different. Intracellular follicle-like cavities were actually demonstrated (Remy et al., 1977), but a closed intercellular space was never observed. A zonula adherens-like membrane coupling was, however, noted between two cells. The zonula adherens is the very reduced intercellular space into which the intracellular cavities open out before fusion. Closing of the intercellular space by a zonula occludens probably takes place after fusion of the cavities.
CONCLUSION
The study of follicular morphogenesis is most rewarding because this phenomenon is directly related to fundamental thyroid physiology. It is generally admitted that thyroglobulin iodination sites are located in the apical membrane of the adult cell. If the follicle lumen originates only in the intercellular space and apical membrane differentiation occurs later, follicular structure would thus be indispensable to thyroglobulin iodination. On the other hand, an intracellular origin for the follicle lumen suggests intracellular iodination sites. Indeed, the histoautoradiography results support this theory. Thus, these investigations seem to favor the existence, during morphogenesis, of thyroglobulin iodination sites other than those of the apical membrane. Those authors who feel that the origin of the follicle lumen is extracellular draw an analogy with the general system of lumen edification in tissues in general. However, the thyroid gland might acquire its specific follicular structure from a morphogenetic process initiated by intracellular lumina. This work was supported by the CNRS, ERA No. 322, and by the INSERM, CRL No. 75 5 039 4. REFERENCES CALVERT,R. (1973)Anat. Rec. 177, 359-375. CALVERT, R., AND PUSTERLA, A. (1973) Gen. Comp. Endocrinol. 20, 584-587. CAU, P. (1974) Thesis Medicine, Marseille. CAU, P., MICHEL-BECHET,M., AND FAYET,G. (1976) Advan. Anat. Embryol. Cell Biol. 52, 1-66. EAGLE, H. (1955)J. Exp. Med. 102,595-600. EAGLE, H. (1959) Science 130, 432-437. FAYET, G., PACHECO, M., AND TIXIER, R. (1970) Bull. Soc. Chim. Biol. 52, 299-318. FAYET, G., AND HOVSEPIAN, S. (1977). Mol. Cell. Endocrinol. 7, 67-78. FAYET, G., AND LISSITZKY,S. (1970) F E B S Lett. 2, 185-188. FAYET, G., MICHEL-BECHET, M., AND LISSITZKY, S. (1971) Eur. J. Biochem. 24,100-111. FISHER, D. A., AND DUSSAULT, J. H. (1974) in GREEP, R. O., AND ASTWOOD, E. B. (Eds.), Endocrinology, Vol. 3, pp. 21-38, American Physiological Society, Washington, D. C. GAILLARD,J. P. (1953) Int. Rev. Cytol. 2, 361-367.
