Immunohistochemical localization of carboxypeptidases D, E, and Z in pituitary adenomas and normal human pituitary.

Carboxypeptidases may play important role(s) in prohormone processing in normal and neoplastic adenohypophyseal cells of the pituitary. We have recently demonstrated carboxypeptidase E (CPE) and carboxypeptidase Z (CPZ) in the majority of adenohypophyseal cells with carboxypeptidase D (CPD) immunoreactivity largely confined to adrenocorticotrophs. This study evaluated the expression patterns of CPE, CPD, and CPZ immunoreactivity in 48 pituitary adenomas. Our immunohistochemistry demonstrated extensive intracytoplasmic immunoreactivity for CPE, CPD, and CPZ in adrenocorticotrophic hormone (ACTH)-producing adrenocorticotroph cells, prolactin-producing lactotroph cells, and growth hormone (GH)-producing somatotroph cell adenomas, all of which require carboxypeptide processing of prohormones to produce active endocrine hormones. In contrast to the restricted expression in the normal adenohypophysis, CPD appeared to be widespread in the majority of adenomas, suggesting that CPD levels are increased in adenomas. In luteinizing hormone/follicle-stimulating hormone (LH/FSH)-producing gonadotroph adenomas, which do not require carboxypeptidases to produce gonadotropins, only CPZ immunostaining was demonstrated. In null-cell adenomas, CPE immunoreactivity was detected in the majority of tumors, but CPD and CPZ were identified only in a minority of cases. CPE in these cells may process other peptides critical for pituitary cell function, such as chromogranin A or B. These findings suggest that CPs participate in the functioning of pituitary adenomas.     

Co-existence of gonadotrophins (FSH,LH) and thyrotrophin (TSH) in single anterior pituitary cells of the musk shrew,Suncus murinus

According to recent immunocytochemical studies of anterior pituitary cells, it is obvious that the one cell-one hormone theory must be modified. Many pituitary morphologists have demonstrated that there are some cells that contain two hormones. In this study, we demonstrate by means of immuno-electronmicroscopy the co-existence of gonadotrophins (FSH and LH) and thyrotrophin (TSH) in the same anterior pituitary cells of the musk shrew. These cells were remarkably altered in their ultrastructural features by either gonadectomy or thyroidectomy. Double labeling for gonadotrophins and thyrotrophin was present not only in the same cells but also in the same secretory granules. Our ability to demonstrate co-existence of gonadotrophins and thyrotrophin in the same cell may be due to our selection of fixative and embedding media for electron-microscopic immunocytochemistry. Our conclusion that gonadotrophins and thyrotrophin are produced in a single cell type of the anterior pituitary gland in the musk shrew, i.e., thyrogonadotrophs, suggests the need to consider a modification of the classic scheme for classification of anterior pituitary cells.     

Ultrastructural immunocytochemical localization of prolactin and growth hormone in the porcine pituitary

Summary The immunocytochemical peroxidase-antiperoxidase technique was used to identify prolactin- and growth hormone-producing cells in the porcine pituitary at the ultrastructural level. The growth hormone-producing cells contain round secretory granules (300 nm to 500 nm in diameter). The prolactin-producing cells can be identified by their distinct round and ovoid secretory granules which vary in size. Most of these cells contain large granules (450 nm to 750 nm in diameter), but some prolactin-producing cells display smaller secretory granules (250 nm to 500 nm). The two hormones were localized exclusively in the secretory granules. Staining for prolactin was observed in round and ovoid granules, as well as in small and polymorphic granules within the Golgi complex. This study confirmed (i) that the two hormones are located in different cells, and (ii) that under normal physiological conditions no one cell can synthesize and store both hormones simultaneously.     

Human relaxin increases cyclic AMP levels in cultured anterior pituitary cells

Although relaxin acts at several abdominal sites and mammary tissue associated with pregnancy and parturition, the scope of target tissues and the signals conveying the relaxin message into the cell are poorly defined. We found that human relaxin rapidly elevates the cyclic AMP content of cultured rat anterior pituitary cells. This is a graded response (EC50 0.3 nM relaxin) that can be blocked by anti-relaxin antibodies or the hormones somatostatin and dopamine. Furthermore, other hormones with some sequence homology to relaxin, such as insulin and insulin-like growth factor-I, have no such action. We conclude that the anterior pituitary may be a target tissue for relaxin and that cyclic AMP may act as an intracellular messenger for relaxin in these cells.     

