In situ hybridization study of pro-opiomelanocortin (POMC) gene expression in human pituitary corticotrophs and their adenomas

Summary Pro-opiomelanocortin (POMC) mRNA was detected on paraffin sections by in situ hybridization (ISH) in corticotrophs of 12 nontumorous pituitaries, 11 functioning corticotroph, and 11 silent pituitary adenomas. ISH combined with immunocytochemistry for adrenocorticotrophic hormone (ACTH), a POMC-derived peptide, was also performed. ACTH immunoreactive cells of the anterior lobes and those invading the posterior lobe showed a high or moderate level of POMC mRNA that was not correlated with the intensity of ACTH immunoreactivity. Variable levels of POMC gene expression were present in Crooke's cells, corticotrophs suppressed by glucocorticoid excess. Most functioning corticotroph adenomas and silent subtype 1 adenomas had an intense hybridization signal and ACTH immunoreactivity. In silent subtype 2 and 3 adenomas, POMC mRNA had a diffuse low level or was absent; in these adenomas ACTH immunoreactivity was diffuse, restricted to some cells, or negative. The results indicate that POMC gene is expressed in both normal and suppressed nontumorous corticotrophs. Intense signals for POMC mRNA are found in most functioning corticotroph adenomas. The difference between POMC gene expression in silent 1 and silent 2 and 3 adenomas suggests that different mechanisms are responsible for the lack of endocrine activity.     

Corticotroph (Basophil) invasion of the pars nervosa in the human pituitary: Localization of proopiomelanocortin peptides,galanin and peptidylglycine α-amidating monooxygenase-like immunoreactivities

Corticotroph (basophil) invasion or the migration of corticotroph cells into the pars nervosa of the human pituitary gland was found in 35 of 767 (4.4%) consecutive pituitaries obtained at autopsy. The degree of invasion increased with patient age and extensive invasion was more common in men than in women. Immunoreactive ACTH, β-MSH, α-MSH, and galanin were detected both in the anterior lobe and invading corticotroph cells in approximately equal frequency. Fewer cells stained positively for α-MSH than for the three other peptides in both the anterior lobe and invading corticotrophs. Twelve corticotropic pituitary adenomas obtained surgically from patients with Cushing’s disease were also examined and expressed varying degrees of immunoreactivity for ACTH, α MSH, β-MSH and galanin. Staining for all major pituitary hormones revealed only ACTH in the invading basophil cells. Peptidylglycine α-amidating monooxygenase (PAM) was present in the anterior pituitary, in invading corticotroph cells, and in some cells lining the cysts of the pars intermedia zone. PAM immunoreactivity was also detected in 4/12 corticotroph adenomas. These results indicate that corticotroph cells invading the pars nervosa are immunohistochemically similar to anterior lobe corticotrophs and have the ability to amidate various peptides such as proopiomelanocortin cleavage products and galanin with PAM.     

Immunohistochemical and histochemical studies of pituitary β-lipotrophs

The presence of β-lipotrophin or an immunologically similar substance was demonstrated in the intermediate lobe cells and some basophils of the anterior lobe by both fluorescent and peroxidase-conjugated antibody methods. These basophils were found to be corticotrophs, i.e., reactive with anti-ACTH. ACTH, when added to anti-β-LPH, decreased the fluorescence and peroxidase reactions of intermediate lobe cells and the anterior lobe corticotrophs. The addition of MSH to anti-β-LPH produced a slight decrease in the reaction between the intermediate lobe cells and anti-β-LPH. The intermediate lobe cells also reacted with anti-ACTH. Both Somatotrophs and lactotrophs reacted with anti-β-LPH to give positive fluorescence or peroxidase reactions. However, the addition of STH to anti-β-LPH abolished the reaction between anti-β-LPH and somatotrophs. Similarly, LTH, when added to anti-β-LPH, abolished the positive reaction of lactotrophs to anti-β-LPH. When STH, LTH and ACTH were added to anti-β-LPH, the fluorescence of the somatotrophs and lactotrophs was abolished, whereas the corticotrophs maintained their fluorescence at a reduced intensity. These results indicate that the somatotrophs and lactotrophs do not contain β-LPH and that corticotrophs contain β-LPH.     

