Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.     

Effect of dibutyryl cyclic adenosine 3′5′-monophosphate on mitotic activity in the thyroid of hypophysectomized rats

Summary Effect of dibutyryl cyclic adenosine 35-monophosphate (dbcAMP) on mitotic activity in the thyroid of hypophysectomized rats has been examined. It has been demonstrated that dbcAMP stimulates the incidence of mitoses in the thyroid follicular cells. It is therefore suggested that cAMP may be a mediator of the proliferogenic effect of TSH on the thyroid in vivo. Cyclic AMP could also release some unidentified growth-promoting factors for the thyroid. A direct stimulating effect of dbcAMP on the proliferation of the thyroid follicular cells is assumed to be possible as well.     

Correlation between thyroid-follicle fusion and structural modifications of the epithelial cells

Summary Changes in thyroid structure induced by a decrease in TSH or iodide-dependent stimulation were quantified by stereological analysis of light micrographs. Studies were carried out on intact (R5) and hypophysectomized (R 5H) rats receiving 5 g iodide, and on intact rats (R5O) receiving 50 g iodide daily.For R 5H- and R5O-thyroids, the mean parameters of the epithelial cells, height, volume and lateral membrane area, were smaller than those of R 5-thyroids. An inverse shift was observed for the apical membrane area, whereas the peripheral membrane area was unchanged. The number of epithelial cells was similar in each of the three groups; however, the number of follicles was greater in R 5-thyroids, suggesting that follicular fusion occurs in R 5O- and R 5H-thyroids. This was confirmed by direct observation.The present results demonstrate that in adult rats a lack of TSH or an increased iodide diet (insufficient to produce a physiopathological state) induce follicle fusion probably by means of cellular reorganization. This increase in follicle size could be involved in the regulation of thyroid iodine turnover.     

Localization of 3′, 5′-cyclic nucleotide phosphodiesterase activity in isolated thyroid cells and intact thyroid tissue

Summary Cytochemical localization of 3,5-cyclic nucleotide phosphodiesterase (cPDEase) has been investigated by light and electron microscopy in dissociated bovine thyroid cells and in intact bovine thyroid tissue. By light microscopy in isolated thyroid cells reaction product deposition associated with cPDEase activity was localized at the level of the plasma membrane. In intact cryostat cut thyroid tissue, the activity was primarily observed in the cytoplasm and to a lesser extent at the level of the plasma membrane. By electron microscopy, cPDEase activity in isolated cells was found on the plasma membrane and was also encountered on the inner surface of membrane bound vacuoles, presumably pinocytic in origin. In intact tissue, cPDEase activity appeared mostly localized on the apical and lateral plasma membranes and was also present on the outer surface of the endoplasmic reticulum (ER).Even though cPDEase and 5-AMPase did share the same plasma membrane localization, the inhibitory response to theophylline and stimulation with Imidazole permitted the dissociation of their respective activities. 5-AMPase failed to respond to either theophylline or Imidazole suggesting absence of cross reactivity between 5-AMP and cyclic AMP. Thyrotropin (TSH) had no effect on cPDEase activity.We conclude that: (1) regardless of the nature of the material used, the cytomembranes of thyroid cells possess cPDEase activity; and that (2) the variability in distribution as well as in staining intensity recorded by light and electron microscopy between isolated thyroid cells and cryostat cut thyroid tissue is probably inherent to the methodology used.This paper was presented, in part, at the 60th Annual Meeting of the International Academy of Pathology, Montreal, Canada, March 1971 and was initiated in the Department of Pathology, Rhode Island Hospital, Providence, R.I., supported from a Grant-in-Aid of the American Cancer Society, Rhode Island Division, Inc. and the Brown-Hazen Fund.     

Detection of Oxidant Producing-sites in Glutaraldehyde-fixed Human Neutrophils and Eosinophils Stimulated with Phorbol Myristate Acetate

The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure. In biochemical analyses, the detectable O2– generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10sec followed by O2– production. The maximal rate reached was 3.18±0.07nmol/min/1×106 cells (mean±S.D.; n=4) after 30sec of stimulation. PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2– without a lag period at a rate of 0.35±0.05nmol/min/1×106 cells (mean±S.D.) by the addition of NADPH as substrate to the cell suspension. In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells). Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium. No oxidant-reaction product was seen in pre-fixed cells stimulated for 10sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20sec. The fact that the pre-fixed PMNs stimulated for 30sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30sec after start of stimulation with PMA. These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B. The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths. The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.     

