The thyrotropic cells of the guinea pig pituitary

Summary Three different immunocytoenzymatic techniques were used to identify and characterize the thyrotropic cells in the pituitary of normal guinea pigs at the ultrastructural level (superimposition technique, immunocytochemical technique using P.A.P. and indirect immunohistoenzymatic method before embedding).These cells are characterized by a dark cytoplasm with granules ranging from 1500 to 2000 Å in diameter. The appearance of these granules is very variable: some display a marked electron density and are homogeneous but some have a less marked electron density with a more electron dense peripherally situated region.The TSH molecules are essentially confined to the granules but when the immunocytochemical reactions are carried out before embedding, positive staining is also seen in the cytoplasm and the outer surface of most of the rough endoplasmic reticulum membranes. These results are discussed.We thank D. Quief for technical assistance. This work was supported by a grant from U.E.R. III LilleAttaché de Recherche INSERM     

On the identity of the thyrotropic cell in the red-spotted newt

Summary Although the pars distalis of the red-spotted newt has previously undergone extensive cytological examination, the identity of its thyrotropic cells has remained uncertain. From the present ultrastructural study, cells of type 3 (Masur, 1969) containing granules 150–180 nm in diameter are concluded to be the thyrotropes. Such cells were found to be present in the regions of the pars distalis where thyroidectomy cells arise after ablation of the thyroid gland. Cells of type 3 are probably identical with a population of cells containing granules which stain with silver methenamine indicating the presence of a glycoprotein such as thyroid stimulating hormone (TSH). Thyroidectomy cells containing a few residual granules 150–180 nm in diameter were occasionally found in partes distales from newts killed 3 or 7 days after ablation of the thyroid gland, and were abundant in pituitaries 21 days after thyroidectomy. Only cells of type 3 responded (by vacuolation of granules) when animals were immersed in water containing 10 g/l of thyroxine. No cells of the pars distalis showed cytological change after administration of synthetic thyrotropic releasing hormone (TRH) giving additional support to the view that this hormone has no stimulatory role in amphibians.Work supported in part by research grant 1 R01 AM 16731-01 from the Institute of Arthritis, Metabolism and Metabolic Diseases and in part by grant PCM 75-17637 from the National Science Foundation     

Light- and electron-microscopic studies on the secretory cytology of the adenohypophysis of the Japanese quail,Coturnix coturnix japonica

The effects of thyroidectomy, adrenalectomy, and castration on the pars distalis of male Japanese quail, and of injection of LH-RH on sexually inactive females, were investigated by light and electron microscopy. Correlation between light and electron microscopy was attained by use of alternate thin and thick sections. Six types of secretory cells were identified and the ultrastructural characteristics described. Putative endocrine functions have been designated on the basis of responses to experimental interventions and on other criteria. The putative STH cells are characterized by the presence of large dense secretory granules (250-300 nm) that are stained with orange-G by the trichrome method. They occur only in the caudal lobe and appear to be unchanged by castration, thyroidectomy, adrenalectomy and LH-RH injection. The putative prolactin cells are characterized by large (400-600 nm), spherical or polmorphic, dense secretory granules stainable with acid fuchsin and aniline blue; prominent Golgi apparatus and well developed endoplasmic reticulum with densely packed, regularly parallel lamellae. They are found mainly in the cephalic lobe. The prolactin cells develop some vacuolization after adrenalectomy and undergo some degeneration after castration. The ACTH cells, which are restricted to the cephalic lobe, are identified by the dense, spherical granules (250-300 nm) that are stained with acid fuchsin. After adrenalectomy, they lose their secretory granules and are transformed into large, chromophobic adrenalectomy cells. TSH cells are so designated by their response to thyroidectomy including loss of their fine secretory granules and transformation to large, vacuolated thyroidectomy cells. We have found TSH cells and thyroidectomy cells only in the cephalic lobe. Basophilic cells, considered to be gonadotropes, occur in both the cephalic and caudal lobes. The gonadotropes of the cephalic lobe appear to have slightly larger (120-200 nm) granules than the caudal lobe (120-150 nm). However, after castration, the gonadotropes in both lobes become hypertrophied and vacuolated and are transformed into mutually indistinguishable castration cells. Twenty minutes after injection with LH-RH, the gonadotropes of both lobes increase in size and number, degranulate, develop vacuoles in the cytoplasm, and appear very similar to castration cells.     