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Exp. Cell Res. 51, 68-78. GARCIA-BUNUEL,R., ANTONE., AND BRANDES, D. OLIN, P., EKHOLM, R., AND ALMQVIST, S. (1970) (1972) Endocrinology, 91, 438-449. Endocrinology 87, 1000-1014. HEIMANN, P. (1966) Acta Endocrinol. 53, (suppl.), PETROVIC,A., ANDPORTE,A. (1961) C. R. Soc. Biol. 1-102. 155, 1848-1855. HEYNINGEN, H. E. VAN (1961)Endocrinology 69, PORTE, A., ANDPETROVIC,A. (1961)C. R. Soc. Biol. 720-727. 155, 1701-1705. HILFER, S. R. (1964)J. Morphol. 115, 135-152. REMY, L., MICHEL-BECHET,M., ATHOUEL-HAON,A. HILFER, S. a., ANDHILFER,E. K. (1966)J. Morphol. M., HOVSEPIAN, S., AND FAYET, G. (1977)Ann. 119, 217-231. Sci. Nat. 18, 21-35. LARRA, F., AND DROZ, B. (1970) J. Microsc. (Paris), ROUSSET, S., PONCET, C., AND MORNEX, a. (1976) 9, 845-88O. Biochim. Biophys. Acta 437, 543-561. MICHEL-BECHET,M., ATHOUEL-HAON,A. M., REMY, SHEPARD, T. H. (1967) J. Clin. Endocrinol. 27, 945L., HOVSEPIAN, S., AND FAYET, G. (1976) Bull. 958. Assoc. Anat., in press. MICHEL-BECHET, M., BOTTINI,J., FAYET, G., AND SHEPARD, W. H. (1968) Gen. Comp. Endocrinol 1O, 174-181. HOVSEPIAN,S. (1976)J. Microsc. Biol. Cell. 26, 33a. SHEPARD, T. H., ANDERSEN,H., AND ANDERSEN,H. MICHEL-BECHET,M., CAU, P., ANDFAYET,G. (1973) J. (1964)Anat. Rec. 149, 363-380. C. R. Acad. Sci. Paris 277, 1029-1032. MICHEL-BECHET, M., COTTE, G., AND ATHOUEL- STOLL, R., MARAUD,R., BLANQUET,P., MOUNIER, P., AND LACHAPELE,A. (1958) Ann. Endocrinol. HAON, A. M. (1969) C. R. Soc. Biol. 163, 233819, 183-201. 2341. THIERY,J. P. (1967)J. Microsc. (Paris), 6, 987-1017. MICHEL-BECttET, M., COTTE, G., AND HAON, A. M. TIXIER-VIDAL, A., PICART,R., RAPPAPORT,L., AND (1968) Bull. Assoc. Anat. 142, 1238-1246. NUNEZ, J. (1969) J. Ultrastruct. Res. 28, 78-101. NEVE, P., AND DUMONT,J. E. (1970) Z. Zellforsch. TONG, W., KERKOF,P., AND CHAIKOFF,I. L. (1962) 103, 61-74. Biochim. Biophys. Acta 60, 1-19. NEVE, P., RODESCH, R., AND DUMONT,J. E. (1968)
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243-253 (1977)
The Role of Intracellular Lumina in Thyroid Cells for Follicle Morphogenesis in Vitro LIONEL REMY, MARC MICHEL-BECHET, CLAUDE CATALDO, J A N I N E BOTTINI, SONIA HOVSEPIAN,* AND GUY FAYET* Laboratoires d'Histologie I e t de *Biochimie M~dicale, Facult~ de Mddecine, 27, boulevard Jean-Moulin, 13385 Marseille Cedex 4, France Received March 22,1977, and in revised form, August 2, 1977 In a previous study, the existence of intracellular cavities was demonstrated in porcine thyroid follicle cells in culture. Utilization of the polysaccharide characterization technique with silver proteinate, and the histoautoradiographic technique with 125I, led to the conclusion that these intracellular structures contain an iodinated glycoprotein, probably thyroglobulin. The functional aspect and morphology of the intracellular lumen suggest that it undoubtedly plays a part in follicular morphogenesis. Research workers are divided as to whether the follicle lumen originates from the intercellular space or from an intracellular cavity. In this study, it is suggested that the follicle lumen results from the coalescence of several intracytoplasmic cavities with the participation of a very special intercellular space, the zonula adherens.