Mise en évidence par immunofluorescence des cellules à hormones glycoprotidiques dans l'hypophyse d'un Rongeur d'Iran,Ellobius lutescens (Th): cellules gonadotropes et cellules thyréotropes

Résumé Les cellules à hormones glycoprotidiques (thyréotropes et gonadotropes) ont pu être indentifiées grâce aux réactions d'immunofluorescence qu'elles donnent avec des anticorps préparés à partir de LH et FSH ovine, de HCG et de PMSG, ainsi que de TSH bovine. Tandis que l'anticorps anti TSH brut révèle la totalité des cellules à hormones glycoprotidiques tout comme l'anticorps anti HCG, l'anticorps anti TSH saturé par LH ne laisse subsister la fluorescence qu'au niveau des grandes cellules anguleuses (Bleu Alcian et AF positives) qui fixent l'anticorps rendu ainsi spécifique pour TSH. Les deux autres types de cellules glycoprotidiques (PAS positives), qui présentent un dimorphisme très accentué, réagissent positivement avec l'anticorps anti LH ainsi qu'avec l'anticorps anti FSH: nous pouvons donc attribuer la fonction gonadotrope à ces deux catégories cellulaires sans qu'il soit cependant possible d'y distinguer les secrétions de type LH ou FSH.

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Immunofluorescent demonstration of gonadotrophic and thyrotrophic cells in the hypophysis of Ellobius lutescens (Th.), an iranian rodent

Summary Glycoprotein-containing cells (thyrotrophic and gonadotrophic cells) have been identified by immunofluorescence reactions, with antibodies to ovine LH and FSH, HCG, PMSG, and bovine TSH, in the hypophysis of the rodent Ellobius lutescens. Whereas raw anti-TSH antibody (as well as anti-HCG antibody) gives a positive reaction with all glycoprotein-containing cells, LH- saturated anti-TSH antibody fluoresces only at the site of large, angular cells (Alcian Blue- and Aldehyde Fuchsin-positive) which retain the antibody thus rendered TSH-specific. The two other types of glycoprotein (PAS-positive) cells, which exhibit a marked dimorphism, give a positive reaction with anti-LH and anti-FSH antibodies; thus we can attribute gonadotrophic functions of these two cell-types although we cannot discriminate between LH or FSH secretion.

     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions

Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.     

Polarization of thyroid cells in culture: evidence for the basolateral localization of the iodide "pump" and of the thyroid-stimulating hormone receptor-adenyl cyclase complex

When cultured in collagen gel-coated dishes, thyroid cells organized into polarized monolayers. The basal poles of the cells were in contact with the collagen gel, whereas the apical surfaces were facing the culture medium. Under these culture conditions, thyroid cells do not concentrate iodide nor respond to acute stimulation by thyroid-stimulating hormone (TSH). To allow the free access of medium components to the basal poles, the gel was detached from the plastic dish and allowed to float in the culture medium. After release of the gel, the iodide concentration and acute response to TSH stimulation were restored. Increased cAMP levels, iodide efflux, and formation of apical pseudopods were observed. When the thyroid cells are cultured on collagen-coated Millipore filters glued to glass rings, the cell layer separates the medium in contact with the apical domain of the plasma membrane (inside the ring) from that bathing the basolateral domain (outside the ring). Iodide present in the basal medium was concentrated in the cells, whereas no transport was observed when iodide was added to the luminal side. Similarly, an acute effect of TSH was observed only when the hormone was added to the basal medium. These results show that the iodide concentration mechanism and the TSH receptor-adenylate cyclase complex are present only on the basolateral domain of thyroid cell plasma membranes.     

Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council     

Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells

Summary Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing the intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.     

Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated,perfused rabbit cortical collecting tubule

Summary Antidiuretic hormone increases the water permeability of the cortical collecting tubule and causes the appearance of intramembrane particle aggregates in the apical plasma membrane of principal cells. Particle aggregates are located in apical membrane coated pits during stimulation of collecting ducts with ADHin situ. Removal of ADH causes a rapid decline in water permeability. We evaluated apical membrane retrieval associated with removal of ADH by studying the endocytosis of horseradish peroxidase (HRP) from an isotonic solution in the lumen. HRP uptake was quantified enzymatically and its intracellular distribution examined by electron microscopy. When tubules were perfused with HRP for 20 min in the absence of ADH, HRP uptake was 0.5±0.3 pg/min/m tubule length (n=6). The uptake of HRP in tubules exposed continuously to ADH during the 20-min HRP perfusion period was 1.3±0.8 pg/min/m (n=8). HPR uptake increased markedly to 3.2±1.1 pg/min/m (n=14), when the 20-min period of perfusion with HRP began immediately after removal of ADH from the peritubular bath. Endocytosis of HRP occurred in both principal and intercalated cells via apical membrane coated pits. We suggest that the rapid decline in cortical collecting duct water permeability which occurs following removal of ADH is mediated by retrieval of water permeable membrane via coated pits.     

Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH-regulated channel mediating iodide efflux selectively across the apical domain of the plasma membrane.

The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid-stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle.     

Observations ultrastructurales sur la parathyroïde de souris

Résumé La parathyroïde de souris ne diffère pas essentiellement par sa structure générale de celles des autres espéces étudiées. Les cellules glandulaires se caractérisent cependant par une plus grande richesse en matériel sécrétoire figuré. La présence de ce matériel figuré dans les cavités golgiennes permet de suivre l'élaboration de granules sécrétoires typiques d'un diamètre de 160 à 200 m qui sont spécialement abondants chez la souris. Ces granules ne sont pas excrétés au pôle vasculaire des cellules; ils migrent vers les parois cellulaires apicales et latérales, fusionnent avec la membrane plasmique et semblent être excrétés sous forme soluble dans les espaces intercellulaires. Il n'existe dans la parathyroïde de souris, comme dans celle du hamster et du rat, qu'un seul type cellulaire fondamental. Les éléments clairs et sombres ne diffèrent que par leur densité hyaloplasmique; cette différence s'accuse plus ou moins selon la qualité du prélèvement.

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Summary The structure of the mouse parathyroid does not differ essentially from other species studied. However, the glandular cells are characterized through an abundance in visible secretory material. The presence of this material in the cavities of the Golgi apparatus makes it possible to observe the formation of the typical secretory granules (160–200 m in diameter) which are particularly abundant in the mouse parathyroid. These granules are not secreted directly at the vascular pole; they migrate to the apical and lateral walls, fuse with the plasma membrane, and seen to pass into the cellular space in a soluble form. In the mouse parathyroid, as in the hamster and rat, there is only one fundamental cell type. The difference between dark and clear cells lies in the density of their hyaloplasm, which, however, is a consequence of the pretreatment of the material (fixation).

     

Localization of adenylate cyclase and 5′-nucleotidase activities in human thyroid follicular cells

Summary The localization of adenylate cyclase and 5-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultanous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the other hand, the histochemical localization of 5-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.     

Activité cholinestérasique des corpuscules sensoriels cutanés de Herbst et de Grandry

Résumé L'activité cholinestérasique (ChE) a été étudiée dans les corpuscules de Herbst et de Grandry du bec de canard par la méthode de Karnovsky. Le BW 284 C 51, l'iso-OMPA et l'ésérine ont été utilisés comme inhibiteurs. Une activité cholinestérasique non spécifique (nsChE) a été identifiée dans les corpuscules. Elle est présente dès le début de leur différenciation.Corpuscule de Herbst: l'activité nsChE est localisée dans les cellules du bulbe interne (citernes périnucléaires, reticulum endoplasmique granulaire, vésicules associées à l'apppareil de Golgi) et à la surface de la membrane plaamique des cellules du bulbe interne et de la terminaison nerveuse.Corpuscule de Grandry: l'activité nsChE est localisée dans les cellules satellites (citernes périnucléaires, reticulum endoplasmique granulaire) et à la surface de la membrane plasmique des cellules satellites, des cellules de Grandry et de la terminaison nerveuse.Une forte activité nsChE a été mise en évidence dans certaines zones de contact entre les cellules dites sensorielles (cellules du bulbe interne et de Grandry) et les terminaisons nerveuses. Une réaction positive a été observée au niveau des mitochondries des cellules satellites et de la terminaison nerveuse du corpuscule de Grandry, et dans des vésicules du réticulum endoplasmique lisse des terminaisons nerveuses.Aucune activité ChE n'a été détectée dans les cellules capsulaires, les cellules de l'espace interne, les cellules de Grandry, et dans les vésicules synaptiques.Ces résultats ont été discutés en relation avec ce que l'on sait sur l'activité ChE des autres types de corpuseules sensoriels. La signification fonctionnelle de cette activité ChE et le problème de l'origine des différentes catégorics cellulaires qui constituent les corpuscules de Herbst et de Grandry ont été envisagés.

