Satellite cells in the normal human adrenal gland and in pheochromocytomas

In light of the recent finding of the S-100 antigen in satellite cells of the rat adrenal medulla, we looked for S-100-labelled cells in both the normal human adrenal medulla and in pheochromocytomas. Immunostaining enabled us to detect S-100-labelled satellite cells by both light and electron microscopy in a significant number of pheochromocytomas and, as expected, in the normal human adrenal medulla.     

Intercellular communications within the rat anterior pituitary XII: immunohistochemical and physiological evidences for the gap junctional coupling of the folliculo-stellate cells in the rat anterior pituitary

Since Farquhar [1957. "Corticotrophs" of the rat adenohypophysis as revealed by electron microscopy. Anat. Rec. 127, 291] was the first to report the presence of agranular folliculo-stellate cells as corticotrophs in the anterior pituitary gland, there were no reports about electro-physiological characteristics of the folliculo-stellate cells because of its no hormonal activity and the chaotic distribution of the parenchyma cells. Male Wistar rats, aged 7 weeks with weighing 250--300 g, were separated into two groups. One group was used for immunohistochemical and light microscopical studies to detect S-100 protein and connexin 43. The other group was used for the electro-physiological study and then for the electron microscopical study to know the fine structural character of folliculo-stellate cells after the electro-physiological experiment. Clusters of S-100 protein cells (agranulated folliculo-stellate cells) and numerous connexin 43 positive sites on S-100 protein cells were clear in the "transitional zone" at which the pituitary tissue made the transition from the pars tuberalis to the proximal part of the anterior lobe. Penetration of electrodes to the cells distributed in the transitional zone showed stable membrane potential ranged between--27 and--67mV with no spontaneous activity. Random penetration of electrode showed that larger populations of cell ( approximately 80%) had membrane potentials with -55.6+/-5.1 mV, and less than 20% of cells had the resting membrane potential with -36.0+/-4.4 mV. There were two types of cell couplings; one major group for the recordings from cells with similar deep resting membrane potentials and the other for the recordings from cells with different resting membrane potentials. The former indicated that two cells were electrically coupled while the latter no electrical couples were observed. Carbenoxolone depolarized the membrane by 12.3+/-5.5 mV and reduced the amplitude of electrotonic potentials, and the response recovered by removal of carbenoxolone by the superfusate. The transitional zones of the pituitary glands examined the electrical coupling were observed by an electron microscopy. Almost cytological profiles were observed as intact. The results clearly indicated that the folliculo-stellate cell system deeply participated in the regulation of the anterior pituitary parallel with the portal vessel system, which was the main regulatory system for pituitary hormone secretion.     

Immunohistochemical study of cells positive for glial fibrillary acidic protein (GFAP) in the human pituitary gland,with special reference to folliculo-stellate cells

Summary The cytology and the distribution of cells which contain glial fibrillary acidic protein (GFAP) were studied immunohistochemically in thick frozen sections of human pituitary glands. Immunoreactive cells were constantly demonstrated in both neuro- and adenohypophysis. In the neural lobe, an irregular network of long GFAP-positive pituicyte processes was revealed. Within this network, some asymmetric pituicytes became visible. A variable number of cells was stained in cell cords and follicles of the pars distalis and the intermediate zone. The morphology of these cells could be studied in detail, providing strong evidence to support the hypothesis that adenohypophyseal GFAP-immunoreactive cells belong to the folliculo-stellate (FS) cell system. Cells with similar cytological features in the pars distalis or the intermediate zone were found to share common immunoreactivities against GFAP and the presumable FS cell markers vimentin and S-100 protein. Our results corroborate the notion that, in the human pituitary, GFAR can be regarded as a marker protein of pituicytes and FS cells, which is expressed at varying degrees.     