In a previous study (Remy et al., 1977), we demonstrated the existence, in porcine thyroid cells cultures, of an intracellular lumen containing an osmiophilic substance. This cavity seems to occur simultaneously with the appearance of numerous finger-like protrusions, which probably give rise to the future microvilli of the intracellular lumen. This led to the hypothesis that during the cellular reassociation the cells can pool their cavities to form the follicle lumen. In the first part of this work the nature of the substance inside the cavity was determined, and subsequently an attempt was made to elucidate the follicular morphogenesis mechanism. MATERIALS AND METHODS
Cell Culture Primoculture. Adult porcine thyroid glands were collected aseptically at the abbatoir and transported to the laboratory at 0°C. Muscular, fatty tissues and the thyroid capsule were removed. The glands were rinsed with Earle's saline solution, then minced with scissors, rinsed once more, and placed in a flask containing prewarmed trypsin at 35°C (0.25% proteolytic enzymes, pH 7.0, from the Institut Pasteur). The thyroid cells Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
were dissociated by means of the continuous trypsinization method derived from the methods described by Tong et al. (1962) and Fayet et al. (19701 and recently modified (Fayet and Hovsepian, 1977). The cells were placed in Falcon plastic dishes (Falcon 3024) filled with Eagle's medium (Eagle, 1955, 1959) supplemented with 20% calf serum, 200 U of penicillin G/ml, 50 t~g of streptomycin/ml, and N ~2'-O-dibutyryl cyclic AMP (DBC) at a final concentration of 0.4 mM and incubated at 35°C in a 95% air-5% CO2 atmosphere. After 36 and 48 hr, some cultures were fixed for electron microscopy and the remainder were subcultured. Subcultures. After 48 hr, follicle-reassociated cells were dissociated once more by treatment wit:h 3 mM ethylene glycol bis (fl-aminoethyl ether-N#V"tetraacetic acid (EGTA) in divalent ion-free Earle's saline solution for 14 hr at 35°C, as previously described (Fayet and Lissitzky, 1970; Fayet and Hovsepian, 1977). The isolated cells were postcu][tured in Falcon plastic dishes (Falcon 3001) wit:h crude bovine thyroid-stimulating hormone (TSH) (40 mU/ml) and fixed at 0.2, 4, 6, 8, 10, 12, and 24 hr of culture in a serum-free Eagle's medium. Control cultures without TSH were grown simultaneously.
Morphology The cells were washed three times with Earlds saline and the cell layers were fixed in situ. The first solution used was composed of 47.5 ml of 0.2 M sodium cacodylate buffer at pH 7.4, 47.5 ml of 0.4% ruthenium red, and 5 ml of 50% glutaraldehyde 243 ISSN 0022-5320
244
REMY
(Fayet et al., 1971); the cultures were fixed for 2 hr. After overnight washing with 0.1 M sodium cacodylate buffer at pH 7.4 containing 0.05 M sucrose, the cultures were postfixed for 1.5 hr in a solution of 4% osmium tetroxide, 4% ruthenium red, and 0.2 M sodium cacodylate buffer at pH 7.4 (1:1:2, v/v/v). All these procedures were carried out at 4°C. The cultures were dehydrated with successive ethanol baths (50, 70, 80, 90, and 100%) and embedded in Epon. After polymerization, ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 101 electron microscope.
Polysaccharide Cytochemistry:Periodic acid-Thiocarbohydrazide--Silver Proteinate Method (Thiery, 1967) The reaction was performed on 0.1-tLm-deep sections, collected by capillarity in small plastic rings. These rings were placed on the surface of the various solutions used in the reaction. Culture sections were oxidized with 1% periodic acid for 25 min at room temperature. The sections were washed meticulously in distilled water, twice for 15 min and once overnight (10-12 hr). The sections were then treated at room temperature with a solution composed of 0.2% thiocarbohydrazide in 20% acetic acid, washed for 20 min with 20, 10, 5, 2, and 1% acetic acid, and rinsed three times, 20 min each time, in distilled water. Subsequently, the sections were placed successively for 30 min on a 1% silver proteinate solution in the dark at room temperature and washed three times, 20 min each time, with distilled water. They were placed on grids and examined without contrast in an electron microscope. Reference sections were oxidized with 5% hydrogen peroxide instead of periodic acid. Sections that had not been oxidized were also examined.
ET AL. room, dried, and stored with P205 in opaque, airtight, plastic boxes, for 30 days. The emulsions were developed over 4 min in Microdol X at 18°C. The autoradiographs were rinsed 10 sec in distilled water, fixed in 30% thiosulfate for 5 min then washed three times for 3 min in distilled water and kept in a humid atmosphere. The celloidin films bearing the sections were removed from the slides, grids were placed on the sections, and the film was lifted off with filter paper. Film thickness was reduced by treatment with isoamyl acetate for 2 to 3 min. The sections were then examined with the electron microscope. RESULTS
Polysaccharide Cytochemistry I n a 12-hr s u b c u l t u r e , t h e i n t r a c e l l u l a r l u m i n a present silver proteinate deposits against microvilli membranes and inside t h e c a v i t y (Figs. 1 a n d 2). T h e i n t e r c e l l u l a r spaces a n d f i n g e r - l i k e p r o t r u s i o n s do n o t e x h i b i t a n y deposits. In the intracellular cavity the silver proteinate deposits forms small clumps. A c c u m u l a t i o n s of d e p o s i t s a r e o b s e r v e d running along the cavity against the microvilli, w h o s e m e m b r a n e s a r e also outl i n e d b y d e p o s i t s (Fig. 2). No s i l v e r prot e i n a t e d e p o s i t is s e e n i n t h e c o n t r o l sections.