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Cholinesterase activity in the Herbst and Grandry cutaneous sensory corpusclesHistochemical study at the light and electron microscope

Summary Cholinesterase activity (ChE) was localized in the Herbst and Grandry cutaneous sensory corpuscle of the duck beak with Karnovsky's method. Controls with BW 284 C 51, iso-OMPA and eserine were carried out. A non-specific cholinesterase activity (nsChE) was identified in the corpuscles. This enzyme activity is present from the beginning of their differenciation.Herbst corpuscle: nsChE activity was localized in the inner bulb cells (perinuclear cisterna, granular endoplasmic reticulum, vesicles associated with the Golgi complex) and along the plasma membrane of inner bulb cells and nerve ending.Grandry corpuscle: nsChE activity was localized in the satellite cells (perinuclear cisterna, granular endoplasmic reticulum) and along the plasma membrane of satellite cells, Grandry cells, and nerve ending.A high nsChE activity was evident in certain zones of contact between the so-called sensory cells (inner bulb and Grandry cells) and the nerve endings. A positive reaction was seen in mitochondria of satellite cells and nerve ending for the Grandry corpuscle, and in smooth endoplasmic vesicles of the nerve endings.No ChE activity was detected in capsular cells, inner space cells, Grandry cells and in synaptic vesicles.These results are discussed in relation with what is known about ChE activity of other sensory corpuscles. The functional significance of this ChE activity and the problem of the origin of the different cells of Herbst and Grandry corpuscles have been considered.

     

Données cytologiques et autoradiographiques sur la glande thyroïde de quelques Mammifères hibernants. Activité thyroïdienne et présence de cellules C à calcitonine

Résumé L'étude histologique et autoradiographique de glandes thyroïdes de Lérots à diverses périodes de l'année indique un fonctionnement réduit pendant la préhibernation et le maintien d'une certaine activité glandulaire en Décembre et Janvier, durant l'hibernation, s'accentuant fortement en Février et Mars avant le réveil et au cours de celui-ci; on note ultérieurement un retour à une activité modérée, cycle en accord avec les données biochimiques et isotopiques recueillies par Lachiver sur ces mêmes animaux.Cette stimulation liée à l'approche du réveil n'apparaît pas en Mars si l'hibernation est différée, l'animal étant maintenu en chambre froide jusqu'en Mai.L'aspect qualitatif de ces processus mérite aussi d'être envisagé: pendant l'hibernation et parfois avant celle-ci, on peut observer des autoradiogrammes avec un noircissement en anneau et non plus homogène, indiquant que seule la partie périphérique de la masse de thyroglobuline, en contact avec l'épithélium, participe au métabolisme des hormones, la zone centrale apparaissant peu active.Les cellules C sont toujours abondantes et volumineuses chez les Lérots en préhibernation et en hibernation; elles sont souvent dégranulées, parfois vacuolisées à la fin de celle-ci. Après le réveil et jusqu'en Juillet, elles deviennent plus petites, granulées et moins aisément identifiables avec certaines techniques. A une phase d'excrétion semble succéder une prépondérance des processus de synthèse et d'accumulation des granules.La mise en hypothermie profonde en Juillet induit leur réapparition en nombre élevé, leur hypertrophie et leur dégranulation partielle, des mitoses sont observées. De même, si l'hibernation est différée, ces cellules restent nombreuses et peu granulées jusqu'en Mai alors qu'elles régressent numériquement dès la fin Mars, quand le réveil a lieu.Deux espèces de Mammifères hibernants, originaires de Madagascar, ont aussi été étudiées: le Tenrec, qui est entré spontanément en léthargie en Juin-Juillet en France (hiver austral) et dont les cellules C sont extrêmement abondantes, volumineuses, peu granulées, et l'Ericulus, qui présente en Février, en France, des cellules C un peu moins nombreuses que le Tenrec, après une léthargie de plus courte durée. Un nombre élevé de cellules C actives s'observe dans la glande de Paraechinus en léthargie, en Février, l'animal provenant d'Afrique du Nord (région de Beni-Abbes).Chez la Marmotte, nous retrouvons une stimulation thyroïdienne lors du réveil, mais les cellules C ne sont jamais nombreuses dans la glande et les données actuelles ne permettent pas d'établir l'existence d'un cycle net.Les données bibliographiques sur les cellules C des hibernants sont comparées à celles recueillies sur le Lérot et discutées en fonction de l'état des glandes parathyroïdes et du métabolisme calcique au cours du cycle annuel. L'hypothèse d'une sécrétion de calcitonine par ces cellules semble probable, en liaison possible avec l'immobilité prolongée pendant le sommeil qui pourraît entraîner une décalcification osseuse.

