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.     

Occurrence of both growth hormone- and prolactin-immunoreactive material in the cells of human somatotropic pituitary adenomas containing Mammotropic elements

Summary With the use of immunoperoxidase staining, both growth hormone- and prolactin-immunoreactivity were demonstrated in the cells of 6 pituitary adenomas removed because of frank or suspected acromegaly. By double immunostaining of individual sections or comparison of adjacent immunostained sections, partial to almost complete identity of the cell populations containing growth hormone and prolactin was found in 5 of the tumors.     

Fluorescamine fluorescence detection of growth hormone-producing cells in human pituitary adenomas

Summary Formalin-fixed and paraplast-embedded tissue specimens of human pituitary, thyroid, and pancreas were investigated using fluorescamine fluorescence and immunohistochemical methods. Growth hormone-producing cells present in normal and neoplastic pituitary tissue exhibited fluorescamine fluorescence. The other tissues examined showed no fluorescamine binding.The authors are indebted to Mr. Klaas van der Ham for preparing the mierophotographs.     

Immunohistochemical colocalization of growth hormone (GH) and α subunit in human GH secreting pituitary adenomas

Summary This immunohistochemical study disclosed that 9 of 15 GH secreting pituitary adenomas contained subunit positive cells. These cases also contained PRL positive adenoma cells, but LH was negative. Of these 9 cases, 4 cases showed occasional FSH containing cells, 2 of these also contained a few TSH positive cells. By mirror section technique, variable numbers of adenoma cells were found to contain both GH and subunit. Immunoelectron microscopically, both GH and subunit were localized in secretory granules which suggested their co-release from the tumour cells. The presence of GH and subunit in rough endoplasmic reticulum indicated their active production in the tumour. In the normal adult anterior pituitary gland, about 10% of GH cells contain FSH , and LH subunits and had appearances suggesting the co-production of GH and FSH as well as LH. The colocalization of GH and FSH is considered to be associated with the neoplastic transformation GH cells which possess the intrinsic potentiality of differentiation toward subunit. However, the mechanism for the lack or deficiency of subunits in the neoplastic condition remains to be further investigated.     

Ultrastructure,immunohistochemistry and hormone release of pituitary adenomas in relation to prolactin production

Summary Fifteen cases of pituitary adenoma, 14 of which were associated with hyperprolactinemia, were studied by observation and granule morphometry of electron micrographs, immunohistochemistry and sequential observation of in vitro release with regard to hormone production, storage and secretion. Adenoma cells of 6 cases with marked elevation of plasma prolactin were sparsely granulated, showed characteristic ultrastrucures including the presence of small secretory granules, well developed Golgi and rough membranes, misplaced exocytosis, and positive or negative immunostaining for prolactin. These adenomas also showed vigorous release of the hormone into the circulation and/or culture medium. In vitro studies showed that negative immunostaining of adenoma cells did not preclude the production and secretion of the hormone. One densely granulated adenoma containing cells with numerous lactotroph type granules showed moderate release of prolactin into the circulation. In an acromegalic case associated with both high plasma growth hormone and prolactin, some cells were shown by immunohistochemistry to store both hormones. There were 4 adenomas which could not be shown to produce, store and secrete prolactin by any method available.Abbreviations Used in this Paper ACTH adrenocorticotropic hormone - -MSH -melanocyte stimulating hormone - hGH human growth hormone - hPRL human prolactin - LH luteinizing hormone - FSH follicle stimulating hormone - TSH thyroid stimulating hormone - TRH Thyrotropin-releasing hormone This work was supported in part by Grants-in-Aid for Cancer Research (No. 50-14) and for Specific Diseases (Disorder of Hypothalamic and Pituitary System) from the Ministry of Health and Welfare, and for Cancer Research (No. 401034) from the Ministry of Education, Science and Culture, Japan     

Immunoelectron microscopic observation of prolactin (PRL) in human PRL secreting pituitary adenomas and non-neoplastic pituitary PRL cells

Immunoelectron microscopic localization of prolactin (PRL) was studied in pituitary adenoma cells and in non-neoplastic PRL cells. The adenoma cells showed characteristic concentration of PRL in Golgi region which was ultrastructurally in Golgi saccules and in vesicular RER. In contrast, the non-neoplastic PRL cells showed lamellated RER which contained PRL. These results suggested disproportional production of PRL and its secretion in the adenoma cells.     