Histoautoradiography with ~25I
I n i s o l a t e d cells, t h e i n t r a c e l l u l a r cavities p r e s e n t c o n s i d e r a b l e s i l v e r d e p o s i t s (Fig. 3). H o w e v e r , a n a l o g o u s s t r u c t u r e s i n c o n t r o l cells, t r e a t e d w i t h T a p a z o l e w h i c h Histoautoradiography with 125I i n h i b i t s i o d i n e o r g a n i f i c a t i o n , do n o t exTwo-day-old DBC-stimulated cells were dissociated by EGTA in the presence of 10 t~Ci/ml of 12~I h i b i t a n y s i l v e r deposit. with or without Tapazole (2 mM final concentration). The histoautoradiographic technique was Morphology then used (Larra and Droz, 1970). Glass slides were D u r i n g s u b c u l t u r e , t h e f i r s t c o n t a c t s beimmersed in a solution of 2% celloidin in isoamyl t w e e n ceils occur a f t e r 2 or 4 h r . A n acetate and dried. Ultrathin sections were then placed on the slides so as to stick to the celloidin a d h e s i o n o v e r a s m a l l s u r f a c e c a n b e obfilm. The slides were immersed in a solution of s e r v e d b e t w e e n opposite sectors of t h e cell 2.5% uranyl acetate in 50% ethanol, stained in the m e m b r a n e s (Fig. 4). T h e s e c o n t a c t s s p r e a d dark for 15 to 30 min, and then rinsed in three 50% o u t o n b o t h sides f r o m t h e i n i t i a l p o i n t of ethanol baths for 5 min each time. After drying, contact and entail membrane coupling the slides were plunged into a lead citrate solution for 1 to 3 min, washed with distilled water, dried, a l o n g a m o r e or less e x t e n s i v e surface. A f t e r t h e f o r m a t i o n of a follicle l u m e n and overlaid with a thin carbon layer. The slides were then soaked in Ilford L 4 emulsion in a dark- b e t w e e n t h e t w o cells, t h e j u n c t i o n a l zone
FIG. 1. Polysaccharide localizations, 12-hr culture. Intracellular lumen (IL) with silver deposits on microvilli and inside the cavity. No silver deposits in intercellular spaces (IS). x 28 000. FIG. 2. Polysaccharide localizations, 12-hr culture. Magnification of previous figure. Details of membrane and lumen deposits (arrowheads). x 100 000. FIG. 3. Histoautoradiography. Isolated cell with deposits on the intracellular lumen, x 10 000. 245
THYROID FOLLICLE MORPHOGENESIS I N V I T R O
seems to be of zonula adherens type (Figs. 7 and 12) and may present an inconstant membrane densification (Fig. 8). The intercellular space membranes exhibit ruthenium red deposits (Fig. 7). Intracellular lumina with microvilli and osmiophilic substance are also observed. These cavities are often voluminous, and frequently two cells in contact each possess a similar cavity which is often located on both sides of the junctional zone at about the same distance from this junction (Fig. 5). Certain pictures show two cells, each containing a lumen; the cavities are separated by a cytoplasmic zone which belongs exclusively to one cell (Fig. 6). After 24 hr of culture, the follicular structures are numerous. In the same plane, they can be constituted of two or more cells (Figs. 7, 8, and 11). Under certain circumstances, cells are observed which do not seem to form a follicle and possess a strictly intracellular cavity. This is borne out by our observations of ultrathin serial sections which provide the basis for Fig. 10 (Michel-B6chet et al., 1976). DISCUSSION FUNCTIONAL ASPECT OF THE INTRACELLULAR LUMINA
Polysaccharide Localizations The presence of silver proteinate deposits in intracellular cavities indicates the existence of a polysaccharide substance. This observation agrees with that concerning the glands of Brfinner cells in human duodenum (Thiery, 1967) which show silver deposits against the microvilli and on the secretion products in the lumen. H istoautoradiograph y The presence of i25I in the intracellular
