Demonstration of stellate cells of the pars intermedia of the pituitary gland using a new silver impregnation technique

The pars intermedia of the pituitary gland in the rat and other species contains, in addition to secretory cells, stellate cells first characterized by using electron microscopy. The distribution and relationships of these cells is difficult to assess at the ultrastructural level. We have developed an ammoniacal silver nitrate method for stellate cells of the pars intermedia. Staining is carried out in 15 micron sections of buffered formalin fixed, paraffin embedded pituitaries. This method shows that in the rat pars intermedia, stellate cells showing numerous cytoplasmic projections are abundant in, and evenly distributed throughout, pars intermedia lobules.     

Permeability of the amphibian pars intermedia to peroxidase injected intravascularly

To study the penetration of protein from the blood into and through the pars intermedia of anurans, the electron-dense tracer, horseradish peroxidase (HRP), was injected into the vascular system of the frog, Rana pipiens. Pinocytotic vesicles were seen along plasma membranes of the stellate cells abutting the pericapillary spaces between the pars intermedia and pars nervosa. The HRP was seen within both the pericapillary spaces and these pinocytotic vesicles. In addition, the HRP was very prominent in the extracellular spaces between adjacent stellate and/or MSH cells and around nerve endings and thus, appeared to move from the vascular spaces and into the interior of the pars intermedia. Therefore, the poorly vascularized pars intermedia may have such a route for transfer of materials throughout the gland.     

The pars intermedia of the mink,Mustela vison

Summary The pars intermedia of intact and experimental female minks has been studied by light, electron and fluorescence microscopy. The general structure of the mink intermediate lobe is described. Two main cell types are recognized. One, termed glandular cell, predominates in number and is characterized by the presence of electron-dense granules about 200 nm in diameter and numerous large vesicles up to 300 nm in diameter. The other, termed stellate cell, is devoid of cytoplasmic vesicles and granules and possesses microfilaments, junctional complexes and elongated processes inserted between the glandular cells. Different treatments (photostimulation and administration of hypertonic saline and metopirone) induced morphological reactions in the glandular cells. The significance of these changes and the possibility of a functional relation between the pars intermedia and ACTH secretion are discussed.Numerous nerve fibres and axon terminals containing electron-dense granules (60–120 nm) and electron-lucent vesicles (30–40 nm) are observed throughout the pars intermedia.With the histochemical fluorescence method of Falck-Hillarp a rich system of delicate fluorescent varicose fibres, sometimes provided with irregular swellings or droplets, is observed in the pars intermedia and also in the pars nervosa. Microspectrofluorometrically these fibres exhibit the spectral characteristics of catecholamines. All the cells of the pars intermedia and a large number of cells in the pars distalis show a yellowish weak fluorescence, which becomes much stronger after combined formaldehyde-ozone treatment. This cellular fluorophore shows the same microspectrofluorometric characteristics as does the fluorophores of tryptamine and of certain peptides with NH₂-terminal tryptophan.Supported by the Swedish Fur Breeders' Association and the Swedish Natural Science Research Council (grant No. 2124). Thanks are due to Miss W. Carlsson and Miss Y. Lilliemarck for their helpful technical assistance.Supported by the Harald and Greta Jeanssons Stiftelse and by the Ford Foundation. The skilful technical assistance of Mrs. Eva Svensson and Miss Annika Borgelin is greatfully acknowledged.     

Observations on the functional cytochemistry of pars intermedia of the rat hypophysis

Summary Light and electron microscopic histochemical reactions were studied in the cells of pars intermedia of the rat. The possible correlations between enzymatic reactions and endocrine functions of these cells were discussed. By combined formaldehyde and chloral vapour treatment the cells of the pars intermedia exhibited a strong yellow fluorescence suggesting the presence of a peptide or peptides with NH₂-terminal tryptophan. Masked metachromasia after acid hydrolysis was probably due to these peptides. Only a weak or no α-glycerophosphate dehydrogenase and nonspecific esterase activity was observed in the cells of pars intermedia compared to the cells of pars distalis suggesting low production rate of hormone synthesis. Specific and non-specific cholinesterases were demonstrated light and electron microscopically constantly in the cells bordering the lobules. These cells probably represent a certain type of glial cells. In the other cells the enzymatic activities varied markedly in intensity and distribution showing different ultrastructural localizations. Thus cholinesterase activities in the cells of pars intermedia reflect possibly different functional stages of the cells in their hormone production, storage and secretion processes.     