N-cadherin loss in POMC-expressing cells leads to pituitary disorganization

Pituitary tumors are the third most common intracranial tumor in humans and can cause altered hormone secretions leading to hypercortisolism, acromegaly, and infertility. Reduced expression of the cell adhesion molecule N-cadherin has been linked with the formation of pituitary tumors, but its role in normal pituitary gland physiology or tumor initiation is unknown. In the murine pituitary, N-cadherin expression is detected in virtually all cells of the posterior, intermediate, and anterior lobes. N-cadherin may function to initiate important cues such as controlling proliferation, directing cell placement, and promoting formation of cell networks that coordinately release hormones into the bloodstream. To address this, we generated mice lacking N-cadherin in proopiomelanocortin-expressing melanotrope and corticotrope cells of the intermediate and anterior lobes of the pituitary. We observed that intermediate lobe cells can aberrantly displace SOX2-containing progenitor cells in the N-cadherin conditional knockout mice at postnatal d 1. By postnatal d 30, although a reduction in α- and β-catenin membrane staining occurs, there is little effect on intermediate lobe architecture with N-cadherin loss. Also, despite these changes in adherens junction molecules, no alterations in cell proliferation occur. In contrast, loss of N-cadherin in the corticotropes leads to aberrant cell clustering and a reduction in Pomc mRNA. Taken together, our data reveal important roles of N-cadherin in pituitary cell placement and that loss of N-cadherin alone does not lead to pituitary tumor formation.     

Effect of LH-RH, gonadotrophins or sex hormone treatment on RNA and DNA concentration in hypothalamus, pituitary, ovary and uterus in the immature female rat.

RNA and DNA concentration was measured in hypothalamus, pituitary, ovary and uterus of immature female rats after treatment with 5 doses of LH-RH, PMSG, HCG, FSH + LH, estradiol benzoate or progesterone. All assayed hormones decreased DNA concentration and increased the [RNA]/[DNA] ratio in their target organs. These findings were interpreted as increases in cell volume and RNA synthesis in target organs after treatment. Gonadotrophins and sex hormones decreased DNA concentration and increased RNA synthesis in hypothalamus and pituitary, which revealed the stimulatory effect of both hormonal groups on the above mentioned organs.     

Chromogranin A in pituitary adenomas: immunohistochemical detection and plasma concentrations

Forty one pituitary adenomas excised surgically were immunostained to reveal pituitary hormones and chromogranin A (CgA). In 23 patients, plasma CgA concentration was determined before surgery by ELISA method. The CgA immunopositivity was found in 70.7% of investigated tumors. It was observed in all tumors of gonadotropinoma type and in the majority of null cell adenomas. Elevated (>18 U/L) plasma CgA concentration was observed in approx. a half of the examined patients, being more frequent in gonadotropinomas and null cell adenomas. It may have some, although limited, diagnostic value in these types of pituitary tumors.     

Vitamin D sites of action in the pituitary studied by combined autoradiography-immunohistochemistry

Adult male and female mice under normal diet were injected with 3H 1,25(OH)2 vitamin D3 and sacrificed 3.5 h afterwards. Autoradiograms were prepared according to our thaw-mount technique and stained with antibodies to pituitary hormones. Thyrotropes showed strong and extensive nuclear concentration of radioactivity: about 90% of the immunostained thyrotropes were labeled. Lactotropes, somatotropes and gonadotropes showed no or only weak nuclear radioactivity: a subpopulation of 5%-10% of each of these immunostained cell types displayed nuclear labeling that was weak when compared to thyrotropes. Neural lobe pituicytes also showed weak to intermediate nuclear labeling. The results indicate a presence of nuclear receptors for 1,25(OH)2 vitamin D3 in pituitary cell types and suggest direct but differential genomic effects of 1,25(OH)2 vitamin D3 on pituitary hormone secretion. Evidence further suggests the existence of a vitamin D regulated brain-pituitary-thyroid axis.     