Corticotropin-releasing factor and forskolin increase proopiomelanocortin messenger RNA levels in rat anterior and intermediate cells in vitro

The effect of ovine corticotropin-releasing factor (CRF) on messenger ribonucleic acid (mRNA) level encoding proopiomelanocortin (POMC) was studied in serum-free primary cultures of intermediate (IL) and anterior lobe (AL) cells of rat pituitary. Levels of POMC mRNA were quantitated by hybridization to a 32P-labeled, single-stranded POMC complementary deoxyribonucleic acid (cDNA) probe. This effect was time dependent and after 48 h of treatment, POMC mRNA levels in IL cells and AL corticotrophs were increased by 116 +/- 9% and 118 +/- 2% of control values, respectively. Forskolin (1 microM) induced a similar increase in POMC mRNA in both pituitary cell types. These data suggest that CRF might stimulate the gene expression of POMC in pituitary melanotrope and corticotrope cells. Moreover, our findings are consistent with the role of cAMP as a second messenger for CRF in IL and AL corticotrophic cells.     

A study of intermediate lobe differentiation in the human pituitary gland

The immunohistochemical demonstration of neurofilament (NF) polypeptide was used to identify nerves in a series of 17 pituitary adenomas. NF-positive fibres were present in two out of five corticotroph adenomas sited deep in the anterior lobe, in one out of five sited in the intermediate zone and in two out of seven non-corticotroph adenomas. Such nerve fibres were often seen in relation to blood vessels. The distribution of alpha-MSH immunoreactive cells was examined in 25 normal pituitaries and in 23 cases of Cushing's disease. Such cells were scattered throughout the normal gland and there was no increase in numbers in pregnancy. alpha-MSH was demonstrated in 18 corticotroph adenomas in Cushing's disease. There was no correlation with the site of the tumour or the presence of nerve fibres. alpha-MSH cells were distributed normally in the para-adenomatous gland. Crooke's hyaline change and alpha-MSH coexisted in some corticotrophs. These findings support the concept that 'intermediate lobe' function, as found in animals, has no discrete anatomical location in man.     

Pulmonary cryptococcosis and pituitary Cushing's disease

Pulmonary cryptococcosis was diagnosed by examining smears obtained by fine-needle aspiration (FNA) in a patient with pituitary Cushing's disease. FNA allowed for rapid diagnosis and prompt treatment of a potentially serious infection. The patient fully recovered from her pulmonary disease. Although opportunistic infections may occur in patients with endogenous Cushing's syndrome, it is rare to see such infections in the subset of patients with pituitary Cushing's disease. Hypercortisolism associated with Cushing's syndrome appears to induce a transitory immune deficiency state and opens a window of opportunity for certain infectious agents such as Cryptococcus neoformans to exploit. To our knowledge, this is the third such case reported in this clinical setting, and the first diagnosed by FNA. Diagn. Cytopathol. 1998;18:365–367. © 1998 Wiley-Liss, Inc.     

Cushing's syndrome and nocardiosis associated with a pulmonary carcinoid tumor: Report of a case and review of the literature

Ectopic hormone production is an uncommon complication of neoplastic lung disease. Rarely, patients may present with signs and symptoms of systemic endocrine dysfunction related to a hormone‐secreting tumor. Bronchopulmonary carcinoids are the most common neoplasm implicated in ectopic ACTH‐dependent Cushing's syndrome. Persistent hypercortisolism, such as that which occurs in Cushing's syndrome, causes immunosuppression and makes patients vulnerable to opportunistic infections. We present a case of a 42‐year‐old woman diagnosed with ACTH‐dependent Cushing's syndrome which was originally thought to stem from a pituitary lesion as interpreted on magnetic resonance imaging. Her symptoms persisted after undergoing hypophysectomy, and further work‐up involving a fine needle aspiration of the left lung revealed an ACTH‐producing carcinoid tumor. Before treatment could be administered, the patient developed several new suspicious nodules in the left lung that were shown by fine needle aspiration to be infectious in nature. A Gram stain revealed numerous Gram positive branching organisms, and culture of the specimen grew Nocardia asteroides. Her pulmonary infection was treated with antibiotics and she underwent successful ablation of the carcinoid tumor. Diagn. Cytopathol. 2011. © 2010 Wiley‐Liss, Inc.     