Phagocytic activity of the stellate cells in the anuran pars intermedia

Summary In an attempt to study further the stellate cell and its functions, the ultrastructure of this cell type in the neurointermediate lobe of the bullfrog, Rana catesbeiana, was examined in both organ and dissociated-cell culture. The cytoplasmic activity of stellate cells from neurointermediate lobes incubated 3 1/2 or 5 1/2 h was greater than that of those in vivo. Mitochondria and bundles of cytoplasmic filaments were numerous, in addition to prominent, well-developed Golgi complexes with associated vesicles. The most striking ultrastructural feature was the presence of phagocytic vacuoles that contain cellular debris. The stellate cells were seen to form cytoplasmic processes that phagocytosed this extracellular debris identifiable as belonging to the secretory cells of the pars intermedia. The stellate cells from the dissociated-cell preparations were also seen to contain debris within phagocytic vacuoles. In those neurointermediate lobes transplanted for 3 1/2 to 4 days into the anterior chamber of the eye, the stellate cells demonstrated similar phagocytic ability, but the phagocytic vacuoles contained material that seemed to be at a later stage of degradation. In all three of these conditions, the stellate cells were not seen to release this cellular debris nor were they seen to undergo cell division. These glial-like stellate cells of the pars intermedia acted as macrophages in all three of these experiments. There is now, therefore, a need to determine under what conditions, if any, these stellate cells function in vivo as macrophages.Supported by NSF Program for Small College Faculty Engaged in Research at Larger Institutions and Department of Energy — Associated Western Universities Faculty Participation Program. The authors thank Dr. W. Ferris and Dr. J. Berliner for the use of the electron microscopy facilities at the University of Arizona and Nuclear Medicine Laboratory, UCLA, respectively. Warm thanks are due to Ms. Ruth Cole for technical assistance     

Observations on the functional cytochemistry of pars intermedia of the rat hypophysis.

Light and electron microscopic histochemical reactions were studied in the cells of pars intermedia of the rat. The possible correlations between enzymatic reactions and endocrine functions of these cells were discussed. By combined formaldehyde and chloral vapour treatment the cells of the pars intermedia exhibited a strong yellow fluorescence suggesting the presence of a peptide or peptides with NH2-terminal tryptophan. Masked metachromasia after acid hydrolysis was probably due to these peptides. Only a weak or no alpha-glycerophosphate dehydrogenase and nonspecific esterase activity was observed in the cells of pars intermedia compared to the cells of pars distalis suggesting low production rate of hormone synthesis. Specific and non-specific cholinesterases were demonstrated light and electron microscopically constantly in the cells bordering the lobules. These cells probably represent a certain type of glial cells. In the other cells the enzymatic activities varied markedly in intensity and distribution showing different ultrastructural localizations. Thus cholinesterase activities in the cells of pars intermedia reflect possibly different functional stages of the cells in their hormone production, storage and secretion processes.     

Existence of noradrenalin cells and serotonin cells in the pituitary gland of Rana catesbeiana

The localization of catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)], of serotonin (5-HT), and adrenocorticotropin (ACTH) in the pituitary of bullfrog (Rana catesbeiana), rat, hamster, and dog was examined by the immunofluorescence method. Many TH- and DBH-positive but PNMT-negative noradrenalin cells and 5-HT-positive serotonin cells were first observed in the pars distalis of the frog pituitary together with small numbers of ACTH-positive cells, in marked contrast to our previous findings that TH-positive but DBH-negative dopamine cells are rare in the anterior lobe of rat and dog pituitary. The entire population of cells of the pars intermedia showed a weak ACTH-like immunoreaction. Although most of these cells were TH- or 5-HT-negative, TH-positive but DBH-negative dopaminergic varicose fibers surrounded these cells. Among cells of the pars intermedia of the frog, 5-HT-positive cells with processes were also scattered. In the neural lobe, TH- and 5-HT-immunoreactive fibers were rarely seen.     

An immunohistochemical study of adenohypophyseal cells in the viviparous reptile Chalcides chalcides

The morphology of the hypophysis and the immunocharacteristics of the adenohypophyseal cells in the viviparous reptile Chalcides chalcides were studied by light microscopy, using conventional staining methods and an indirect antibody technique (ABC method), respectively. The general morphology of the C. chalcides hypophysis was comparable to that of other reptiles, showing three main regions: the pars distalis, the pars intermedia and the pars nervosa. The gland appeared as an elongated body in a cephalic-caudal direction and was almost completely enclosed in the sella turcica. For this reason, the hypophysis was studied in toto with the brain in decalcified specimens. The pars distalis accounted for most of the whole organ. The pars intermedia surrounded the pars nervosa as a goblet. The pars tuberalis was lacking.The immunohistochemical identification of the adenohypophyseal cells was performed using rabbit antisera against mammalian/synthetic hypophyseal hormones. Prolactin cells were clustered in small cellular cordons in the rostral pars distalis and in the medial pars distalis in both male and female specimens. Somatotropic cells were found in the caudal pars distalis. Corticotropic cells were observed in the medio-rostral pars distalis, as well as in the pars intermedia, where melanotropic cells were also present. Melanotropic cells were confined to the pars intermedia. Gonadotropic cells were mostly distributed in the ventral and lateral portions of the pars distalis, where they were found isolated or in small clusters. Thyrotropic cells were detected in the pars distalis with a distribution similar to that of the gonadotropic cells; however, atypically, they were also found in the pars intermedia. Therefore, the cytological characteristics of the adenohypophyseal cells appeared mostly conserved.     