247
lumen demonstrates that iodine organification occurs in a molecule, probably iodinated thyroglobulin. Thus, the intracellular cavity seems to be a functional structure. It seems that a follicle composed of several cells is not necessary for thyroglobulin iodination. An intracellular structure appears to be sufficient for glycoprotein iodination, even in isolated cells. This indicates that, under precise conditions, isolated thyroid cells are able to synthetize and iodinate thyroglobulin (Michel-B6chet et al., 1976). Tixier-Vidal et al. (1969) observed, by histoautoradiography, silver deposits on the ergastoplasm of freshly isolated ovine thyroid cells. The existence of intracellular iodination sites is supported by the conclusions of Rousset et al. (1976). In addition, the results obtained by Van Heyningen (1961) strengthen the hypothesis of intracellular colloid formation prior to follicular lumen formation. In our cultures, morphofunctional similarity between the intracellular cavity and the follicle lumen suggests that the iodination sites are located in the membrane of the intracellular lumen as well as on the apical membrane of the cell involw~d in the formation of a follicle lumen. MORPHOGENESIS OF THE THYROID FOLLICULAR LUMEN
The cultures were fixed in a solution containing ruthenium red to facilitate the identification of intracellular structures since ruthenium red settles only on membranes which are in contact with the stain (Fayet et al., 1971). Intercellular Contacts During the first hours of culture, contacts involving only small parts of opposite
FIo. 4. Four-hour culture. Cellular aggregation. Small and continuous contacts in the center of opposite surfaces (arrows). Golgi activity, x 80 000. FIo. 5. Ten-hour culture. Two cells with intracellular l u m i n a (IL). Zonula adherens-type contacts with m e m b r a n e densification (arrowheads). × 20 000. FIo. 6. Twenty-four-hour culture. Contact between two cells, A and B. First degree of fusion. T]~e intracellular lumen of cell A is opened against the plasma m e m b r a n e of cell B (arrowheads). Opening border (arrows). × 50 000.
|
~
rm
i
THYROID FOLLICLE MORPHOGENESIS I N V I T R O
I
~2 A
1~
I
1,
~
: 2' 1
I'
2 B
2' 1
I"
I'" = ' ~ '
2,pp
F[o. 9. Hypothetical mechanism of follicular lumen morphogenesis. (A) Two cells each with a n intracellular lumen. 1-2: E s t a b l i s h m e n t of a zonula adherens-like contact. Growth of the intracellular cavity by Golgi secretions. 1'-2': Opening of the 1' cell cavity against the 2' cell surface. M e m b r a n e continuity between p l a s m a l e m m a and cavity membrane of 1' cell. l"-g': Opening of g' cell cavity by the same mechanism as before; follicular l u m e n constitution by fusion. 1" - 2 " . Growth and extension of the follicular lumen. (B) Only one cell with a n intracellular lumen. 1-2: E s t a b l i s h m e n t of a zonula adherens-like contact. Growth of the 1 cell cavity by Golgi secretions in 2 cell, in front of the contact. 1'-2': Opening of the 1' cell cavity against the 2' cell surface. M e m b r a n e continuity between plasmalemma and cavity m e m b r a n e of 1' cell. Differentiation of microvilli on the 2' cell plasmalemma; formation of the follicular lumen. 1"-~': Growth of the follicular lumen, particularly in ~' cell, by intensive Golgi secretions. 1 " - 2 " : Growth and extension of the follicular lumen.
membranes of two cells are observed. These contacts then spread out and resemble zonulae adherentes. After 24 hr of culture, which is the most advanced stage we observed, the membrane contacts have the appearance ofzonulae adherentes with or without membrane densification. This latter may be characteristic of evolution.