Immunohistochemical study of cells positive for glial fibrillary acidic protein (GFAP) in the human pituitary gland, with special reference to folliculo-stellate cells

The cytology and the distribution of cells which contain glial fibrillary acidic protein (GFAP) were studied immunohistochemically in thick frozen sections of human pituitary glands. Immunoreactive cells were constantly demonstrated in both neuro- and adenohypophysis. In the neural lobe, an irregular network of long GFAP-positive pituicyte processes was revealed. Within this network, some asymmetric pituicytes became visible. A variable number of cells was stained in cell cords and follicles of the pars distalis and the intermediate zone. The morphology of these cells could be studied in detail, providing strong evidence to support the hypothesis that adenohypophyseal GFAP-immunoreactive cells belong to the folliculo-stellate (FS) cell system. Cells with similar cytological features in the pars distalis or the intermediate zone were found to share common immunoreactivities against GFAP and the presumable FS cell markers vimentin and S-100 protein. Our results corroborate the notion that, in the human pituitary, GFAP can be regarded as a marker protein of pituicytes and FS cells, which is expressed at varying degrees.     

Differential expression of the neural cell adhesion molecule NCAM 140 in human pituitary tumors

We have analyzed the expression of the intracellular marker protein neuron specific enolase (NSE), synaptophysin (SYN) and of the cell surface marker NCAM (neural cell adhesion molecule) in both normal hypophysis and in pituitary adenomas in order to explore their potential use as diagnostic tools. All adenomas (4 prolactinomas, 3 growth hormone (GH) producing adenomas and 4 inactive adenomas) showed SYN and NSE immunoreactivity on tissue sections and this was confirmed by immunoblots. NCAM 140 (an isoform of NCAM with molecular mass 140 kDa) was detected by immunoblotting in normal human adenohypophysis, in all GH adenomas, and in three out of four inactive adenomas, but not in prolactinomas. Using highly sensitive techniques, NCAM immunoreactivity was observed by electron microscopy in all adenomas. These data indicate that NCAM 140 is a constituent of the cell surface of endocrine cells in both normal human adenohypophysis and its tumors. Since prolactinomas express very low levels of NCAM 140 compared to other hypophyseal tumors its virtual absence could be used for differential diagnosis. A combined analysis of NCAM, SYN and NSE could be useful to characterize inactive adenomas which are not immunoreactive for pituitary hormones and which may contain no or only low levels of the alpha chain of the glycoprotein hormones.     

Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland

In the present study we have localized immunohistochemically the intermediate filament proteins of the human pituitary gland (adenohypophysis, pars intermedia and pars tuberalis) by an indirect immunoperoxidase technique or by double immunofluorescence methods and analysed the individual cytokeratin polypeptides using two-dimensional gel electrophoresis. We found that the expression of cytokeratins in different epithelial cells of the human anterior pituitary gland was heterogeneous. Whereas the endocrine cells only expressed cytokeratins 8 and 18, the folliculo-stellate cells exhibited a reactivity for cytokeratins 7, 8, 18 and 19 as well as for GFAP and vimentin. The squamous epithelial cells of the pars tuberalis and the Ratke's cysts showed a more complex cytokeratin pattern of both squamous and simple type. Whereas in may cystic epithelial cells including the "pseudo-follicles" a triple expression of cytokeratin, vimentin and GFAP could be observed, only some basal cells of squamous epithelial nests coexpressed cytokeratin and vimentin. The differences in the intermediate filament protein distribution are discussed in the light of embryological relationships of the different parts of the human pituitary gland.     

Localization of metallothionein I-II immunoreactivity in bovine pituitary gland

Metallothioneins belong to a family of shock proteins characterized by an unusual high content of cystein, absence of aromatic amino acids and high metal content (Zinc and Copper). Metallothioneins are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far. To the best of our knowledge this is the first report describing the presence of metallothioneins in the pituitary gland. Metallothioneins were identified immunohistochemically and chromatographically both in the neuro and adenohypophysis of the bovine pituitary gland. Metallothioneins are highly expressed in the neurohypophyseal glial cells, and in a subpopulation of folliculo-stellate cells located in the pars intermedia of the adenohypophysis. While the specific role of these proteins in the pituitary gland remains to be established, we hypothesize that, besides their protective action against free radicals, hypophyseal metallothioneins might be involved in the regulation of metal ion homeostasis with putative implication in release of hypothalamic peptide hormones in the neurohypophysis and synthesis/release of alpha-MSH by POMC-cells located in the pars intermedia of the adenohypophysis.     