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells

We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.     

An electron microscopic study of stellate cells and cavities in the pars distalis of frog pituitary

Stellate cells in the pars distalis of adult Rana ridibunda were observed electron microscopically under normal and experimental conditions (TRH injection). The stellate cells have lengthy processes extending into the intercellular spaces between the secretory cells and scanty cytoplasm surrounding the nucleus. Occasional desmosomes link stellate cells to adjacent secretory cells. In the pars distalis of animals injected with thyrotropic-releasing hormone (TRH), the stellate cells form large cavities (2-6 mum) filled with heterogeneous material. Their cytoplasm contains well-developed Golgi complexes and some lysosomes; these are the principal morphological alterations as compared to those observed in control animals. It is suggested that stellate cells could play an active role in addition to providing a structural framework for the pars distalis.     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Cytodifferentiation of the adenohypophysis of the domestic fowl

Summary In the chick embryo the first membrane-bound secretory granules occur in the cytoplasm of occasional cells in the cephalic lobe of pars distalis at the 7th day of incubation. On the 8th day most of the cells in both the cephalic and caudal lobes contain secretory granules that are variable in size, form and density.On the 9th day at least two types of glandular cells are distinguishable in the cephalic and in the caudal lobes; however, these cells are not comparable with those of the adult gland. Differentiation of acidophils and basophils occurs, apparently simultaneously, in 11-day embryos.The cells of the cephalic and caudal lobes are morphologically distinct from their first appearance. Thus it is concluded that these two lobes develop independently and differently from an early stage of ontogenesis.The secretory granules are formed in the Golgi area of the hypophysial cells after the 8th day of incubation. However, secretory material may be synthesized also by a process not involving the Golgi apparatus.Nerve fibers containing granules first appear in the superficial layer of the median eminence on the 8th embryonic day and by the 12th day three types of granules and two types of clear vesicles are identifiable.The investigation reported herein was supported by grant from Japan-U.S. Cooperative Science Program of Japan Association for Science Promotion to Professor Mikami and by U.S.-Japan Cooperative Science Program Grant No. GF-33334 to Professor Farner.     

Ultrastructural pathology of the thyroid folliculo-papillary and follicular carcinoma

Twenty cases of thyroid carcinoma (10 follicular and 10 folliculo-papillary) were ultrastructurally studied. In the follicular carcinoma the most striking features were: microfollicular cavities with microvilli from the apical surface of the tumorous cells, intracellular microlumens, swollen mitochondria sometimes containing electrondense bodies and tightly packed filaments. In the solid sheaths light and dark cells were present. Golgi complexes were disposed in small dense cristae. The nuclei were large, round, oval or with a folded appearance. In the folliculo-papillary carcinoma were found nuclei with an irregular shape containing stage I and stage II inclusions, dilated endoplasmic sacks, closely packed, sometimes dystrophic mitochondria, dense bodies or tightly packed parallel filaments and numerous phagolysosomes. The peroxidase activity wa present as black precipitates in the nuclear envelope or around colloid droplets. The acid phosphatase activity was found as unhomogeneous precipitates inside the lysosomes. From this study it could be concluded that the follicular and folliculo-papillary carcinomas have some common ultrastructural features; the ultrastructural and cytoenzymological patterns suggest marked alteration of the synthesis, storage and secretion of thyroid hormones.     

Localization of calcium in the secretory cells of the mammary glands in response to oxytocin

Distribution of Ca2+ ions, precipitated by means of pyroantimonate potassium, has been investigated electron microscopically in secretory cells of the mammary gland of lactating white mice. In the glandular cells, that are at the state of inhibition of secretory activity, the cytochemical reaction product is localized on the internal side of the basal, lateral and apical parts of the plasmolemma, in mitochondrial matrix, in cisterns and in the Golgi complex vesicles, in the nuclear areas, occupied by euchromatin. Oxytocin effect produces a certain complex of ultrastructural changes in the cell accompanied by redistribution of Ca2+ ions. Amount of precipitate in mitochondria decreases. It is revealed in the lumen of dilated canals of the granular endoplasmic reticulum, in the zone of decondensated nuclear chromatin, in the Golgi complex vesicles. The vesicles become larger and fuse with each other. The changes mentioned demonstrate increased synthetic and transport processes, occurring in the glandular epithelium of the mammary gland after oxytocin effect.     