249
The Role of the Intracellular L u m i n a
Our observations showed that 40% of the cells present intracytoplasmic cavities (Remy et al., 1977). However, this phenomenon was not universal. Some cells never presented such structures from the time they were isolated to the time they reconstituted a follicular structure. So, it may be envisaged that the way in which the follicle lumen is constituted is different for each of these cell types. The two cells present one intracellular l u m e n each. After coupling along, or almost along, their whole opposite faces and then forming one zonula adherens-type contact, the cytoplasmic zone located between the lumen and the plasma membrane of each cell is greatly reduced. At a certain moment the cavity membrane is directly in contact with the plasmalemma. A membrane adherence then occurs which allows the cavity to open outside the cell, like an exocytosis vesicle (Fig. 6). The neighboring cell behaves more or less simultaneously in a similar fashion and the two cavities become connected and give rise to an early follicular structure comprising only two cells (Figs. 5-7, and 9A). It can be noted that the zonula adherens limits the extension of the ruthenium red deposits (Fig. 7). Only one cell presents an intracellular l u m e n . The process of follicle lumen constitution is different from the previous case as one of the cells has no intracellu.lar c a v i t y . Membrane adhesion of zonula adherens type also occurs between the two cells, and the cell with a cavity behaves like each of the cells in the previous instance, i.e., its cavity opens out. On the opposite area of the other cell's surface, microvilli are built up by membrane foldings similar to those observed in Golgi stacks (Remy et al., 1977).
FIG. 7. Twenty-four-hour culture. Follicular l u m e n (FL), result of the fusion of two intracellular cavities. Zonulae adherentes (arrows). Intercellular space (IS). R u t h e n i u m red (RR). x 40 000. FIG. 8. Twelve-hour culture. Follicular l u m e n (FL) resulting from the intracellular lumen opening (cell A) against the surface of cell B, which shows microvilli (MV). Hypothetical follicular formation using only one intracellular l u m e n per two cells. Zonulae adherentes with m e m b r a n e densification (arrows). × 30 000.
c~
THYROID FOLLICLE MORPHOGENESIS I N V I T R O
Thus a cavity is formed by two cells and is entirely bordered by microvilli; it subsequently enlarges to become a primitive follicular lumen and the result is similar to that in the previous case. However, this follicular lumen is initially assymetrical and extends preferentially at the expense of the cell which formerly enclosed an intracellular cavity (Michel-B~chet et al., 1976). Neither cell encloses an intracellular lumen. Observation of two cells in contact, both lacking an intracellular lumen, suggests at first sight that no follicle morphogenesis could occur between these two cells. However, formation of the follicular cavity from a very modified Golgi complex in the process of becoming a follicle-like intracellular lumen (Remy et al., 1977) can be envisaged. Under these circumstances, a follicle lumen could be constituted directly from one or two very modified Golgi complexes without previous formation of an intracellular cavity. This is an allowable hypothesis although it does not correspond with any observation in our material. Two requirements would then have to be fulfilled: the presence of one or several very modified Golgi complexes close to the cell membrane and a contact between two cells before the Golgi complexes are changed into intracellular cavities. This hypothesis is possible because the Golgi activities, and the biological properties of the apical membrane, under our culture conditions, are not necessarily the same as they would be under other culture conditions or in vivo. According to these observations the intracellular lumen really seems to be the
251
source of follicular lumen formation. This hypothesis is supported by the nature of its components, such as microvilli and osmiophilic substance. Follicle Growth
After two cells have formed a follicular lumen, follicle growth occurs by the addition of other cells to the primitive structure (Fig. 7). Contacts are established between the early follicular lumen and cells having no intracellular lumina or between several early follicular lumina. Depending on the circumstance, either a fusion of follicular structures occurs or there is adjunction of cells, without intracellular cavities, which would "benefit" from the follicular structure previously elaborated by the neighboring cells. The follicle growth could then be dependent on continuous adjunction of cells as well as glycoproteins of Golgi origin, provided by vesicles blending with the apical membrane and pouring their contents into the colloid lumen. Relationship with the previous studies. At the present time, two very different hypotheses have been postulated for thyroid follicle morphogenesis: According to the first hypothesis, the follicle lumen is of extracellular origin. These authors state that junctional complexes delimit parts of intercellular spaces resulting in closed cavities bordered with microvilli which are subsequently enlarged by Golgi secretion. These studies, on porcine thyroid cell cultures, should be compared with those on embryonic chick organ cultures (Porte and Petrovic, 1961; Petrovic and Porte, 1961). Many other authors consider that the follicle lumen orig-
Fro. 10. Twenty-four-hour culture. Follicular lumen (FL). One cell, playing no role in follicular constitution, presents a strictly intracellular lumen (IL). Micrograph derived from consecutive serial sections. Ruthenium red deposits (RR). × 15 000. FIG. 11. Twenty-four-hour culture. '~Cloverleaf" follicular lumen (FL) using five cells in the plane and resulting from true intracellular cavity fusion. × 8000. Fia. 12. Twenty-four-hour culture. Magnification of previous figure. Intercellular contact detail; zonula adherens (arrowheads). × 40 000.