Growth hormone and prolactin response to rehydration during exercise: effect of water and carbohydrate solutions

Summary The effect of progressive rehydration with either water or a carbohydrate solution on the plasma growth hormone (GH) and prolactin (PRL) response to exercise was examined together with plasma somatostatin. Five subjects underwent four 3-h experimental sessions at 36°C in which 25-min exercise periods alternated with 5-min rest periods. The sessions were conducted without fluid replacement (DH) or under rehydration with either water or isosmotic carbohydrate solutions AISO (acid) or NISO (neutral). The fluid was given every 10 min after the 1st h of exercise. Plasma GH increased significantly (p < 0.01) under DH after 2 and 3 h of exercise; this increase was prevented by rehydration with water, AISO and NISO. Plasma glucose was significantly higher following AISO and NISO rehydration compared with DH. This possibly influenced the GH response, but there was no difference between plasma glucose levels under DH and water rehydration at any time. The solutions tended to attenuate the increase in heart rate, rectal temperature and plasma cortisol, suggesting that the lack of GH response under rehydration conditions is a result of decreasing physiological stress levels. The GH response could not be explained by plasma somatostatin, which tended to decline in all sessions. Plasma PRL did not increase in any of the sessions, confirming that exercise without rehydration is a more potent stimulator of GH than of PRL. It is concluded that progressive rehydration with water is sufficient to prevent the exercise-induced increase in plasma GH.     

Immunohistochemical demonstration of cytokeratins in endocrine cells of the human pituitary gland and in pituitary,adenomas

Summary Ten non-neoplastic pituitary glands and 22 pituitary adenomas producing different hormones were studied by immunofluorescence microscopy as well as peroxidase-antiperoxidase and biotin-avidin techniques on frozen sections and formalin-fixed, paraffin-embedded material using antibodies to cytokeratin, vimentin, GFAP, neurofilament protein and different pituitary hormones. The endocrine cells in non-neoplastic pituitary glands as well as in most pituitary adenomas were cytokeratin-positive. The cytoplasmic cytokeratin distribution patterns of non-neoplastic and tumor cells were similar and typical of the type of hormone produced: GH-producing normal cells showed a paranuclear condensation of cytokeratin-reactive intermediate filaments; this accumulation was even further accentuated in GH-producing adenomas resulting in fibrous bodies (Kovacs and Horvath 1978) decorated by cytokeratin antibodies. Prolactin-producing cells showed a less intense cytoplasmic cytokeratin-specific staining with focal paranuclear accentuation in non-neoplastic as well as in neoplastic glands. ACTH-producing cells in normal pituitary glands as well as in adenomas exhibited a strong and more uniform cytoplasmic cytokeratin staining. The cytokeratin reactivity in glycoprotein hormone-producing cells of non-neoplastic tissue and adenomas was weak. Vimentin and GFAP reactivity was confined to agranular folliculo-stellate cells. The specific and different distribution patterns of cytokeratins in pituitary cells can, therefore, provide an (indirect) indication to the production of a specific hormone if immunocytochemistry fails to demonstrate hormone production.Dedicated to Prof. Dr. J.H. Holzner on the occasion of his birthday     

Growth hormone (GH) action in early embryogenesis: expression of a GH-response gene in sites of GH production and action

Growth hormone (GH) may act as a local growth factor in early embryonic development, since GH- and GH-receptor (GHR) immunoreactivity is present in all tissues and most cells of embryonic chicks during organogenesis. However, as GHR-immunoreactivity could, alternatively, reflect the presence of GH-binding proteins (GHBPs) rather than authentic receptors linked to signal transduction mechanisms, GHR immunoreactivity may not be indicative of GH target sites. The possibility that GH may act as an autocrine or paracrine factor during embryogenesis was therefore assessed in the present study by determining the presence and cellular localization of mRNA for a GH-responsive gene. The mechanism of GH action involves the induction of a number of specific GH-response genes. In chickens a novel GH-responsive gene (GHRG-1) has been identified as a marker of GH action. In situ hybridization, using a 860 bp probe for GHRG-1 mRNA, demonstrated widespread expression of the GHRG-1 gene in embryonic tissues known to contain GH- and GHR-immunoreactivity (e.g. in the spinal cord, skin, heart, liver, muscle, bone and lung). GHRG-1 mRNA was not, however, present in all cells of each tissue. It was, furthermore, not present in subepithelial cells of the esophagus and bronchus and was lacking in many spinal cord ependyma, which are also known to lack GH immunoreactivity. These results therefore support the possibility that GH acts as an autocrine/paracrine factor during early chick embryogenesis, which was hitherto thought to be a ”growth-without-GH” syndrome. Accepted: 26 September 2001     

Detection of gonadotropin-releasing hormone receptor in normal human pituitary cells and pituitary adenomas using immunohistochemistry