Localization of Carboxyl Ester Lipase in Human Pituitary Gland and Pituitary Adenomas

Carboxyl ester lipase (CEL) is an enzyme that hydrolyzes a wide variety of lipid substrates, including ceramides, which are known to show inhibitory regulation of pituitary hormone secretion in experimental models. Because no studies on CEL expression in human pituitary and pituitary adenomas have been reported in the literature, we investigated CEL expression in 10 normal pituitary glands and 86 well-characterized pituitary adenomas [12 FSH/LH cell, 17 α-subunit/null cell, 6 TSH cell, 21 ACTH cell, 11 prolactin (PRL) cell, and 19 GH cell adenomas] using IHC, immunoelectron microscopy, Western blotting, and quantitative RT-PCR. In normal adenohypophysis, CEL was localized in GH, ACTH, and TSH cells. In adenomas, it was mainly found in functioning GH, ACTH, and TSH tumors, whereas its expression was poor in the corresponding silent adenomas and was lacking in FSH/LH cell, null cell, and PRL cell adenomas. Ultrastructurally, CEL was localized in secretory granules close to their membranes. This is the first study demonstrating CEL expression in normal human pituitary glands and in functioning GH, ACTH, and TSH adenomas. Considering that CEL hydrolyzes ceramides, inactivating their inhibitory function on pituitary hormone secretion, our findings suggest a possible role of CEL in the regulation of hormone secretion in both normal and adenomatous pituitary cells. (J Histochem Cytochem 58:881–889, 2010)     

Intercellular communication within the rat anterior pituitary: relationship between LH-RH neurons and folliculo-stellate cells in the pars tuberalis

The distribution of LH-RH-positive nerve fibers in the median eminence was demonstrated in the 1970s and 1980s. A few LH-RH fibers have been reported to be present in the adjacent pars tuberalis of the pituitary, but their functional significance has not been clarified and still remains enigmatic. Adult male Wistar-Imamichi rats were separated into two groups: one for immunohistochemistry of LH-RH and S-100 protein (for the identification of folliculo-stellate cells) and the other for electron microscopy. For both immunohistochemistry and electron microscopy, the specimens obtained contained the pituitary gland connected with the hypothalamus. Numerous LH-RH-positive fibers were observed as tiny lines with several varicosities both on the primary vascular plexus and in the hypothalamus corresponding to the posterior half of the portal vein area. LH-RH-positive fibers were also noted around S-100-positive cells in the pars tuberalis. Weakly reactive S-100 cells were scattered in the pars tuberalis in the midsagittal plane, while clusters of strong reactive elements occurred 100–300 m from the center. Similar observations were made using fluorescence immunohistochemistry for LH-RH and S-100, and at the electron-microscopic level. At the posterior portion of the portal vein system, bundles of the LH-RH-immunoreactive fibers invaded the pars tuberalis and terminated on agranular cells. Gap junctions were clearly seen among agranular cells corresponding to folliculo-stellate cells. It is postulated that the LH-RH message might be transmitted not only by the established hypophyseal portal vein system but also via the folliculo-stellate cells in the pars tuberalis to aid in the modulation of LH release.     