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Cytological and autoradiographic study of the thyroid gland in some hibernating mammals. Thyroid activity and presence of C cells (calcitonin cells)

Summary In the garden dormouse (Eliomys quercinus L.) a cyclic thyroid function was evidenced by histological and autoradiographic studies at various months of the year. The gland shows the maintenance of a low activity in December and January, before and during hibernation, greatly increased in February and March, preceding and accompanying the arousal; then, a slow return to a moderate activity is noted. This cycle is in agreement with biochemical data and radioiodine studies (Lachiver, 1952) on the same animals.When the hibernation is delayed and the animals are kept in a cold room until May, without food, the thyroid stimulation which naturally occurs at the time of arousal is simultaneously delayed.The qualitative aspect of these processes also needs to be investigated: during hibernation, and sometimes before it, the blackening of the photographic emulsion is not homogeneous above the colloid, the autoradiograms show several rings corresponding to the peripheral zone of the thyroglobulin at its junction with the epithelium. Only a thin rim seems to be participating in the hormonal metabolism, the central area being rather inactive.Abundant and large C cells, (calcitonin cells), always occur in the dormouse thyroid gland in prehibernation and hibernation, at the end of which they often appear degranulated, sometimes vacuolated. After the arousal, and until July, they become smaller, granulated, less easy to detect with light microscope only. After a period of excretion without storage during hibernation, a phase of synthesis and granule accumulation seems to occur.During experimental lethargy, with a low body temperature, induced in July, C cells reappear in large number; they are hypertrophied and partly degranulated, some mitoses are observed. Also, when hibernation is delayed, these cells remain numerous and lightly granulated until May, although normally their number is reduced by the end of March or the beginning of April, after dormouse arousal.Two hibernating mammals from Malghasia Republic were studied: 3 Tenrecs, Centetes ecaudatus, were spontaneously lethargic in July in France (austral winter). C cells were extremely abundant, voluminous, poorly granulated. In the Ericulus ericulus, from the same area, C cells were less numerous in February, in France, after a lethargy of a short duration. C cells appear numerous and active in the thyroid gland of a lethargic Paraechinus aethiopicus from North Africa (Beni-Abbes) in February.In the marmot (Marmota marmota) a thyroid stimulation also occurs around arousal, but C cells are not as numerous as in the other hibernating mammals, and a cyclic activity in the C cells seems difficult to demonstrate actually.Data from the literature on C cells of hibernating mammals are compared with results obtained on dormice and discussed in correlation with the condition of parathyroid glands and calcium metabolism during the annual cycle. The hypothesis of a secretion of calcitonin by C cells seems probable, perhaps in relation with a prolonged immobility during winter sleep which might induce a bone decalcification.

Nous adressons tous nos remerciements au Professeur M. Fontaine qui a bien voulu mettre à notre disposition ce matériel important et varié et Mr. F. Lachiver pour les documents qu'il nous a aimablement communiqués sur le comportement de ces animaux au Laboratoire de Physiologie du Muséum. Les espèces provenant de Madagascar et de Beni-Abbes ont été procurées au Pr Fontaine grâce à l'obligeance du Professeur F. Petter que nous remercions. Une partie du travail histologique a été réalisée avec la collaboration de Melle J. Olivereau, du CNRS, dont les excellentes techniques sont vivement appréciées.     