CHANGES IN FINE STRUCTURE AND ACID PHOSPHATASE LOCALIZATION IN RAT THYROID CELLS FOLLOWING THYROTROPIN ADMINISTRATION

Shortly after the administration of 1/40 unit thyrotropin to rats, 24 hours post-hypophysectomy, the following sequence of changes has been observed within thyroid follicular epithelial cells: (1) the appearance of apical cell surface activity consisting of pseudopods projecting into the follicular lumen; (2) apparent phagocytic engulfment of colloid droplets lacking indications of acid phosphatase activity; (3) close association and probable fusion of newly formed colloid droplets and dense granules, the latter cytochemically positive for acid phosphatase activity; (4) the appearance of presumptive acid phosphatase activity within colloid droplets; and, (5) further colloid droplet changes, viz., basipetal migration and decrease in size, accompanied by an increase in density and in demonstrable acid phosphatase activity. These changes appeared to represent the resorption and degradation of follicular colloid. Comparable results were obtained using intact and more heavily stimulated animals. Colloid biosynthesis was tentatively visualized in these cells as a separate mechanism involving small vesicles prominent in the Golgi region and beneath the apical plasma membrane of some, but not all, thyroid follicular cells in each specimen.     

Ultrastructure of the APUD-like cells of the frog thymus gland

Within the thymus gland of the European common frog, Rana temporaria, cells with endocrine- like appearance have been found. At the ultrastructural level the most characteristic feature of their cytoplasm is the presence of secretory granules. Some cells possess irregular electron lucent granules with an eccentrically located dense core while others possess smaller electron dense granules. The cytoplasm contains also cisterns of rough endoplasmic reticulum, free ribosomes, and small mitochondria. The cells possess irregular nuclei with the pronounced nucleoli. These endocrine-like cells are connected by desmosomes with neighbouring non-granulated epithelial cells. Ultrastructural features of the cells described here resemble those seen in polypeptide hormone-secreting cells belonging to the family of cells of the APUD (Amine Precursor Uptake and Decarboxylation) series.     

Immunocytochemical and ultrastructural study of the frog (Rana pipiens) pars distalis with special reference to folliculo-stellate cell function during in vitro superfusion

Summary The colloidal gold immunocytochemical technique was used to determine the ultrastructural features of the glandular cells in the pituitaries of male frogs, Rana pipiens, both in vivo and after superfusion in vitro. Specific reactions to antisera against bullfrog gonadotropins, human prolactin, and synthetic 1–39 corticotropin allowed identification of the 3 corresponding types of glandular cells. No immunoreaction was obtained with antisera against human or ovine-growth hormone, human -thyrotropin hormone, and bovine S-100 protein. General morphological features of these immunocytochemically identified glandular cells were similar to those of equivalent cells previously described in other amphibian species. Non-glandular folliculo-stellate cells were distinctive. In freshly removed pituitaries, these folliculo-stellate cells contained lysosome-like structures, but did not show phagocytic vacuoles in the cytoplasm; they contained many mitochondria, and the Golgi complex and endoplasmic reticulum were relatively undeveloped. After 4 or 18 h of superfusion, some immunoreactive gonadotropic, prolactin, and corticotropic cells showed degeneration and destruction. In the same gland, folliculo-stellate cells retained a viable appearance, but showed phagocytic vacuoles containing secretory granule-like structures which were immunoreactive to gonadotropic, prolactin, and corticotropic antibodies. Some folliculo-stellate cells showed phagocytic vacuoles containing complete glandular cells. These results suggest that superfusion causes a destruction of some of the glandular cells, and that folliculo-stellate cells act as phagocytes when cellular debris or moribund cells are present in the intercellular space in the pituitary parenchyma.Supported by grant DCB 8710462 from the National Science Foundation, grant 2148-83 from the CAICYT (Spain) and the Junta de Andalucia (Spain)     