Extravascular circulation in the pituitary of Mugil cephalus (Teleostei)

Summary Extravascular circulation in the pituitary of Mugil cephalus was investigated by injecting live fish with horseradish peroxidase and studying the distribution of the enzyme in the gland. The principal components of the extravascular circulatory system are the pericapillary spaces, and, arising from them, the interlobular and circumhypophyseal spaces. Extensions of these spaces penetrate the glandular parenchyma of the pars distalis, where they merge with pericellular spaces. In the neurohypophysis, pericapillary spaces are connected to the periaxonal spaces.Capillaries penetrating from the proximal neurohypophysis into the pars distalis are accompanied by neurosecretory axons. These axons form a mass of tissue which is limited near the capillaries by the pericapillary spaces and near the adenohypophysis by the interlobular spaces. Toward the interior of the adenohypophysis the amount of nervous tissue accompanying the capillaries progressively diminishes, thus reducing the distance between pericapillary and interlobular spaces. Within the pars distalis, the neurosecretory axons accompanying the capillaries are sparse, and the secretory and stellate cells are mostly located directly adjacent to the pericapillary spaces. In the neuro-intermediate lobe, interlobular spaces outline the neuro-adenohypophyseal boundary.The relationship between extravascular spaces and hormone-secreting cells varies in the different regions of the adenohypophysis depending upon the type of neurosecretory innervation in the respective region. In the regions of prolactin and gonadotropin cells, where neurosecretory axons are in direct contact with the secretory cells, the hormone-secreting and stellate cells are adjacent to the pericapillary spaces. In the regions of ACTH and STH cells, secretory and stellate cells are found adjacent to the interlobular spaces, which are interposed between the cells and the neurosecretory axons.Abbreviations AH adenohypophysis - CH circumhypophyseal - DNH distal neurohypophysis - HRP horseradish peroxidase - NH neurohypophysis - NS neurosecretory - PD pars distalis - PI pars intermedia - PPD proximal pars distalis - RNH rostral neurohypophysis - RPD rostral pars distalis This research was supported by a grant from the National Council for Research and Development, Israel, and the GKSS Geesthacht-Tesperhude, Federal Republic of Germany     

Immunocytochemical localization of vimentin in the posterior lobe of the cat, rabbit and rat pituitary glands

The presence and distribution of vimentin, a subunit of intermediate filaments, in the neural lobe and in the pars intermedia of the cat, rabbit and rat pituitary glands were investigated immunocytochemically. In the pars intermedia, our study revealed the presence of vimentin in glial-like cells located between glandular secretory cells of the three species and in the cells of the marginal layer of the cat and rat hypophyseal cleft. In the neural lobe of the cat and rabbit pituitary glands, there was a large amount of cell processes and immunoreactive pituicytes. In contrast, in the rat neural lobe, few pituicytes exhibited immunoreactivity, and these were located principally in the posterior region near the pituitary stalk. The significance of immunoreactive vimentin in these cells is discussed.     

Ultrastructure of the pars intermedia of the adult sheep hypophysis

Summary Light microscopy of coronal sections of the sheep pars intermedia revealed a compact, incompletely lobulated V-shaped region about 15–20 cells thick, situated between the pars distalis and the pars nervosa. A prominent hypophysial cleft and follicles containing a colloid-like substance were seen.Using electron microscopy, five cell types could be distinguished: pars intermedia glandular cells, pars distalis-like glandular cells, interstitial cells, follicular cells and cleft lining cells. The polyhedral to pear-shaped pars intermedia glandular cells predominated. They contained dense-cored, membrane-bound granules near the Golgi complex, and larger, irregular vesicles with finely granular contents of varying electron density throughout the remaining cytoplasm; exocytotic release of granules was occasionally observed. Smaller numbers of cells resembling those seen in the pars distalis were scattered throughout the pars intermedia. Interstitial cells usually possessed elongated cytoplasmic processes which extended between the glandular cells, and were characterized by deeply indented nuclei, elaborate junctional complexes and an absence of cytoplasmic granules. Cells lining the follicles resembled the interstitial cells. The major cells bordering the hypophysial cleft were triangular in section and bore irregular microvilli on their free surface. The pars intermedia appeared to be less vascular than the remainder of the hypophysis and only occasional fenestrated capillaries were seen. Nerve profiles were rare.     