Lysosomal membrane recovery following labilization by thyroid stimulators

Lysosomal membrane permeability was assessed by measuring freely available naphthylamidase activity in intact preparations of guinea pig thyroid follicular cells following exposure of thyroid tissue to sequential stimulation by two thyroid stimulators, thyrotrophin (TSH) and thyroid stimulating immunoglobulins (TSI). These investigations showed that following labilization by TSH, the lysosomal membranes recovered and were capable of responding to a second thyroid stimulator (TSI). That such recovery represented restabilization of lysosomal membranes was confirmed by the finding that latent naphthylamidase activity was restored without a change in total activity of the enzyme.     

Etude ultrastructurale d'une formation de soutien épidermo-conjonctive inédite chez les Insectes

Résumé Une formation de soutien (formation hypopharyngienne transverse ou FHT) est tendue transversalement dans la cavité hypopharyngienne de Blabera craniifer BURM. (Dictyoptère). Elle est constituée par les épidémies droit et gauche hypertrophiés reliés l'un à l'autre par un matériel conjonctif.Le cytoplasme épidermique est envahi par des microtubules qui s'attachent sur les membranes plasmatiques apicale et basale; les zones d'attache de microtubules sont comparables aux hémidesmosomes connus chez les Vertébrés.La jonction épidermo-cuticulaire est assurée par l'association des zones d'attache apicales et de fibres à structure périodique traversant toute la cuticule. La jonction épidermo-conjonctive est réalisée par l'association des zones d'attache basales et d'une couche de matériel dense extra-cellulaire par l'intermédiaire de laquelle le conjonctif (et en particulier les fibres collagènes) s'attache sur la membrane plasmatique.Cette liaison conjonctivo-épidermo-cuticulaire est comparable à la liaison myo-épidermo cuticulaire des Arthropodes.De telles formations de soutien semblent communes et il est probable que le rôle squelettique du conjonctif a été jusqu'ici sous-estime chez les Insectes.

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Ultrastructure of a supporting structure in insects

Summary A supporting structure (FHT) is stretched transversely in the hypopharyngeal cavity of Blabera craniifer Burm (Dictyoptera); the right and left epidermal cell layers are hypertrophied and connected by connective tissue.The cytoplasm of the epidermal cells is filled with an unusually large number of microtubules inserted both on the apical and basal plasma membranes; the attachment zones of microtubules are similar to the hemidesmosomes of Vertebrates.The epidermo-cuticular junction is formed through the association of intracuticular banded fibers with the apical attachment zones and the epidermo-connective tissue junction through the insertion of connective tissue structures (collagen fibers and others) on an extra-cellular dense material layer apposed to the basal attachment zones.This connectivo-epidermo-cuticular connection can be compared to the myo-epidermocuticular connection of Arthropods.Supporting formations of this type are not infrequent, so it appears that the skeletal functions of the connective tissue of Insects have been underestimated previously.

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Nous tenons à remercier le Prof. Ch. Noirot et Madame Noirot-TimothÉe pour leur aide efficace et les suggestions qu'ils nous ont apportées au cours de la réalisation de ce travail.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

Tissue-specific knockout of TSHr in white adipose tissue increases adipocyte size and decreases TSH-induced lipolysis

Background

The primary function of TSH is to activate TSH receptors (TSHr) in the thyroid gland and thereby stimulate thyroid hormone synthesis and secretion. TSHr are also expressed in other organs, but their physiological importance is still unclear. We have previously shown that TSHr, expressed in adipocytes, are of potential importance for lipolysis and extrauterine adaptation of the neonate.

Methodology

To further study the role of TSHr in adipocytes we selectively removed the TSHr gene in mice adipocytes by using the Cre-loxP recombination system (B6.Cg-Tg (Fabp4-Cre) 1Rev/J. TSHr knockout (KO) newborn mice were phenotypically characterized. Isolated adipocytes from 8-week-old male mice were studied in term of adipocyte size and metabolism.

Results

Mice lacking TSHr in adipocytes were apparently normal at birth and no differences in thyroid gland function or histology were observed. Sensitivity to TSH-induced lipolysis was ten times lower in adipocytes from targeted animals compared to wild-type. This indicates that adipocytes from targeted animals are refractory to stimulation of physiological concentrations of TSH. Catecholamine-induced lipolysis and insulin-induced inhibition of lipolysis were unaltered. Adipocyte size was increased in the targeted animals. Basal lipolysis was increased as an effect of the increased adipocyte size.

Conclusion

Our results indicate that adipocyte TSHr under normal conditions affects adipocyte growth and development.