Multiple pigmented adrenal cortical nodules associated with cushing's syndrome

Multiple pigmented adrenocortical nodules were found in a 25?year-old woman associated with Cushing's syndrome, whose laboratory data indicated that the adrenal cortex had been functioning autonomously and adrenocorticotropic hormone (ACTH) from the pituitary gland as suppressed. The surgically removed left adrenal gland disclosed multiple black nodules measuring up to 3 mm in diameter and histologically consisting of large “compact cells” which contained numerous yellow-brown pigments, but adjacent cortical cells were not atrophied. This kind of adrenal lesion is generally regarded as nodular hyperplasia of the cortex. The present case revealed scanty lipid and markedly increased activity of 3β-hydroxysteroid dehydrogenase (3β-HSD) and glucose?6?phosphate-dehydrogenase (G6PD). Ultrastructural study showed abundant cytoplasm with a large number of mitochondria, well-developed smooth-surfaced endoplasmic reticulum (SER), less rough-surfaced endoplasmic reticulum (RER), lysosomes, and numerous granules in cells of the nodules. Mitochondria varied in size and shape up to occasional giant mitochondria. SER was vesicular or tubular forming a network of anastomosing tubules. Granules varied greatly in size from 400 mm? to 6 m? in diameter, with diverse electron densities, mostly exhibiting the structural features of lipofuscin. The ultrastructural features resembled those in black adenoma associated with Cushing's syndrome ever reported. Concentration of cortisol was increased in the tissue where numerous black nodules were contained. Acta pathol. jpn. 34: 827 ～ 837, 1984.     

分泌颗粒素Ⅱ和促肾上腺皮质激素在脑损伤大鼠垂体腺细胞共存(英文)

采用荧光双标记技术研究了脑损伤时大鼠垂体前叶促肾上腺皮质激素和分泌颗粒素 的定位。垂体前叶细胞呈现分泌颗粒素 染色强阳性 ,阳性物质位于胞浆中。免疫荧光双标记技术用于鉴定分泌颗粒素 和促肾上腺皮质激素共存的细胞 ,共聚焦显微镜观察显示促肾上腺皮质激素和分泌颗粒素 共存于同一细胞。结果表明 :分泌颗粒素 可在大鼠垂体前叶促肾上腺皮质激素阳性细胞表达 ,其生理作用可能是调节分泌泡的 p H值以利于促肾上腺皮质激素从其前体裂解。     

Immunocytological study of the differentiation of chick and quail adenohypophysis epithelial rudiments,grafted or cultivated in vitro: Evidence for polypeptidic hormones

Epithelial rudiments of adenohypohysis were removed from chick and quail embryos between days 3 and 5 of development. Chick rudiments were grafted for 11–13 days onto the chorioallantoic membrane of decapitated chick embryo hosts. Quail rudiments were cultivated in vitro for 6 days. Both grafted and cultivated Rathke's pouches differentiated into adenohypophyseal tissue. The adenohypophyseal tissue cultured on chorio-allantoic membrane exhibited cells reacting with the following immune sera: anti-β-(1–24)ACTH, anti-α-(17–39)-ACTH, anti-α-endorphin, anti-β-endorphin and anti-β-LPH, which also gave a positive reaction when applied to adenohypophysis of corresponding age which had differentiated in situ. In situ, corticotrophs were located exclusively in the cephalic lobe of adenohypophysis. Therefore, the differentiation of corticotrophs in the whole graft, i.e., from both cephalic and caudal lobes of Rathke's pouch, showed that the cells of the caudal lobe, or at least some of them, were uncommitted when the rudiment was removed. In vitro, tissue derived from Rathke's pouch contained cells reacting with antibodies to β-(1–24)-ACTH, α-(17–39)-ACTH, and β-LPH, as did adenohypophysis from quail embryos of corresponding age (9–10 days), differentiated in situ. The differentiation of quail Rathke's pouch in vitro corroborates that differentiation can occur without influence from hypothalamus and, moreover, shows that at least some kinds of cells can differentiate without influence exerted by any other encephalic factors, and in the absence of mesenchyme. The question arises whether fibroblastic cells derived from Rathke's pouch cells act as feeder-cells and/or secrete some factors promoting differentiation.     

Organisation of basement membrane components in the human adult and fetal pituitary gland and in pituitary adenomas