Effects of p-chloroamphetamine, a serotonin-depleting drug, on the median eminence and pituitary pars intermedia

p-Chloroamphetamine (PCA), an agent known to cause depletion of levels of brain serotonin in rodents, was administered to rats in three sequential injections (10mg/kg) to study effects on the hypothalamic median eminence and pituitary gland. One week following the initial sequence of injections of PCA, light and electron micrographs revealed degenerate fibers in the outer zone of the median eminence. Lower drug doses or single 10-mg/kg doses did not lead to morphologic changes. Neuronal processes located in the pituitary intermediate lobe appeared normal although there was a significant increase in the numbers of secretory granules contained within intermediate lobe cells drug-treated rats, as compared to controls. Fluorometric analysis of levels of catecholamine and indoleamine showed a decrease in serotonin in median eminence and pons-medulla, but no change in that of the pituitary. Levels of dopamine and norepinephrine remained unchanged after PCA treatment. The data suggest that fibers affected in the median eminence contain serotonin. Processes in the intermediate lobe may be resistant to the serotonin-lowering effects of PCA observed in brain tissue. In addition, PCA may directly affect granule release from pituitary cells, or may alternatively act on hypothalamic regions which affect the release of intermediate lobe cell hormones.     

Making a pituitary gland in a dish

The adenohypophysis secretes multiple hormones that control vital physiological functions. A recent article in Nature (Suga et al., 2011) describes a 3D protocol for the derivation of several endocrine pituitary cell types from mouse ESCs.     

Dexamethasone increases potassium channel messenger RNA and activity in clonal pituitary cells.

Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.     

Specific subclones derived from a multipotential clone of rat anterior pituitary cells.

Several functional subclones of rat anterior pituitary cells were established from our 2A8 clone which apparently contains a heterogenous population of committed and uncommitted cells. On the basis of the hormones secreted into the culture media, as measured by radioimmunoassay, these subclones were divided into four categories, i.e., subclones which secrete (1) ACTH only, (2) prolactin only, (3) prolactin and GH or (4) ACTH, prolactin and GH. None of the subclones produced detectable amounts of thyrotrophic or gonadotrophic hormones. Subclones which secrete a single hormone have shown no change in the type of hormone produced, indicating that these subclones were each derived from a committed cell. The cells of all subclones exhibit a normal diploid karyotype and show good growth characteristics. The cells of the different subclones can be classified by phase contrast microscopy into four categories. However, no clear-cut ultrastructural features have been observed which can be correlated with the different categories of subclones. On the basis of the results a hypothesis is proposed relative to the functional cytodifferentiation of anterior pituitary cells.     

Immunohistochemical identification of cells in the pars distalis of the pituitary of the lizard Anolis carolinensis

Immunocharacteristics of the pars distalis cells of the pituitary of the male lizard A. carolinensis are determined by employing the immunoperoxidase technique with antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, 5 different cell types with characteristic anatomical distribution are recognized. ACTH cells are found in the rostral half of the pars distalis, and PRL cells in the rostral two thirds of the pars distalis. GH and TSH cells are located in the caudal half of the pars distalis. GTH cells are distributed throughout the gland. When consecutive sections are stained with antiserum to ovine FSH or its beta-subunit and to ovine LH, the same cells show immunoreactivity to all the three antisera. None of the GTH cells show positive immunoreactivity to ovine beta-LH antiserum. The results suggest the existence of one gonadotropic cell type in the pituitary of this lizard.     

Combined expression of different hormone genes in single cells of normal rat and mouse pituitary

Cells displaying combined expression of different pituitary hormone genes (further referred to as 'multi-hormone mRNA cells') were identified in normal rat and mouse pituitary by single cell RT-PCR. These cells do not seem to produce or store all the respective hormones the mRNAs encode for. The cells are already developed at day 16 of embryonic life (E16) in the mouse. Different peptides, such as gamma3-melanocyte-stimulating hormone (gamma3-MSH) and gonadotropin-releasing hormone (GnRH), affect different subsets of these cells. In culture, estrogen and GnRH increase the number of 'multi-hormone mRNA cells' that contain prolactin (PRL) mRNA or mRNA of the alpha-subunit of the glycoprotein hormones (alpha-GSU) but not the number of 'multi-hormone mRNA cells' not containing PRL or alpha-GSU mRNA. 'Multi-hormone mRNA cells' may function as 'reserve cells' in which a particular hormone mRNA may be translated under a particular physiological condition demanding a rapid increase of that hormone.     

