Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis

Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence in immunocytochemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N-acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they form the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa.     

Tissue distribution of Ca2+-dependent activator protein for secretion family members CAPS1 and CAPS2 in mice.

The family of Ca2+-dependent activator proteins for secretion (CAPS) is involved in dense-core vesicle exocytosis. CAPS1/CADPS1 and CAPS2/CADPS2 have been identified in mammals. CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of brain-derived neurotrophic factor and neurotrophin-3 from cerebellar granule cells. CAPS1 and CAPS2 are predominantly expressed in brain. Here we show the immunohistochemical localization of the CAPS family proteins in various mouse tissues. In the pituitary gland, CAPS1 and CAPS2 were localized to the pars nervosa and the pars intermedia, respectively. In non-neural tissues, CAPS1 was observed in the islets of Langerhans, minor cell types of the spleen and stomach, and medullary cells of the adrenal gland, whereas CAPS2 was present in bronchial epithelial cells, thyroid parafollicular cells, chief cells of the stomach, ductal epithelium of the salivary gland, kidney proximal tubules, and minor cell types of the thymus, spleen, and colon. These results suggest that secretion from distinct cell types in various tissues involves either or both members of the CAPS family.     

Electrical correlates of secretion in endocrine and exocrine cells

Many types of secretory cells including neurons and cells of endocrine and exocrine glands show changes in electrical potential and resistance when secretion is stimulated. These electrical correlates result from the movement of ions across the cell membrane through specific ion-selective channels. In neurons and certain endocrine cells (such as pancreatic beta cells and certain cells of the anterior pituitary), these channels are voltage dependent and open transiently upon depolarization leading to action potentials. Thus some endocrine cells are electrically excitable, a property previously held to occur only in nerve and muscle. In other nonexcitable endocrine and exocrine cells (such as the pancreas and parotid), ion channels are responsive to either occupancy of specific membrane receptors or changes in intracellular metabolites and second messengers. Ion fluxes through these latter channels also lead to changes in the electrical potential and resistance, but these changes are generally more sustained and action potentials are not seen. The entry of Ca2+ through both voltage-dependent and voltage-independent ion channels plays a major role in the activation of secretion via exocytosis.     

Distribution of 7B2 (secretogranin V)-like immunoreactivity in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary

In this study, we examined 7B2 (secretogranin V)-like immunoreactivity (IR) in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary. Results showed that the pars nervosa was filled with immunoreactive granules. In the pars intermedia, all melanotrophs showed 7B2-IR. In the pars distalis, immunoreactive cells were dispersed, and the 7B2-immunoreactive cells were also immunopositive for the β-subunit of bullfrog luteinizing hormone (fLHβ). 7B2-IR co-localized with fLHβ-IR in the same secretory granules. Our results suggest that 7B2 may participate in the secretion processes of gonadotropins in the pars distalis.     

Immunohistochemistry of the golden hamster pituitary during chronic administration of diethylstilbestrol: a quantitative analysis using confocal laser scanning microscopy

The aim of this study was to examine by immunohistochemistry the morphologic changes affecting pituitary cell populations in male Syrian hamsters undergoing chronic exposure (3 days to 9 months) to diethylstilbestrol (DES). Cell proliferation in the hypophysis was monitored by the immunohistochemical demonstration of S-phase cells after pulse labeling with 5-bromo-2'-deoxyuridine. Cell proliferation analysis was combined with the identification of different cell populations by immunostaining with antisera raised against hypophyseal hormones. Sections processed for double-label immunofluorescence were examined by confocal microscopy. In the adenohypophysis, the relative surface occupied by gonadotrophs and thyrotrophs decreased rapidly during the first months of treatment while corticotroph and somatotroph populations remained unaffected. Accordingly, the incidence of S-phase cells in these four cell populations was lower than or similar to control values. In contrast, lactotrophs increased gradually during the first month of exposure to DES to reach a maximum value at 2-4 months. At the beginning, this increase was primarily due to hyperplasia but later on it also involved cellular hypertrophy. Somatomammotrophs did not seem to be involved in this model. In the pars intermedia, the labeling index of melanotrophs rose rapidly to reach values 5-6 times higher than controls. After 4 months, neoplasms originating from the pars intermedia were seen invading both the neuro- and the adenohypophysis. At the end of treatment, the pituitary was markedly enlarged resulting from the development of an adenoma of the pars intermedia.     