 Cell–matrix interactions undoubtedly have a role in the development and maintenance of the complex nonrandom structure of the human pituitary gland. We have extended previous studies by documenting the patterns of immunoreactivity for type IV collagen, laminin and fibronectin in the fetal gland, comparing these with the adult patterns. In both we have examined the differences between the anterior lobe and intermediate zone in an attempt to elucidate the apparent differences in functional response between corticotrophs in the two areas. We have also examined expression of these proteins in a series of pituitary adenomas. Finally, we have immunolocalised β₄ integrin, a component of the α₆β₄ laminin receptor, in the adult gland and in adenomas. In the anterior lobe of the adult gland, type IV collagen and laminin were present in both epithelial and vascular basement membrane. Fibronectin was related to the basement membrane but showed a less continuous distribution. β₄ Integrin was expressed on the basal aspects of pituitary cells, in association with laminin, suggesting that this did identify the α₆β₄ laminin receptor. In addition, immunoreactivity was present on the lateral margins of some pituitary cells, which might indicate a role in cell–cell adhesion. None of the proteins showed specific association with any particular cell type, suggesting that these specific interactions do not regulate differentiation. This pattern of expression had developed in the fetal gland by the second trimester, with expression relating to vessels preceding that in epithelial basement membrane. Type IV collagen, laminin and fibronectin were also expressed in epithelial and vascular basement membrane in the intermediate zone of the adult gland, and around Rathke’s cleft in the fetal gland. However, the organisation differed, with larger groups of cells enclosed within a single basement membrane. Possible vascular connections demonstrated between the posterior lobe and the intermediate zone would permit access of posterior lobe hormones to this zone. Our data confirmed disruption of expression in pituitary adenomas, type IV collagen, laminin and β₄ integrin having a mainly perivascular distribution, with more variable immunoreactivity for fibronectin. Received: 17 February 1997 / Accepted: 17 May 1997     

An unusual primary biphasic neuroendocrine carcinoma of lung associated with ectopic ACTH production and Cushing's syndrome

A 62-year old female presented with signs and symptoms of Cushing's syndrome. The source of the ectopic ACTH was a left lung lower lobe neuroendocrine tumour composed of two cell types: epithelioid and spindle shaped cells and; a second component of smaller, uniform round neuroendocrine cells. The tumour did not conform to any of the recognized lung neuroendocrine categories and an atypical carcinoid was the best fit based on mitotic count (6 per 10 high power field).This case highlights unusual histology for a pulmonary neuroendocrine tumour and also the nomenclatural difficulties associated with the current classification.     

Presence of neurophysins in the human pituitary corticotrophs, Cushing''s adenomas, and growth hormone-producing adenomas detected by immunohistochemical study.

Neurophysins have been recognized as the carrier proteins of vasopressin and oxytocin. The distribution of neurophysins is immunohistochemically confirmed in the hypothalamus, median eminence, and posterior lobe of the pituitary gland. The authors detected neurophysins in the human corticotrophs and pituitary adenomas with the use of the immunohistochemical method with antiserum to human neurophysins, which did not cross-react with adrenocorticotropic hormone (ACTH), beta-endorphin, and corticotropin-releasing factor. All of ten pituitary glands obtained by autopsy revealed the presence of neurophysin-positive cells in the anterior, intermediate, and the posterior lobes. The neurophysin-positive cells were similar to the corticotrophs in shape and distribution. Simultaneous staining for ACTH and neurophysins in the serial sections revealed that neurophysin-positive cells were also ACTH-positive. One hundred twenty-four cases of pituitary adenoma operated upon were investigated. All of 7 Cushing's adenomas were composed of neurophysin-positive cells. Six tumors with giantism showed sparsely distributed neurophysin-positive cells. No neurophysin-positive cells were observed in any other cases. This study is the first reported evidence of the presence of neurophysins in the human corticotrophs and pituitary adenomas.     

Lack of neuronal NOS has consequences for the expression of POMC and POMC-derived peptides in the mouse pituitary

The relevance of NO in neuroendocrine signalling has been investigated by analysis of cellular expression of pro-opiomelanocortin (POMC) and the POMC-derived peptides beta-endorphin, alpha-melanocyte stimulating hormone and adrenocorticotropin. Expression patterns were studied in the pituitary gland of 150-day old wild-type and neuronal-NOS (nNOS) knock-out mice by using immunohistochemistry, in situ hybridization and Northern blot analysis. Remaining NO-generating capacities in the knock-out mice were demonstrated by immunohistochemical localization of inducible, endothelial and neuronal NOS isoforms. Quantitative analysis revealed that cellular expression of POMC mRNA was drastically reduced in the pituitary of knock-out mice in comparison to controls. In situ hybridization studies demonstrated that this reduction was most pronounced in the intermediate lobe, while the anterior lobe was much less affected. Immunostaining for the proteolytic fragments of POMC was significantly reduced in the intermediate lobe cells of knock-out mice. A moderate reduction of immunostaining for these peptides was also observed in adenopituitary cells of nNOS knock-out mice. Our data demonstrate that the lack of nNOS substantially affects cellular levels of pituitary opioid peptides, which may have consequences for the response of these animals to stress and pain.     