Gonadotropin-releasing hormone (GnRH), which is a well-known regulator of gonadotroph function, has recently been considered to be a paracrine factor involved in the control of somatotroph, lactotroph, and corticotroph cells. GnRH action is initiated by binding to a specific cell surface receptor, the gonadotropin-releasing hormone receptor (GnRHR), which is expressed by follicle-stimulating hormone/luteinizing hormone (FSH/LH) cells. Using in situ hybridization techniques, GnRHR messenger ribonucleic acid (mRNA) has recently been detected in normal human anterior pituitary gland and in various pituitary adenomas, including FSH/LH-cell, growth hormone (GH)-cell, adrenocorticotropic hormone (ACTH)-cell, and null-cell adenomas. However, immunohistochemical studies indicating the specific cell distribution of GnRHR in normal pituitary cells have never been reported. The aim of the present investigation was to evaluate the immunohistochemical expression of GnRHR in different types of normal pituitary cells and related tumors. Using double-label immunohistochemical techniques on formalin-fixed and paraffin-embedded tissues and specific antibodies directed against pituitary hormones and GnRHR, we found GnRHR immunoreactivity not only in FSH/LH cells, but also in GH- and thyroid-stimulating hormone (TSH) cells. GnRHR was detected in FSH/LH-cell, GH-cell, mixed GH- and prolactin (PRL)-cell, and α-subunit (α-SU)/null-cell adenomas. The findings of this study suggest that the interaction between GnRH and GnRHR may play a role in paracrine/autocrine regulation of different types of normal pituitary cells and pituitary adenomas. Received: 24 January 2000 / Accepted: 12 April 2000     

Adenoma of the human pituitary producing growth hormone and thyrotropin

Summary A pituitary adenoma removed by surgery from a 22-year-old man was studied by histology, immunocytology, transmission electron microscopy and immunoelectron microscopy. Clinically, the patient had acromegaly and euthyroidism with elevated blood GH concentrations. Blood TSH and T₄ levels were within the normal range. Histologically, the adenoma was chromophobic and exhibited no PAS, lead hematoxylin, aldehyde thionin or Grimelius silver positivity. By the immunoperoxidase technique GH, -TSH and -subunit but no PRL, ACTH, -endorphin, -FSH or -LH were demonstrated in the adenoma cells. Electron microscopy revealed adenoma cells which were similar to TSH cells and showed no resemblance to GH cells of nontumorous pituitaries or GH-secreting tumors. Immunoelectron microscopy demonstrated GH and -TSH in the secretory granules.It is concluded that pituitary adenomas composed of TSH-like cells may secrete GH, resulting in acromegaly. Production of GH by adenomatous TSH cells cannot be explained on the basis of the one cell- one hormone theory. The question is raised whether bihormonal or multihormonal clones, capable of synthesizing more than one hormone, exist in the human pituitary. These cells are apparently dormant under normal conditions, but in the course of neoplastic transformation may undergo functional dedifferentiation and acquire the ability to produce two or more different hormones.     

Immunocytochemical detection of the neurokinin B receptor (NK3) on melanin-concentrating hormone (MCH) neurons in rat brain

The presence of the neurokinin B receptor (NK3 receptor) in the rat lateral hypothalamus and the zona incerta was previously reported. The aim of the present study was to define its cellular localization in these areas. Investigations, coupling immunocytochemical and in situ hybridization techniques, focussed on two neuron populations: the melanin-concentrating hormone (MCH) neurons and a population of neurons recognized by an ovine prolactin antiserum (PRL-ir neurons). While PRL-ir neurons did not exhibit NK3 immunoreactivity, 57%±6% of MCH neurons were strongly stained by the NK3 antiserum. These results suggest that neurokinin B is involved in the regulation of MCH neuron activity via the NK3 receptor; they provide new bases for further investigations on MCH role in the control of food and water intake.     

GH-, PRL-, POMC-, beta-TSH-, beta-LH-, beta-FSH-mRNA in gonadotroph adenomas of the pituitary by in situ hybridization in comparison with immunostaining and clinical data

In situ hybridization (ISH) enables the visualization of specific mRNA for pituitary hormones. Our collection consists of 40 surgically removed pituitary adenomas that were classified as follicle stimulating hormone/luteinizing hormone (FSH/LH) cell adenomas by structure and by immunostaining (IH) for all pituitary hormones. All forty adenomas were regarded as clinically inactive. The aim of our study was to examine nonfunctioning adenomas by ISH for demonostration of mRNAs for all pituitary hormones. The results were compared with proliferation markers, invasiveness and clinical data. ISH detected signals for all pituitary hormones at a range of 30% for prolactin (PRL) to 85% for proopiomelanocortin (POMC). mRNA for β-FSH was detected in 70% and β-LH mRNA in 43% of adenomas. Thirty-three percent of adenomas revealed negative mRNA detection for β-LH but positive hormone content. The majority of adenomas (75%) expressed more than two mRNAs simultaneously, mostly the combination of POMC mRNA together with β-FSH mRNA and one to four others. Comparison with clinical data showed no significant differences except for one adenoma with a high Ki-67 index (>2.1% positive nuclei). This adenoma showed very high signals for PRL and β-TSH mRNA.     