Localization of fibronectin in the folliculo-stellate cells of the rat anterior pituitary by the double bridge peroxidase-antiperoxidase method

Summary The localization of fibronectin was demonstrated in the rat anterior pituitary by the highly sensitive double bridge peroxidase-antiperoxidase (PAP) method. The fibronectin immunopositive cells were characterized by stellatelike morphology. The cells immunostained for fibronectin were observed to be identical to those for S-100 protein in adjacent mirror sections, whereas the S-100 protein has been specifically immunodetected in the folliculo-stellate (FS) cells of the anterior pituitary. The present study indicates that the fibronectin is present in the FS cells, suggesting that FS cells might play a role in the regulation of pituitary function through the interaction of fibronectin with hormone secreting cells     

Localization of fibronectin in the folliculo-stellate cells of the rat anterior pituitary by the double bridge peroxidase-antiperoxidase method

The localization of fibronectin was demonstrated in the rat anterior pituitary by the highly sensitive double bridge peroxidase-antiperoxidase (PAP) method. The fibronectin immunopositive cells were characterized by stellate-like morphology. The cells immunostained for fibronectin were observed to be identical to those for S-100 protein in adjacent mirror sections, whereas the S-100 protein has been specifically immunodetected in the folliculo-stellate (FS) cells of the anterior pituitary. The present study indicates that the fibronectin is present in the FS cells, suggesting that FS cells might play a role in the regulation of pituitary function through the interaction of fibronectin with hormone secreting cells.     

Intermediate filament expression in non-neoplastic pituitary cells.

Fifty-one non-neoplastic human pituitary glands, including examples with Crooke's hyalinization or amyloidosis, were examined by an immunoperoxidase method using antibodies to keratin, vimentin, neurofilaments (NFs), glial fibrillary acidic protein (GFAP), desmin, actin, S-100 protein and a variety of pituitary hormones. It was confirmed that most of the epithelial cells in the pituitary gland express keratin immunoreactivity. These cells included endocrine cells in the anterior lobe, endocrine cells and squamous metaplastic cells in the pars tuberalis, columnar and ciliated epithelia forming follicular structures and salivary-type epithelium in the pars intermedia, and anterior lobe cells infiltrating the posterior lobe. This study also demonstrated that keratin and NFs may be co-expressed in endocrine cells in the pituitary anterior lobe, that keratin, vimentin and GFAP may be co-expressed in the epithelial cells forming cyst-like follicle in the pars intermedia, and that vimentin and GFAP may be co-expressed in folliculo-stellate cells and pituicytes. In addition, the GFAP and S-100 protein-negative high columnar epithelium in the pars intermedia tended to be positive for adrenocorticotropic hormone and melanocyte stimulating hormone, while the low columnar epithelium with the co-expression of GFAP and S-100 protein was negative for pituitary hormones.     

Intercellular communications within the rat anterior pituitary XI: an immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat

The distribution of the S-100 protein cell (folliculo-stellate cell) is very important to our understanding of the regulation of the anterior pituitary. In this study, 10 intact 60-day-old male Wistar-Imamichi rats, were separated equally into two groups. One was used for immunohistochemical study, and the other for electron microscopic analysis. Immunostained pituitary sections with S-100 protein antibody were photographed using a CCD camera equipped with a computer. The S-100 protein cells were then measured using NIH image software, and the three-dimensional distribution of the cells was analyzed. The distribution of the cells observed in each serial section showed that S-100 protein cells were dense at the basal zone of the gland and at the "transitional zone" where the pars tuberalis adjoined the anterior and intermediate lobes, where they represented over 50% of the total cell population. They then decreased in number with distance from this region to mid-way towards the sagittal axis before increasing again in the tail of the gland. The population of cells also decreased with increasing distance from the "transitional zone" to the wing and with distance from the basal zone. Portal vessels entered the anterior lobe through the "transitional zone" as thick capillaries, ran through the basal surface and penetrated into the central area of the anterior lobe. In all planes, S-100 protein cells encircled the capillaries. Ultrastructural observations confirmed the light microscopic findings indicating that clusters of agranular cells were densely located at the "transitional zone" and in the pars tuberalis. The distribution pattern of the folliculo-stellate cells and the capillaries showed good agreement and the spatial relationship between these two is detailed so as to better understand hypophyseal histophysiology.     