Detection of the messenger RNAs coding for the opioid peptide precursors in pituitary and adrenal by "in situ' hybridization: study in several mammal species

The messenger RNAs coding for opioid peptide precursors have been detected and mapped in histological sections by "in situ' hybridization using specific DNA probes labelled with 32P. Using bovine preproenkephalin A (PPA) cDNA, PPA mRNA was detected in adrenal medulla of bull, hamster and guinea pig. No signal was detected in adrenal of man, rat and cat. The pro-opiomelanocortin (POMC) mRNA was detected in pituitary of man, bull, cat, rat and pig, in all cells of the intermediate lobe as well as in scattered cells of the anterior lobe producing POMC. Adequate controls demonstrated the specificity of the labelling. These results provide evidence of the expression of the gene coding for PPA in the adrenal and for POMC in the pituitary. They show cross-hybridization of one DNA probe with mRNAs of various mammals and then provide evidence that one single probe can be used to analyze expression of a given gene in tissues of several animal species by "in situ' hybridization.     

Glucocorticoid receptor expression in nontumorous human pituitaries and pituitary adenomas

Glucocorticoids have multiple actions, including a suppressive feedback effect on pituitary corticotrophs via the glucocorticoid receptor (GR). By immunocytochemistry, we studied GR expression in 86 surgically removed various pituitary adenoma types. Ten cases contained nontumorous pituitary fragments, which were suitable for immunocytochemical investigation. In addition, 30 autopsy-obtained pituitaries, 10 of them containing incidental microadenomas, were examined as well. Using a polyclonal GR antibody, the streptavidin-biotin-peroxidase complex method revealed nuclear and/or cytoplasmic GR immunoreactivity in many nontumorous corticotrophs and other adenohypophysial cell types and in S-100 protein immunopositive stellate cells. Cellular localization was confirmed by double immunostaining. Pars intermedia corticotrophs, posterior lobe axons, Herring bodies, and pituicytes as well as several endothelial cells lining the capillaries were also immunopositive. GR immunoreactivity was also demonstrated in many GH, PRL, ACTH, TSH, FSH, LH α-subunit producing adenomas, null cell adenomas, and oncocytomas. The extent and degree of immunostaining varied considerably from case to case. Suppressed corticotrophs showing the Crooke’s hyaline change due to glucocorticoid excess were present in the nontumorous pituitaries of patients with Cushing’s disease and in those treated with pharmacologic doses of glucocorticoids. Many suppressed nontumorous corticotrophs exhibited only weak or no GR immunopositivity, indicating GR downregulation accompanied by cellular injury. Study of autopsy obtained pituitaries for GR yielded inconclusive results indicating that autopsy obtained adenohypophyses are not suitable for the immunocytochemical investigation of GR.     

AN AUTOPSY CASE OF ISLET-CELL CARCINOMA WITH CUSHING'S SYNDROME*

A case of islet-cell carcinoma with Cushing's syndrome observed in a 9y-ear-old Japanese girl who expired 8 months after the onset of symptoms was presented. ACTH activity was demonstrated in the tumor by means of biological and immunological methods. Autopsy findings were consistent with the Cushing's syndrome except for a peculiar type of hyaline change of the pituitary cell, which was briefly discussed from the view point of corticotrophogenesis. Cases of islet-cell tumor associated with Cushing's syndrome so far reported in the world literature were reviewed. ACTA PATH. JAP. 18: 333–343, 1968.     

Detection of messenger RNA using a digoxigenin end labelled oligodeoxynucleotide probe.

A synthetic oligodeoxynucleotide sequence complementary to the mRNA for the adrenocorticotrophin (ACTH) precursor pro-opiomelanocortin (POMC) was end labelled using digoxigenin. The probe was used to detect POMC mRNA both on nitrocellulose filters and by non-isotopic in situ hybridisation (NISH) in tissue sections. Digoxigenin was identified using anti-digoxigenin alkaline phosphatase. The model system examined was the rat pituitary gland. Removal of both adrenal glands and dexamethasone administration were used to change the concentrations of POMC mRNA in the rat anterior lobe. The labelled probe reacted with a single band of appropriate molecular weight in Northern blot analysis. The distribution of signal in tissue sections and the changes induced by experimental manipulation were as predicted. The results indicate that this method of NISH will prove useful in the detection of specific messenger RNAs in tissue sections of buffered, formalin fixed, paraffin wax embedded material.