Ultrastructure of the male rat hypophysis chronically grafted under the kidney capsule

Summary Male rats were divided in two experimental groups. In group I two partes distales of the hypophysis were grafted under the kidney capsule and in group II two complete hypophyses were transplanted. Animals were killed 5 to 22 months after the operation. The grafted tissue was excised and processed for light and electron microscopy.The transplanted pars distalis tissue showed a well developed vascularisation in contrast to the pars intermedia which appeared poorly vascularised. Six different cell types were observed in grafted pars distalis. They correspond to the different types of cells found in the rat pars distalis in situ. The predominant cell type in the graft displayed all the morphological characteristics of stimulated prolactotrophs. Pars intermedia cells appeared hypertrophied resembling the MSH cells under stimulation. Two types of syncytial formations were frequently seen. One of them appeared to originate from prolactotrophs and the other from MSH cells. Bodian impregnated fibres and structures resembling either growth cones of axons or typical nerve endings were observed in the pars intermedia of long-term grafted hypophyses. Pituicytes remained as isolated clusters of cells. Canaliculi lined by two or more pituicytes were observed. Saccular formations resembling the hypophyseal cleft appeared in all grafts studied. The present findings suggest that in the male rat the chronically grafted pituitary gland is capable of synthesising most or all the hormones which are known to be produced by the gland in situ. Furthermore, prolactin and MSH seem to be the predominant secretion of the transplanted pituitary.Supported by the National Research Council of Argentina. Grant 7315/74Members of the Research Career of Conicet, Argentina     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates cyclic AMP formation as well as peptide output of cultured pituitary melanotrophs and AtT-20 corticotrophs.

The present study was aimed at investigating whether PACAP stimulates accumulation of cAMP, as well as hormonal secretion of homogeneous populations of pituitary proopiomelanocortin (POMC) cells, namely melanotrophs and AtT-20 corticotrophs. PACAP was shown to enhance cAMP accumulation in a dose-dependent fashion in both cell types (with EC50 values of approx. 10(-10) M) and elicited additive increases of cAMP production with CRF in melanotrophs, but not in corticotrophs. PACAP also stimulated dose-dependently the secretion of alpha-MSH and ACTH, with EC50 concentrations of about 10(-9) M. In melanotrophs, bromocriptine significantly depressed PACAP-induced cAMP formation and blunted by more than 90% stimulated alpha-MSH release. This study shows that (1) pituitary POMC cells did respond to PACAP by enhancing cAMP accumulation and elevating hormone secretion as well; (2) the effect of PACAP was additive with CRF on cAMP production in melanotrophs, but not in corticotrophs, while there was no additivity on peptide output from both cell types; (3) activation of dopamine receptors in melanotrophs dampened both cAMP formation and peptide secretion. These findings are consistent with PACAP playing a possible hypophysiotropic role in the regulation of pituitary POMC cell activity.     

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.     

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.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.     

Dependence of pituitary hormone secretion on the pattern of spontaneous voltage-gated calcium influx. Cell type-specific action potential secretion coupling.

In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In this study, we demonstrate that although rat anterior pituitary lactotrophs, somatotrophs, and gonadotrophs exhibited spontaneous and extracellular calcium-dependent electrical activity, voltage-gated calcium influx triggered secretion only in lactotrophs and somatotrophs. The lack of action potential-driven secretion in gonadotrophs was not due to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization comparable with signals observed in somatotrophs and lactotrophs. The secretory vesicles in all three cell types also had a similar sensitivity to voltage-gated calcium influx. However, the pattern of action potential calcium influx differed among three cell types. Spontaneous activity in gonadotrophs was characterized by high amplitude, sharp spikes that had a limited capacity to promote calcium influx, whereas lactotrophs and somatotrophs fired plateau-bursting action potentials that generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered luteinizing hormone secretion. These results indicate that the cell type-specific action potential secretion coupling in pituitary cells is determined by the capacity of their plasma membrane oscillator to generate threshold calcium signals.     

CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED₅₀ values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.     

