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.     

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.     

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     

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.     

Expression and distribution of vascular endothelial growth factor receptor Flk-1 in the rat pituitary.

Vascular endothelial growth factor (VEGF) acts primarily as an endothelial cell mitogen via the specific receptors Flk-1 and Flt-1. To help further define the possible role of VEGF in the control of pituitary cell function, we examined Flk-1 expression in normal rat pituitaries and in GH3 cells. Flk-1 expression was studied by immunohistochemistry, in situ hybridization, and double-labeling immunofluorescence combined with confocal laser microscopy. In normal rat pituitaries, Flk-1-immunoreactive cells appeared widely distributed only in the anterior lobe and were not detected in the intermediate or posterior lobe. Apart from the adenohypophysial cells, Flk-1 immunopositivity was also evident in endothelial cells of many capillaries distributed throughout the gland. Immunohistochemistry also showed that majority of GH3 cells expressed Flk-1 protein. In situ hybridization showed conclusive staining with the antisense probe and confirmed the immunohistochemical results. The double immunofluorescence method revealed Flk-1 expression in all types of hormone-producing adenohypophysial cells but not in folliculostellate cells. The percentage of immunopositive cells varied among the various cell types. The present study demonstrates that pituitary cells are not only sources of VEGF but also targets of this multifunctional substance, supporting the concept that VEGF functions as an autocrine/paracrine factor in the pituitary.     

Intermediate filament expression in pituitary adenomas.

Seventy-five formalin-fixed and 18 alcohol-fixed pituitary adenomas were studied immunohistochemically using antibodies to keratin, vimentin, neurofilaments (NFs), glial fibrillary acidic protein, desmin, actin, S-100 protein and a variety of pituitary hormones. The pituitary adenoma cells were positive for keratin, vimentin and NFs (68 kDa and 160 kDa) and in a few instances there was co-expression of these three types of intermediate filaments (IMFs). The pattern of keratin-specific staining showed diffuse cytoplasmic or patchy paranuclear reactivity and of NF- or vimentin-specific staining showed fibrillar or patchy paranuclear reactivity. The patchy staining seemed to decorate the fibrous body. There was no correlation between the distribution of IMFs and pituitary hormones in pituitary adenomas except that melanocyte-stimulating-hormone-positive reactivity was limited to the NF-positive adenomas. The pattern of IMF staining did not depend on hormone production in adenomas.     

Immunocytochemical study of S-100 protein in human pituitary adenomas

The S-100 protein was localized by immunocytochemistry in 70 pituitary tumors including 30 prolactin, 16 growth hormone, two corticotropin and 22 non-functioning adenomas. Positive immunostaining was observed in only one case (prolactin adenoma). It is concluded that in functioning and non-functioning pituitary tumors there is no particular involvement of S-100 protein-containing cells, at least under the conditions of this study.     

Lipocortin-1 immunoreactivity in the human pituitary gland

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.     

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.     

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.     

Folliculostellate cells in pituitary pars distalis of male viscacha: immunohistochemical,morphometric and ultrastructural study

Folliculostellate cells (FSC) have been reported in pituitary of several mammalian species. FSC morphology and secreted substances have been instrumental to the understanding of their function. The purpose of this work was to perform an immunohistochemical, morphometric and ultrastructural study of the pituitary pars distalis FSC in adult male viscacha and to analyze their relation with hormone secreting cells. Immunohistochemistry and image analysis were carried out in different sectors of the gland, from the middle (sector 1) to the glandular periphery (sector 5). Transmission electron microscopy with lanthanum as electrodense tracer was used. FSC formed follicles with PAS-positive colloid inside. They expressed S-100 protein mainly in both nucleus and cytoplasm. FSC were stellate-like in shape and exhibited short cytoplasmic processes that contacted with blood vessels and endocrine cells. In addition, some follicular colloids were immunostained with anti-S-100 protein. A few FSC were immunostained with anti-glial fibrillary acidic protein (GFAP) and anti-vimentin. The morphometric parameters analyzed (percentages of S-100-positive total, cellular and colloidal areas) increased from sector 1 to sector 3 and then decreased to sector 5. Hormone secreting cells, mainly lactotrophs, gonadotrophs and corticotrophs were associated with FSC and follicles. The ultrastructural study demonstrated that FSC developed junctional complexes and desmosomes between their lateral membranes. Lanthanum freely penetrated the spaces between granulated cells and FSC, but did not penetrate into the follicular lumen. In conclusion: 1) the differential expression of S-100 protein, GFAP and vimentin may indicate different physiological stages of FSC; 2) the expression of these proteins suggests a neuroectodermic origin of these cells; 3) FSC spatial distribution, association with endocrine cells, and the generation of an intercellular communication network suggest that FSC are involved in the pituitary pars distalis paracrine regulation of the viscacha.Key words: Lagostomus, pituitary, folliculostellate cells, immunohistochemistry, distribution, ultrastructure.     