Ultrastructure and secretory cycle of the gastrin-producing cell

Summary The gastrin-producing cells in the cat have been studied under various experimental conditions indicating the secretory cycle of these cells. Normal gastrin cells in animals fed ad libitum show secretory granules of different electron density. After fasting for 24 hrs the cells are granulated with electron dense secretory granules and after refeeding the cells are degranulated, showing clear secretory granules. The implication of the endoplasmic reticulum and the Golgi apparatus in the secretory cycle is discussed on the basis of the ultrastructural findings.Supported by a grant from the Fonds national suisse de la Recherche scientifique, Berne, Switzerland.     

Immunohistochemical, electron microscopic and morphometric studies of estrogen-induced rat prolactinomas after bromocriptine treatment

To clarify the effects of bromocriptine on prolactinoma cells in vivo, immunohistochemical, ultrastructural and morphometrical analyses were applied to estrogen-induced rat prolactinoma cells 1 h and 6 h after injection of bromocriptine (3 mg/kg of body weight). One h after treatment, serum prolactin levels decreased markedly. Electron microscopy disclosed many secretory granules, slightly distorted rough endoplasmic reticulum, and partially dilated Golgi cisternae in the prolactinoma cells. Morphometric analysis revealed that the volume density of secretory granules increased, while the volume density of cytoplasmic microtubules decreased. These findings suggest that lowered serum prolactin levels in the early phase of bromocriptine treatment may result from an impaired secretion of prolactin due to decreasing numbers of cytoplasmic microtubules. At 6 h after injection, serum prolactin levels were still considerably lower than in controls. The prolactinoma cells at this time were well granulated, with vesiculated rough endoplasmic reticulum and markedly dilated Golgi cisternae. Electron microscopical immunohistochemistry revealed positive reaction products noted on the secretory granules, Golgi cisternae, and endoplasmic reticulum of the untreated rat prolactinoma cells. However, only secretory granules showed the positive reaction products for prolactin 6 h after bromocriptine treatment of the adenoma cells. An increase in the volume density of secretory granules and a decrease in the volume densities of rough endoplasmic reticulum and microtubules was determined by morphometric analysis, suggesting that bromocriptine inhibits protein synthesis as well as bringing about a disturbance of the prolactin secretion.     

Ultrastructural modifications of the cavities formed by folliculo-stellate cells in chicken adenohypophysis under septic shock conditions

Effects of septic shock by repeated inoculations with Escherichia coli on the ultrastructure of the folliculo-stellate cells and cavities of the adenohypophysis of the chicken were investigated in order to determine the function of these cavities. The principal morphological modifications were dilation of the Golgi apparatus, endoplasmic reticulum and autophagic vacuoles, and necrosis phenomena in the stellate cells. The follicular cavities showed dilation, and there was heterogeneous dense material and granular elements in the follicular lumen. Based on results reported in the literature, the observations reported here are evidence of a "cleaning-role", for the removal of cell debris, when there is endocrine disfunction.     

Formation of secretion granules in the Golgi apparatus of plasma cells in the rat

The three-dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanuclear Golgi apparatus was formed by a continuous ribbonlike structure composed of the following stacked elements. On the cis-face of the Golgi stack, there was a tubular membranous network referred to as the cis-element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80-nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or "peeling off" configuration. These last elements consisted of prosecretory granules attached to flattened, empty-looking saccules showing buds at their surface; detached, more-or-less fenestrated, flattened saccules; and shrivelled residual trans-tubular networks. In the trans-region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans-saccules flows toward the dilated portions which become prosecretory granules. The trans-most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans-tubular networks.     