Structural relationships between parenchymal and stromal elements in the pars intermedia of the rat adenohypophysis as demonstrated by extracellular space markers

Summary The pars intermedia (PI) of the rat adenohypophysis was studied by light and transmission electron microscopy after conventional staining as well as ruthenium red staining, and after systemic injection of horseradish peroxidase. The studies disclose a complex and constant system of two channel types (Type I and Type II channels) formed by PI cells with specific relationships to a very rich nerve supply, to each other, and to a stellate cell type proposed here to represent an element of neuroglia. The channel system could perform a function in the movement of fluids and solutes within the PI which is virtually avascular in the rat as well as in other mammals.Supported by the Medical Research Council of Canada Grant # MA6445We thank Mr. J. Jones and Mr. A.R. Morris for their expert technical assistance     

Life span changes in the presence of alpha-melanocyte-stimulating-hormone-containing cells in the human pituitary.

Since recent circumstantial evidence has suggested possible functions of alpha-MSH in intrauterine growth and labour, the presence of this hormone in the human pituitary was determined by means of the indirect immunofluorescence procedure during development and adulthood. Cross reaction of the antibodies with other peptides was measured after which they were purified by solid phase absorption. Experiments on the rat pituitary showed that staining of alpha-MSH- and ACTH-containing cells could be obtained well until 48 h after death. In the pars distalis the ability of ACTH-containing cells to take up stain increased during the period of post-mortem storage. In the youngest human fetus studied (15 weeks) only alpha-MSH-containing cells were found in the pars intermedia and no ACTH-containing cells were observed. In the other fetal pituitaries a distinct pars intermedia containing more alpha-MSH cells than ACTH cells was found. In the pars distalis of the fetuses more ACTH- than alpha-MSH-containing cells were observed. From birth to 19 years, progressively fewer alpha-MSH containing cells could be detected in the 'zona intermedia' and pars distalis, while in adults only a few such cells were found in either area. Irrespective of age, sex, cause of death or therapy, alpha-MSH-containing cells were found in all pituitaries throughout life. The number of ACTH containing cells gradually increased in the zona intermedia and pars distalis and reached a high adult level in the latter structure. In the pituitaries of seven anencephalics, no alpha-MSH-containing cells were present. The presence of alpha-MSH in the fetal pars intermedia, the change in the ratio of the alpha-MSH/ACTH cells during the course of development, and the absence of alpha-MSH in anencephaly all support the possibility that human fetal pituitary alpha-MSH is involved in both intrauterine growth and fetal adrenal function and thus also in parturition.     

Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis.

Immunocytofluorescence techniques with well characterized anti-sera to α-endorphin and β-endorphin show presence of these two peptides in all cellular elements of the pars intermedia of the rat hypophysis, and in discrete cells of the pars distalis (adenohypophysis) at the complete exclusion of the neurohypophysis (pars nervosa, posterior lobe).     

Interrelationships between pars intermedia and posterior pituitary with regard to corticotrophic and thyrotrophic partial function in rats

Functional interrelationships between the pars intermedia and posterior pituitary were proved under different experimental conditions. After stimulation of the thyrotropical axis of rats by an acute intraperitoneal application of 50 microgram TRH/rat the nuclear sizes of the thyroid follicular and the anterior pituitary thyrotropical cells increased according to a monophasic time curve with maximal amplitude at the time of 30 minutes. Interestingly, the nuclear sizes of the posterior pituitary cells were also enhanced. Under the same experimental conditions the nuclear areas of the cells of the external layer of the adrenal zona fasciculata decreased as did the nuclei of the pars intermedia cells (without regard to the cell type or localization of the cells in the intermediate lobe). Stimulation of the adrenocorticotropical axis by an acute injection of 0.2 ml isotonic saline solution/rat was followed by a time-dependent increase of nuclear sizes of the fasciculata cells and pars intermedia, whereas the nuclear volumes of the thyroid follicular cells, the anterior pituitary thyrotropical cells and the posterior pituitary cells decreased. Thus the functional state of the pars intermedia was in accordance with that of the adrenal cortex. Also the posterior pituitary cells responded to stimuli applied to the thyrotropical axis at the same degree as the thyrotropic organs themselves. Between the nuclear sizes of the pars intermedia and posterior pituitary we established the same inverse functional relationships as between the adrenal cortex and the thyroid gland.     