Immunohistochemical detection of ACTH and MSH cells in the hypophysis of the hermaphroditic teleost, Diplodus sargus

Hypophyseal ACTH and MSH cells were immunohistochemically characterised in the teleost fish, Diplodus sargus, using anti-ACTH (1-24) and anti alpha-MSH polyclonal antisera. ACTH cells were found both in the pars distalis and in the pars intermedia. In the former region, they appeared small, round-shaped and clustered; in the latter, they were either small or large and elongated. Moreover, a few ACTH-immunoreactive cells resembling microglia were present in the neurohypophysis. Conversely, MSH cells were found only in the pars intermedia, and were similar to the larger ACTH cells of the same region. In the pars intermedia, co-localisation of ACTH and MSH immunoreactivity in the same cell was revealed by double immunostaining, though the two hormones were also observed in distinct cell types. The distribution of ACTH cells appeared quite uniform, without any marked difference between the specimens tested. Conversely, MSH cell amount varied according to the stage of the sexual cycle of this teleost fish, which is characterised by protandrous hermaphroditism. In fact, a lower amount of MSH cells were observed in females, whereas no significant difference was found between immature and male specimens.     

Calcium and calcium-dependent chloride currents generate action potentials in solitary cone photoreceptors

Vertebrate rod and cone photoreceptors hyperpolarize when illuminated. However, synaptic input from horizontal cells can depolarize cones and even elicit action potentials. Using the whole-cell tight-seal recording technique, we determined that, in solitary cones isolated from a lizard retina, action potentials can be generated by depolarizing current steps under conditions where only two ionic currents are activated. A dihydropyridine-sensitive, inward Ca2+ current that activates at potentials positive to -40 mV can regeneratively depolarize the cell. Subsequently, a SITS-sensitive, Ca2(+)-dependent outward Cl- current repolarizes the cell. We suggest that these ionic currents may help explain lateral inhibition in the retina.     

Corticotrope and melanotrope cells in cat, fox, rat and human fetus adenohypophysis: studies with induced fluorescence, cytoimmunologic technics and lead hematoxyline]

Formaldehyde-induced fluorescence, acid-catalyzed or not, methods, and immunocytology with anti-ACTH (1-24), anti-ACTH(17-39), ANTI-BETA-LPH immunserums were applied on the same preparations of cat, fox, rat and human foetus pituitaries. The superpositions of results showed that the pars intermedia and pars distalis corticomelano-lipotrophic cells of fox and cat pituitary, those of human foetal pituitary, and the purely corticotrophics cells of the rat pars distalis contained a fluorogenic probably granular compound. Moreover, the granules of the same cell types were electively revealed on our lymphilized material by plombic hematoxylin. Only the anti-beta-LPH and/or anti-beta-MSH fixing celpls exhibited hypercyanophilic, PAS-positive and bleu alcian-positive caracteristics.     

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.     

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.     

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.     

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.     

Cadmium induces changes in corticotropic and prolactin cells in the Podarcis sicula lizard pituitary gland

We analyzed the effect of cadmium on corticotropic (ACTH) and prolactin (PRL) cells in the pituitary gland of the Podarcis sicula (P. sicula) lizard under chronic exposure to this metal. Adult lizards were given CdCl₂ in drinking water at the dose of 10 µg/10 g body mass for 120 days. Light microscopy was performed after histological and immunohistochemical staining, and the effects were followed at regular time intervals up to 120 days post-treatment. We detected substantial variations in the general morphology of the pituitary: unlike the control lizards in which the gland appeared compact, the treated lizards showed a glandular tissue with dilated spaces that were more extensive at 90 and 120 days. PRL and ACTH cells showed an increase in occurrence and immunostaining intensity in treated lizards in comparison with the same cells of control animals. This cellular increase peaked for PRL at 30 days in the rostral, medial and also caudal pars distalis of the gland. ACTH cells appeared to increase markedly after 60 days of treatment in both the pars distalis and the pars intermedia. Again, at 60 days small, isolated ACTH cells were also found in the caudal pars distalis in which these cells were generally absent. However, at 120 days both these cellular types showed an occurrence, distribution and morphology similar to those observed in the control lizards. In lizards, protracted oral exposure to cadmium evidently involves an alteration of the normal morphology of the gland and an inhibitory effect of ACTH and PRL cells, since they increase in occurrence and immunostaining. Yet in time the inhibitory effect of cadmium on ACTH and PRL cells falls back and their occurrence appears similar to that of the control lizard.Key words: cadmium, corticotropic and prolactin cells, lizard.