Proliferative activity of cells of the intermediate lobe of the rat pituitary during the postnatal period.

Cellular proliferation was studied in the intermediate lobe (IL) of the pituitary gland of developing rats by labelling cells at the S-phase of the cell cycle with bromodeoxyuridine (BrdU). The number of BrdU-labelled cells in the IL decreased from birth until the 14th postnatal day and was low from that day until the end of the first month after birth. Throughout the postnatal period a large proportion of BrdU-labelled cells was found in the marginal layer (ML) of the IL, suggesting for the ML a role as a germinative layer of the IL during postnatal growth. Double immunostaining with anti-BrdU and anti-MSH showed that MSH cells actively proliferate as from the day of birth. Cells doubly immunostained with anti-BrdU and anti-S100 protein were first seen on the 14th postnatal day. From then onwards, most proliferating cells were labelled with either anti MSH or anti S-100 protein. This, together with the high proportion of proliferating cells found in the ML marks a clear difference with the pattern of cellular proliferation previously reported during a similar period in the anterior lobe of the rat pituitary.     

Distribution of angiotensinogen immunoreactivity in rat anterior pituitary glands

Angiotensin II (AII) has been previously shown to be localized in the gonadotropes of the rat anterior pituitary gland. Renin and angiotensin-converting enzyme, two enzymes that participate in the generation of AII, also have been shown to be present in gonadotropes. To determine whether angiotensinogen, the precursor to AII, is present in the same cells, we have stained rat anterior pituitary sections with an antirat angiotensinogen antiserum. Angiotensinogen staining was observed in cells that had a distinctive distribution at the periphery of the gland; the number of these cells and the intensity of the staining were increased in the pituitaries of rats that had been nephrectomized 24 hr before sacrifice. When double staining was performed, we never observed colocalization of angiotensinogen with any of the known pituitary hormones or with S100 protein. The results show that in the rat anterior pituitary gland, angiotensinogen is present, at least for the most part, in cells that are different from those containing renin, angiotensin-converting enzyme, and AII.     

Dynamics of connexin 43 levels and distribution in the mink (Mustela vison) anterior pituitary are associated with seasonal changes in anterior pituitary prolactin content

Because in mammals the anterior pituitary lacks innervation, we investigated whether gap junctions established between selected cells within the gland are part of an intrapituitary mechanism to ensure physiological synchronization of cells involved in the control of hormone secretion. We report here the dynamics of anterior pituitary connexin 43 (Cx43)-gap junctions throughout the mink (Mustela vison) annual reproductive cycle and its relationship with the anterior pituitary prolactin (PRL) content that parallels variations in serum PRL levels documented in the literature. We found that PRL anterior pituitary levels were maximal in spring and during lactation and that they were minimal in autumn and winter. Anterior pituitary Cx43 levels were maximal during periods of high PRL secretion. During these periods, Cx43-positive gap junctions localized to stellate-shaped cells occupying the center of anterior pituitary follicles and to the rounded cells occupying the remaining follicles. Connexin 43-positive gap junctions were also observed between adjacent follicles. During periods of low PRL pituitary content, Cx43-positive gap junctions localized to the stellate cells but not to the cells of the remaining follicles. Moreover, Cx43 labeling was undetected between adjacent follicles. To assess between which cells within the mink anterior pituitary the Cx43 gap junctions were established, the different anterior pituitary cell populations were separated by a discontinuous Percoll gradient, and Western blot analyses of each cell population using Cx43 antibodies were performed. The immunoblots showed a Cx43 immunoreactive band associated with the cell layer enriched in S-100-positive, stellate-shaped cells. The result was confirmed by fluorescence microscopy studies that showed that Cx43-mediated gap junctions were established preferentially between the cultured S-100-positive, elongated cells. The results show that in mink stellate cells, the junctional machinery associated with the Cx43 protein varies in synchrony with the anterior pituitary PRL content throughout the mink annual reproductive cycle. It is suggested that the Cx43 gap junctions on the stellate cells play an important role in the synchronization of cellular activity within selected follicles of the anterior pituitary, thus contributing to the control of PRL secretion during the annual reproductive cycle.     

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.     

Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.     

Immunohistochemical detection of folliculo-stellate cells in human pituitary adenomas

In the light of recent findings concerning the presence of S-100 antigen in folliculo-stellate cells of the rat adenohypophysis, we investigated the possible presence of S-100-labelled cells in both the normal human adenohypophysis and in pituitary adenomas. Immunostaining enabled us to detect, with both light and electron microscopy, the presence of S-100-labelled folliculo-stellate cells in a significant number of pituitary adenomas, mostly growth-hormone secreting, and, as expected, in the normal human adenohypophysis.