252
REMY E T AL.
inates in the intercellular space. Some research workers have observed structures similar to intracellular cavities in chick (Stoll et al., 1958; Hilfer, 1964; H i l f e r a n d Hilfer, 1966) and in porcine thyroid cell cultures (Michel-B~chet et al., 1973; Cau, 1974; Cau et al., 1976), but they have always inferred that these structures were tangential sections of actual follicular lumina. According to the second hypothesis, the follicle lumen is of intracellular origin with or without participation of the intercellular space; the follicle origin is a genuine intracellular cavity. Intracellular lumina with microvilli and osmiophilic substance have been described by Shepard (1967, 1968), Garcia-Bunuel et al. (1972), and Fisher and Dussault (1974). Analogous observations have been made in the rat embryo (Calvert, 1973; Calvert and Pusterla, 1973), in the human fetus (Olin et al., 1970), in human pathological thyroids (Heimann, 1966; Michel-B~chet et al., 1968, 1969), and in thyroid cell cultures of chicks (Gaillard, 1953), and sheep (Neve et al., 1968; Neve and Dumont, 1970). Some of these authors think that the intercellular space plays a role in morphogenesis. Studies in vivo (Shepard et al., 1964) and in vitro (Shepard, 1967) suggest that in each cell the intracellular lumen is connected to a closed intercellular space by a fine canaliculus and that the follicle lumen is the result of the growth of this structure. In our experimental model the morphogenetic mechanism is different. Intracellular follicle-like cavities were actually demonstrated (Remy et al., 1977), but a closed intercellular space was never observed. A zonula adherens-like membrane coupling was, however, noted between two cells. The zonula adherens is the very reduced intercellular space into which the intracellular cavities open out before fusion. Closing of the intercellular space by a zonula occludens probably takes place after fusion of the cavities.
CONCLUSION
The study of follicular morphogenesis is most rewarding because this phenomenon is directly related to fundamental thyroid physiology. It is generally admitted that thyroglobulin iodination sites are located in the apical membrane of the adult cell. If the follicle lumen originates only in the intercellular space and apical membrane differentiation occurs later, follicular structure would thus be indispensable to thyroglobulin iodination. On the other hand, an intracellular origin for the follicle lumen suggests intracellular iodination sites. Indeed, the histoautoradiography results support this theory. Thus, these investigations seem to favor the existence, during morphogenesis, of thyroglobulin iodination sites other than those of the apical membrane. Those authors who feel that the origin of the follicle lumen is extracellular draw an analogy with the general system of lumen edification in tissues in general. However, the thyroid gland might acquire its specific follicular structure from a morphogenetic process initiated by intracellular lumina. This work was supported by the CNRS, ERA No. 322, and by the INSERM, CRL No. 75 5 039 4. REFERENCES CALVERT,R. (1973)Anat. Rec. 177, 359-375. CALVERT, R., AND PUSTERLA, A. (1973) Gen. Comp. Endocrinol. 20, 584-587. CAU, P. (1974) Thesis Medicine, Marseille. CAU, P., MICHEL-BECHET,M., AND FAYET,G. (1976) Advan. Anat. Embryol. Cell Biol. 52, 1-66. EAGLE, H. (1955)J. Exp. Med. 102,595-600. EAGLE, H. (1959) Science 130, 432-437. FAYET, G., PACHECO, M., AND TIXIER, R. (1970) Bull. Soc. Chim. Biol. 52, 299-318. FAYET, G., AND HOVSEPIAN, S. (1977). Mol. Cell. Endocrinol. 7, 67-78. FAYET, G., AND LISSITZKY,S. (1970) F E B S Lett. 2, 185-188. FAYET, G., MICHEL-BECHET, M., AND LISSITZKY, S. (1971) Eur. J. Biochem. 24,100-111. FISHER, D. A., AND DUSSAULT, J. H. (1974) in GREEP, R. O., AND ASTWOOD, E. B. (Eds.), Endocrinology, Vol. 3, pp. 21-38, American Physiological Society, Washington, D. C. GAILLARD,J. P. (1953) Int. Rev. Cytol. 2, 361-367.
