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.     

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.     

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.     

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. XVI: Postnatal changes of distribution of S-100 protein positive cells,connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5–60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis.     

Granulated 'marginal cell layer' in the rat anterior pituitary gland

A granulated 'marginal layer cell' was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5-20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the 'invading' residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by 'marginal layer cells' had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the 'marginal cell layer' and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.     

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.     

Granulated ‘marginal cell layer’ in the rat anterior pituitary gland

A granulated ‘marginal layer cell’ was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5–20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the ‘invading’ residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by ‘marginal layer cells’ had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the ‘marginal cell layer’ and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.     

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.     

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.     

Immunohistochemical localization of anterior pituitary hormones in S-100 protein-positive cells in the rat pituitary gland

In the anterior and intermediate lobes of the rat pituitary gland, non-hormone-producing cells that express S-100 protein coexist with various types of hormone-producing cells and are believed to function as phagocytes, supporting and paracrine-controlling cells of hormone-producing cells and stem cells, among other functions; however, their cytological characteristics are not yet fully understood. Using a transgenic rat that expresses green fluorescent protein under the promoter of the S100β protein gene, we immunohistochemically detected expression of the luteinizing hormone, thyroid-stimulating hormone, prolactin, growth hormone and proopiomelanocortin by S-100 protein-positive cells located between clusters of hormone-producing cells in the intermediate lobe. These findings lend support to the hypothesis that S-100 protein-positive cells are capable of differentiating into hormone-producing cells in the adult rat pituitary gland.     

Immunohistochemical localization of keratin in bull,goat, and sheep anterior pituitary glands

Summary Immunohistochemical localization of keratin, an intermediate filament protein, was studied in bull, goat, and sheep anterior pituitary glands, i.e., in animals of the order Artiodactyla. Strong immunoreactivity was detected in the cells of the marginal layer of bull and goat, as well as in cysts or large follicles in the anterior lobe of all 3 species. In addition, a number of stellateshape cells were immunoreactive for keratin and were distributed throughout the anterior lobe. The localization of keratin-positive cells in light-microscopic preparations correlated precisely with the localization of folliculo-stellate cells in adjacent ultrathin sections. In ultrastructural studies, many slender and elliptical membranous components which were different from smooth endoplasmic reticulum were observed in the cytoplasm of the some keratin-positive cells. Some of the folliculostellate cells in the 3 species were also immunoreactive for the subunit of S-100 protein, which exists in some epithelial cells. On the other hand, immunolocalization of glial fibrillary acidic protein, a glial cell marker, could not be demonstrated in the anterior pituitary glands of the 3 species studied. These results suggest that keratin-positive folliculo-stellate cells express epithelial-like characteristics.     

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.     

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.     

In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats

Type I and III collagens widely occur in the rat anterior pituitary gland and are the main components of the extracellular matrix (ECM). Although ECM components possibly play an important role in the function of the anterior pituitary gland, little is known about collagen-producing cells. Type I collagen is a heterotrimer of two α1(I) chains (the product of the col1a1 gene) and one α2(I) chain (the product of the col1a2 gene). Type III collagen is a homotrimer of α1(III) chains (the product of the col3a1 gene). We used in situ hybridization with digoxigenin-labeled cRNA probes to examine the expression of col1a1, col1a2, and col3a1 mRNAs in the pituitary gland of adult rats. mRNA expression for these collagen genes was clearly observed, and cells expressing col1a1, col1a2, and col3a1 mRNA were located around capillaries in the gland. We also investigated the possible double-staining of collagen mRNA and pituitary hormones, S-100 protein (a marker of folliculo-stellate cells), or desmin (a marker of pericytes). Col1a1 and col3a1 mRNA were identified in desmin-immunopositive cells. Thus, only pericytes produce type I and III collagens in the rat anterior pituitary gland.     

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.     

Expression of retinaldehyde dehydrogenase 1 in the anterior pituitary glands of adult rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development and is also a regulatory factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine factor is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. Recently, we demonstrated that RALDH2 and RALDH3, but not RALDH1, were expressed in the developing anterior pituitary gland of rats, but the expression of RALDHs in the adult pituitary gland was not determined. Therefore, we have now examined the expression of RALDH1, RALDH2, and RALDH3 mRNAs in the pituitary gland of adult rats. Analysis by quantitative real-time polymerase chain reaction of adult pituitary glands has revealed a high level of RALDH1 mRNA but not of RALDH2 mRNA or RALDH3 mRNA. We have also detected mRNA expression for RALDH1 in the anterior pituitary gland by in situ hybridization with digoxigenin-labeled cRNA probes. Double-staining for RALDH1 mRNA and pituitary hormones or S-100 protein, a marker of folliculo-stellate cells (FS-cells), has revealed RALDH1 mRNA expression in a portion of prolactin-producing cells, marginal layer cells, and FS-cells. Our results suggest that RA is generated in the adult anterior pituitary gland, and that it may act locally on pituitary cells. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18790149) and by the Foundation of Growth Science.     

Cytology of the pituitary gland of the Plains viscacha (Lagostomus maximu)

The anatomy of the pituitary gland of the Plains viscacha ( Lagostomus maximus ) is described. The pituitary gland in various physiological states has been investigated in order to localise the secretory sites of the trophic hormones. The various cell types of the pituitary gland were distinguished on the basis of their morphological characteristics, tinctorial properties and differential solubilities of the protein hormones in trichloroacetic acid solutions. Two cell types were found in the pars tuberalis, three cell types in the pars intermedia and six chromophi1 cell types in the pars anterior. Their possible functions are discussed.     

Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the adenohypophysis in the monkeys Macaca irus and Cercopithecus aethiops

Summary With the use of an antibody against bovine S-100 protein, it was possible to reveal a characteristic cell type in the pars distalis and the pars tuberalis of the monkey Macaca irus. In the adenohypophysis of Cercopithecus aethiops, labeled cells were present in the pars distalis, pars tuberalis, and pars intermedia. These cells, so-called folliculo-stellate cells, were found in all pituitaries studied. Surprisingly, an antibody against human S-100 protein did not label the stellate cells of the adenohypophysis. However, in Macaca irus, this antibody gave a strong positive reaction with various other cell types (interstitial cells of the pineal gland, Müller cells of the retina, autonomic ganglionic cells, glial cells of the central nervous system, Schwann cells, Bergmann glia of the cerebellum, fat cells, reticular cells of lymphoid organs). By use of double immunoenzymatic labeling, it was evident that stellate cells are spatially related either to somatotropes, prolactin cells, corticotropes, or to glycoprotein-containing cells. Thus, a specific relationship to a particular endocrine-cell type could not be observed.Dr. M.P. Dubois died in Tours (France) on March 30, 1986, aged 65