Immunocytochemistry of a “private” luteinizing-hormone-releasing hormone system in the pituitary

Summary Immunocytochemistry of paraffin sections of Bouin-fixed rat pituitaries with antiserum to luteinizinghormone-releasing hormone (LHRH) revealed two types of cells. Type I cells exhibit granular staining throughout their cytoplasm. The immunoreactivity of type II cells is confined to a much smaller area of the cytoplasm. Type I cells are located in the ventral margin of the pars intermedia, the region between the pars intermedia and the pars distalis, and the pars distalis adjacent to this region. Type II cells have a broader distribution in the pars distalis, but tend to concentrate in the region of the pars distalis near the pars intermedia. Type I cells are distinct from gonadotropes. Type II cells appear to comprise a subgroup of gonadotropes. Staining in type I, but not type II, cells in pituitary explants, maintained in serum-free media for seven days, is as intense as that in normal pituitary tissue. The data suggest that the type I cells are producing an intrinsic LHRH-like material and may be responsible, in part, for the regulation of some gonadotropes.Supported by NIH grants HD12932, NS15843 and NS15809 (LAS), National Science Foundation grant BNS 82-05643 (LAS), and a grant from the Phillippe Foundation (JYL)     

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     

Distribution of opioid peptides in the pituitary: a new hypothalamic-pars nervosa enkephalinergic pathway

The distribution of the endorphins, beta-endorphin and enkephalin (Met5-enkephalin and Leu5-enkephalin), was determined in the pars distalis, intermedia, and nervosa of the rat pituitary using both immunocytochemical and radioimmunological methods. Immunoreactive (ir) beta-endorphin was found in pars distalis and pars intermedia. On gel filtration of the pars distalis extracts, beta-endorphin immunoreactivity was eluted in three peaks corresponding to pro-opiocortin (5%), beta-lipotropin (75%), and beta-endorphin (20%). beta-Endorphin was the only component in the pars intermedia. Enkephalin was found in high amount in the pars nervosa. A new enkephalinergic hypothalamic-pars nervosa pathway was observed. Dehydration experiments on normal rats and analysis of the genetically polyuric Brattleboro rat suggest that this enkephalinergic pathway may modulate neurohypophyseal neurosecretion.     

Ontogenesis of the α-MSH, β-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity

Pituitary sections from 15 to 21 day-old rat foetuses have been studied with the immunofluorescence technique, using antibodies anti alpha-MSH, anti beta-MSH and anti beta (1-24) ACTH. The first ACTH cells appear on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia. beta-msh cells have been observed on day 16 in the pars anterior and on day 17 in the pars intermedia, while alpha-MSH cells appear only on day 18 and exclusively in the pars intermedia. The cytodifferentiation of the beta-MSH and ACTH cells occurs in the pars intermedia with about a 24 hours delay in comparison to that of the pars distalis. The first revealed cells are always located in the posterior half of the pituitary gland. The corticostimulating activity of the hypophysis has been tested with the fluorescence intensity of the corticotrophs, the adrenal weight, the adrenal content in corticosterone and the plasma corticosterone level. The fluorescence of the corticotrophs increases on day 18, shows a maximum on day 19 and decreases until term. The adrenal weight rises regularly between day 16 to day 20, thereafer the increase subsides. Adrenal and plasma corticosterone concentrations reach a peak on day 19 of pregnancy. These data suggest that hypophyseal corticostimulating activity is very high between days 18 and 19 and decreases between days 19 and 21.     

Monoamine-containing fibres in the pituitary neuro-intermediate lobe of the pig and rat

Summary A rich system of monoamine-containing fibres is described in the neural lobe and pars intermedia of the pig and rat. a) A rich network of delicate varicose fibres is evenly distributed throughout the parenchyma of the neural lobe and surrounds the cells of the pars intermedia. b) Droplets or clusters of droplets are scattered throughout the neural lobe. Most of them probably constitute terminal swellings or end-apparatuses of smooth or varicose fibres. The number of droplets varies from animal to animal; they are found also in the pars intermedia. c) Coarse varicose fibres are mainly localized around larger vessels. At least some of these fibres are nerve fibres of sympathetic origin. A combination of fluorescence microscopy and aldehyde-fuchsin staining on the same sections demonstrated that the majority at least of these monoamine-containing structures were not identical with aldehyde-fuchsin positive neurosecretory fibres.This research was supported by a grant from the Swedish Medical Research Council (B68-12X-712-03B) and by the Faculty of Medicine, University of Lund.