Morphology of defense glands of the opilionids (daddy longlegs) Leiobunum vittatum and L. flavum (Arachnida: Opiliones: Palpatores: Phalangiidae)

Opilionid defense glands consist of 0.5 × 0.9-mm sacs attached to the underside of low tubercles located on the dorsal side of the cephalothorax, posterior to the first pair of legs. Each gland opens via an elongated slit, located in the posterior floor of a crater that is situated at the summit of the tubercle. The center of the sac, called the reservoir, is lined by a cuticle consisting of epicuticle and endocuticle which is continuous through the slit with the exoskeleton. The layers of cuticle vary in thickness with different locations in the gland. A hemocoelomic (basement) membrane, 0.5–1, μ thick, forms the boundary between glandular cells and hemocoel. The gland has a nonsecretory portion consisting only of cuticle-supporting cells and a secretory portion consisting of secretory and cuticle-supporting cells. The cuticle lining the reservoir in the secretory area is broached by many cuticle-lined ductules, each of which drains an isolated intercellular space called the intercalated cistern. This in turn drains microvilli-lined canaliculi located between and extending into secretory cells. The cisterns are devoid of microvilli. Secretory cell cytoplasm contains a Golgi apparatus, many free ribosomes, rough endoplasmic reticulum (RER), two types of granules (speckled and dense), and mitochondria. Speckled granules are partially filled with fairly large particles and are found in association with the Golgi apparatus. They also surround canaliculi into which they empty. Dense granules are packed with very small particles, have a gray homogeneous appearance, and are scattered throughout the cytoplasm. Mitochondria containing matrix granules tend to scatter throughout the cytoplasm but are concentrated around canaliculi.     

Structural and ultrastructural differentiation of the thyroid gland during embryogenesis in the grass snake Natrix natrix L. (Lepidosauria, Serpentes)

The differentiation of the thyroid primordium of reptilian species is poorly understood. The present study reports on structural and ultrastructural studies of the developing thyroid gland in embryos of the grass snake Natrix natrix L. At the time of oviposition, the thyroid primordium occupied its final position in the embryos. Throughout developmental stages I-IV, the undifferentiated thyroid primordium contained cellular cords, and the plasma membranes of adjacent cells formed junctional complexes. Subsequently, the first follicular lumens started to form. The follicular lumens were of intracellular origin, as in other vertebrate species, but the mechanism of their formation is as yet unclear. At developmental stages V-VI, the thyroid anlage was composed of small follicles with lumens and cellular cords. Cells of the thyroid primordium divided, and follicles were filled with a granular substance. At developmental stage VI, the cells surrounding the follicular lumen were polarized, the apical cytoplasm contained dark granules and the Golgi complex and the rough endoplasmic reticulum (RER) developed gradually. Resorption of the colloid began at developmental stage VIII. At the end of this stage, the embryonic thyroid gland was surrounded by a definitive capsule. During developmental stages IX-X, the follicular cells contained granules and vesicles of different sizes and electron densities and a well-developed Golgi apparatus and RER. At developmental stage XI, most follicles were outlined by squamous epithelial cells and presented wide lumens filled with a light colloid. The Golgi complex and RER showed changes in their morphology indicating a decrease in the activity of the thyroid gland. At developmental stage XII, the activity of the embryonic thyroid gradually increased, and at the time of hatching, it exhibited the features of a fully active gland.     

Seasonal changes of parafollicular and follicular cells of the dormouse thyroid (Myoxus glis): an ultrastructural and immunocytochemical study

The follicular epithelium of dormouse thyroid consists of two distinct cellular types, follicular and parafollicular cells. Parafollicular cells can be easily identified by their high cytoplasmic dye-affinity for phloxine, round to ovoid shape, basal arrangement and lack of contact with follicular colloid. The wide cytoplasmic matrix is clear and contains many secretory granules of variable electron density whose contents histochemically appears to be proteic with a lean glucidic component. Furthermore immunocytochemical reactions with antibodies against calcitonin and somatostatin showed that both hormones are co-stored in the secretory granules of all parafollicular cells. Both follicular and parafollicular cells show seasonal morphological variations in their secretory activity. Follicular cell activity is high in summer, reaches a peak in late fall or prehibernation and progressively slows down throughout hibernation. Parafollicular cells exhibit a fair synthetic activity in summer, in fall, and in the animals captured during winter hibernating sleep and killed after 12 days stay in laboratory. In winter sleep, granules with interrupted membrane and cottony contents are prevalent and the ultrastructural aspects suggest an intense discharge of secretion. The results are compared with those from other hibernating mammalians and discussed in the light of blood calcium values and seasonal balances of other metabolisms.