Substance P enhances the proliferation of rat anterior pituitary cells in vitro

The undecapeptide substanceP(SP) was shown to be intimately involved in both the structural and functional aspects of the anterior pituitary.Yet,in addition to its influences on hormonal secretion,SP may well possess more actions in this master gland.The present study was ftherefore aimed to investigate the effect of SP on the proliferation of rat anterior pituitary cells in primary culture,It was found that SP could dose-dependently increase the incorporation of tritiated thymidine(3H-TdR) into cultured anterior pituitary cells.Other mammalian tachykinins such as neurokinin A and neurokinin B had similar effect but to varying degrees.The equipotent analogue of SP,Norleucine^11-SP(Nle^11-SP),also acted likewise.with its action antagonizable by spantide,a SP receptor blocker.To further characterize the nature of cells responsive to the challenge of SP,immunocytochemical staining against S-100 protein and some adenohypophyseal hormones was performed alone or plus autoradiography.The results showed that the percentage of S-100 proteinimmunorective cells was apparently elevated by the addtion of Nle^11-Sp for 48h,which indicates a preferential proliferation of folliculo-stellate cells under the regime .This was confirmed by increases in immunocytochemical or autoradiographical labelling indices of anterior pituitary Substance P and anterior pituitary cell proliferation.Cells treated similarly.Taken together,These results reveal that the trophic action of SP observed previously in other tissues is also present at least in cultured rat anterior pituitary cells.with responding cells being predominantly folliculo-stellate cells as typified by S-100 proteinimmunoreactivity.Therefore,an intra-pituitary trophicaction of SP in vivo could be anticipated.     

Ultrastructure of pituitary folliculo-stellate cells of lactating rat during treatment with 2-bromo-alpha-ergocriptine

Folliculo-stellate cells of adenohypophysis were studied by transmission electron microscopy in lactating rats during treatment with the dopaminergic agonist 2-bromo-alpha-ergocriptine. Folliculo-stellate cells showed marked activation in control lactating rats, were unaffected by a single injection of the drug (3 mg/kg), regressed to the morphology of those found in non-lactating animals upon 6 days of drug treatment (1 mg/kg/die) both in presence and in absence of the suckling stimulus. However, morphometric analysis showed a proportion of folliculo-stellate cells in the gland which was similar in the four experimental groups (range: 11.3-13.5%), despite the increasing reduction of prolactin cells (55, 44, 32, 30%). In chronically treated animals to which suckling had been denied, extruding and degenerating prolactin cells were found, as well as extracellular material in non follicular spaces; the latter were lined by both folliculo-stellate and endocrine cells. These data suggest a dual reactivity of folliculo-stellate cells in the adenohypophysis according to the functional status of the gland.     

Electron microscopic observations of the anterior pituitary gland. Part I. The neurons in the "transitional zone" of the anterior pituitary gland

Since [Westlud, K.N., Chils, G.V., 1982. Localization of serotonin fibers in the rat adenohypophysis. Endocrinology 111, 1761-1763] initially identified the serotonin nerve fibers in the anterior pituitary gland, attention has been paid to the rostral zone of the anterior lobe into which nerve fibers enter and subsequently spread to deeper regions of the lobe. The rostral zone is the trifurcated junction of the partes tuberalis, intermedia and distalis, and has the important role(s) for hormone secretion via the "transitional zone" [Sato, G, Shirasawa, N, Sakuma, E, Sato, Y, Asai, Y, Wada, I, Horiuchi, O, Sakamoto, A, Herbert, DC, Soji, T, 2005a. Intercellular communications within the rat anterior pituitary. XI: An immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat. Tissue and Cell 37, 269-280.]. The objective of this study was to focus on the ultrastructure of this "zone." All of the animals studied were fixed by perfusion with glutaraldehyde via the left ventricle of the heart and examined by electron microscopy. In the "transitional zone," a cluster of neuronal elements was observed between the folliculo-stellate cell-rich area and the anterior lobe. This cluster consisted of myelinated fibers, unmyelinated fibers, neuroendocrine fibers, large cells, and supporting cells. The large cells were perikarya of neurons which made a "ganglion-like" structure with associated satellite cells. Agranular, folliculo-stellate cells were intermingled among the elements. This is the first report that neuronal elements form clusters in the "transitional zone." A relationship of the unmyelinated and neuroendocrine fibers in the basal layer and in the "transitional zone" is discussed.     

Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland

Summary In the present study we have localized immunohistochemically the intermediate filament proteins of the human pituitary gland (adenohypophysis, pars intermedia and pars tuberalis) by an indirect immunoperoxidase technique or by double immunofluorescence methods and analysed the individual cytokeratin polypeptides using two-dimensional gel electrophoresis. We found that the expression of cytokeratins in different epithelial cells of the human anterior pituitary gland was heterogeneous. Whereas the endocrine cells only expressed cytokeratins 8 and 18, the folliculo-stellate cells exhibited a reactivity for cytokeratins 7, 8, 18 and 19 as well as for GFAP and vimentin. The squamous epithelial cells of the pars tuberalis and the Ratke's cysts showed a more complex cytokeratin pattern of both squamous and simple type. Whereas in many cystic epithelial cells including the pseudo-follicles a triple expression of cytokeratin, vimentin and GFAP could be observed, only some basal cells of squamous epithelial nests coexpressed cytokeratin and vimentin. The differences in the intermediate filament protein distribution are discussed in the light of embryological relationships of the different parts of the human pituitary gland.     

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)     

S-100 protein and neuron-specific enolase in parathyroid glands and C-cells of the thyroid

Normal parathyroid glands and parafollicular cells (C-cells) of man, rat and rabbit, and also human parathyroid adenomas and medullary carcinomas were investigated for the presence of S-100 protein and neuron-specific enolase (NSE). For determination of the proteins immunoperoxidase methods were applied, i.e., the PAP method and the avidin-biotin system. The antisera, of polyclonal origin, were specifically directed against cow S-100 protein and rat or bovine NSE. The respective antisera are known to crossreact with S-100 protein from man, rat, and rabbit, as well as with NSE from man and rat. Surprisingly, the test for S-100 protein was found to be strongly positive in the parathyroid glands of rat and rabbit and was focally positive in normal and adenomatous human parathyroid glands, but completely negative in C-cells and medullary carcinoma cells. NSE was present in C-cells of rat and man, and in medullary carcinoma cells, but was absent in normal and adenomatous parathyroid cells. The results support data that indicate that both parathyroid cells and C-cells are derived from elements of the neural crest, but undergo different maturation processes during embryological development.     

Formation of S-100 containing cells of the hypothalamus and adenohypophysis in normal ontogenesis and in protein deficiency

By means of the indirect immunohistochemical method distribution of S-100 containing cells has been studied in sections of the mediobasal hypothalamus (astrocytes) and adenohypophysis (follicular-stellate cells) in newborn, 10- and 21-day-old rats under normal development and under protein insufficiency. For this the animals are given the diet containing 6% of protein (control--25% of protein). S-100 containing cells are revealed in the hypothalamus and adenohypophysis in 10- and 21-day-old animals. In the brain of the newborn rats S-100 immunoreactive cells are not revealed. At the ultrastructural level the diaminobenzidine (DAB) reaction products in the immunoreactive cells are revealed diffusely along the whole cytoplasm of the cells, in nuclei the DAB reaction products are absent. Part of S-100 containing cells is essentially lowered, comparing with the control. In the rat adenohypophysis part of S-100 containing cells from the 10th up to the 21st day also decreases. Unlike the hypothalamus, however, content of cells, immunopositive to S-100 exceeds the analogous index in the control rats of the corresponding age groups.