Vitellogenin hormonal control in the green frog, Rana esculenta. Interplay between estradiol and pituitary hormones

1. The effect of estradiol and pituitary hormones on the titre of serum vitellogenin has been studied in Rana esculenta by rocket immunoelectrophoresis. 2. Hepatic synthesis of vitellogenin depends on physiological doses of estradiol. 3. Gonadotrophins enhance the uptake, presumably by acting directly on the oocyte plasma membrane. 4. In addition, our data support direct pituitary intervention on liver synthesis and/or release of vitellogenin. 5. Hormonal response, as evaluated by vitellogenin serum titres, tends to increase from November to July. This could be the expression of a modification, throughout the sexual cycle, of liver sensitivity to the hormones.     

Genetics of pituitary tumors: Focus on G-protein mutations

In recent years the demonstration that human pituitary adenomas are monoclonal in origin has provided further evidence that pituitary neoplasia arise from the replication of a single mutated cell in which growth advantage results from either activation of proto-oncogenes or inactivation of tumor suppressor genes. While common oncogenes, such as Ras, are only exceptionally involved, the only mutations identified in a significant proportion of pituitary tumors, and particular in GH-secreting adenomas, occur in the Gsalpha gene (GNAS1) and cause constitutive activation of the cAMP pathway (gsp oncogene). Moreover, pituitary tumors overexpress hypothalamic releasing hormones, growth factors, and their receptors as well as cyclins involved in cell cycle progression. As far as the role of tumor suppressor genes in pituitary tumorigenesis is concerned, reduced expression of these genes seems to frequently occur in pituitary tumors as a consequence of abnormal methylation processes. Although the only mutational change so far identified in pituitary tumors is the gsp oncogene, this oncogene is not associated with a clear phenotype in patients bearing positive tumors. Mechanisms able to counteract the cAMP pathway, such as high sensitivity to somatostatin, and induction of genes with opposite actions, such as phosphodiesterases, CREB end ICER, or instability of mutant Gsalpha, have been proposed to account for the lack of genotype/phenotype relationships.     

Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas.

To investigate the constituents of the matrix of endocrine secretory granules, we analyzed endocrinoilogically silent ("non-functioning") human pituitary adenomas for the occurrence of the chromogranins/secretogranins (granins), a protein family normally stored together with many different hormones. When five non-functioning pituitary adenomas were analyzed by immunoblotting using polyclonal and monoclonal antibodies specific for individual members of the granin family, chromogranin A was detected in four cases and chromogranin B and secretogranin II were detected in all cases. The cellular distribution of the granins and of various hormones known to be expressed in the anterior pituitary was studied by immunocytochemistry in fixed, frozen tissue sections from five additional adenomas. Of the eight hormones investigated, only thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone were detected, occurring in only two of the five adenomas. In contrast, granins were found in all five tumors. Chromogranin B and secretogranin II were detected in each of the adenomas in virtually every cell studied, whereas chromogranin A exhibited such a widespread cell distribution in only three adenomas, being focally present in one and absent from the other tumor. The subcellular localization of the granins and the three glycoprotein hormones was investigated by double immunoelectron microscopy. Chromogranin A and chromogranin B were mainly co-localized in secretory granules, whereas secretogranin II was either co-localized with the other two granins or segregated to different secretory granules. When present, glycoprotein hormones were immunodetected in both the secretory granules containing all three granins and those containing mainly secretogranin II. Our data indicate that in non-functioning pituitary adenomas chromogranin A is differentially expressed from chromogranin B and secretogranin II. Moreover, the granins appear to be the most widespread constituents of endocrine secretory granules known, forming the dense-core matrix irrespective of the presence or absence of hormones.