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Exp. Cell Res. 51, 68-78. GARCIA-BUNUEL,R., ANTONE., AND BRANDES, D. OLIN, P., EKHOLM, R., AND ALMQVIST, S. (1970) (1972) Endocrinology, 91, 438-449. Endocrinology 87, 1000-1014. HEIMANN, P. (1966) Acta Endocrinol. 53, (suppl.), PETROVIC,A., ANDPORTE,A. (1961) C. R. Soc. Biol. 1-102. 155, 1848-1855. HEYNINGEN, H. E. VAN (1961)Endocrinology 69, PORTE, A., ANDPETROVIC,A. (1961)C. R. Soc. Biol. 720-727. 155, 1701-1705. HILFER, S. R. (1964)J. Morphol. 115, 135-152. REMY, L., MICHEL-BECHET,M., ATHOUEL-HAON,A. HILFER, S. a., ANDHILFER,E. K. (1966)J. Morphol. M., HOVSEPIAN, S., AND FAYET, G. (1977)Ann. 119, 217-231. Sci. Nat. 18, 21-35. LARRA, F., AND DROZ, B. (1970) J. Microsc. (Paris), ROUSSET, S., PONCET, C., AND MORNEX, a. (1976) 9, 845-88O. Biochim. Biophys. Acta 437, 543-561. MICHEL-BECHET,M., ATHOUEL-HAON,A. M., REMY, SHEPARD, T. H. (1967) J. Clin. Endocrinol. 27, 945L., HOVSEPIAN, S., AND FAYET, G. (1976) Bull. 958. Assoc. Anat., in press. MICHEL-BECHET, M., BOTTINI,J., FAYET, G., AND SHEPARD, W. H. (1968) Gen. Comp. Endocrinol 1O, 174-181. HOVSEPIAN,S. (1976)J. Microsc. Biol. Cell. 26, 33a. SHEPARD, T. H., ANDERSEN,H., AND ANDERSEN,H. MICHEL-BECHET,M., CAU, P., ANDFAYET,G. (1973) J. (1964)Anat. Rec. 149, 363-380. C. R. Acad. Sci. Paris 277, 1029-1032. MICHEL-BECHET, M., COTTE, G., AND ATHOUEL- STOLL, R., MARAUD,R., BLANQUET,P., MOUNIER, P., AND LACHAPELE,A. (1958) Ann. Endocrinol. HAON, A. M. (1969) C. R. Soc. Biol. 163, 233819, 183-201. 2341. THIERY,J. P. (1967)J. Microsc. (Paris), 6, 987-1017. MICHEL-BECttET, M., COTTE, G., AND HAON, A. M. TIXIER-VIDAL, A., PICART,R., RAPPAPORT,L., AND (1968) Bull. Assoc. Anat. 142, 1238-1246. NUNEZ, J. (1969) J. Ultrastruct. Res. 28, 78-101. NEVE, P., AND DUMONT,J. E. (1970) Z. Zellforsch. TONG, W., KERKOF,P., AND CHAIKOFF,I. L. (1962) 103, 61-74. Biochim. Biophys. Acta 60, 1-19. NEVE, P., RODESCH, R., AND DUMONT,J. E. (1968)