PTTG基因蛋白的表达与垂体腺瘤侵袭性和增殖能力的关系

目的探讨人垂体腺瘤中垂体肿瘤转化基因(PTTG)蛋白的表达与肿瘤侵袭性和增殖程度的关系。方法采用免疫组织化学染色方法检测手术切除石蜡包埋的63例垂体腺瘤(侵袭组45例,非侵袭组18例)组织中PTTG蛋白的表达,染色增殖细胞核抗原(PCNA),同时计数组织内微血管数量(MVD)。结果 PTTG在侵袭性垂体腺瘤中的表达水平显著高于非侵袭性垂体腺瘤。侵袭性垂体腺瘤中PCNA标记指数和微血管密度也显著高于非侵袭性垂体腺瘤。相关分析显示PTTG表达与垂体腺瘤内PCNA标记指数和微血管密度呈正相关(P<0.05)。结论 PTTG在垂体腺瘤形成过程中起重要作用,并与垂体腺瘤的侵袭性和增殖程度密切相关。     

人血清泌乳素放射受体分析及垂体腺瘤患者血清泌乳素分子形式的研究

本研究利用大鼠肝膜泌乳素受体为特异竞争结合蛋白，在国内首次建立了泌乳素放射受体分析法。该方法对泌乳素测定具有高度特异性，灵敏度为５．０±１．４ｍＩＵ／ｄｌ，批内和批间变异系数分别不超过８．８％和１１．３％，泌乳素的血清回收率为９８．６±５．０％。对垂体腺瘤的研究发现，血清泌乳素至少存在三种不同的分子形式。其表现分子量分别为９８．６ｋＤａ、５０．９ｋＤａ和２４．６ｋＤａ，它们的受体结合活性分别为５．４２ＩＵ／ｍｇ、１１．２２ＩＵ／ｍｇ和３９．６０ＩＵ／ｍｇ。与垂体非泌乳素腺瘤相比，在垂体泌乳素腺瘤患者血清中“巨”泌乳素和“大”泌乳素所占比例较高，而“小”泌乳素显著降低。     

Repeated electroconvulsive shocks cause transient changes in rat hippocampal somatostatin and neuropeptide Y immunoreactivity and mRNA in situ hybridization signals

Increased levels of somatostatin (SS) and neuropeptide Y (NPY) have been demonstrated in the hippocampal formation after kindling. The increase might be specifically associated with kindling, or be an effect of repeated seizures per se. In order to separate these two components we studied the effects of repeated electroconvulsive shocks (ECS) on hippocampal SS-like and NPY-like immunoreactivity and SS mRNA and NPY mRNA in situ hybridization. ECS elicit seizures without having a demonstrable kindling effect. Rats were subjected to 10, 20, or 36 ECS (50 mA, 0.5 s), given as one shock per day, 5 days per week. One, 2 and 30 days after the last ECS, the rats were killed, together with sham-treated control rats, and processed for immunocytochemistry and non-radioactive in situ hybridization. There was a bilateral increase in SS-like and NPY-like immunoreactivity 1 and 2 days after the last ECS in the outer part of the dentate molecular layer. This is the terminal field of the hilar SS-containing and NPY-containing neurons, which displayed both increased immunoreactivity and hybridization signal of the cell bodies. There was also a bilateral de novo expression of NPY-like immunoreactivity in the mossy fiber system, but this was not accompanied by the appearance of a detectable NPY hybridization signal over the parent dentate granule cell bodies. The increase in SS-like immunoreactivity and hybridization signal was most pronounced in the rats that had received the largest number of ECS. This was not observed for the NPY-like immunoreactivity and hybridization signal, where the increase appeared similar after 10, 20 and 36 ECS. One month after the last ECS, both the SS-like and NPY-like immunoreactivity and the in situ hybridization signals had decreased towards normal levels. Since increased SS and NPY levels are also induced by repeated ECS, these changes are accordingly not specific to kindling-induced seizures. In a second experiment, the perforant path to the fascia dentata was transected 1 month prior to the ECS treatment. Removal of such major afferent input did not abolish the ECS-induced increase in hippocampal SS-like and NPY-like immunoreactivity, suggesting that the neuropeptide changes were not caused by afferent stimulation via the perfant path fibers, but rather may be an effect of direct electrical activation of the relevant cells.     

Cyclins D1 and D3 and topoisomerase II alpha in inactive pituitary adenomas

The oncogenes cyclin D1 and D3 are overexpressed in many tumors. Topoisomerase IIα is found in proliferating cells. The immunohistological expression of cyclin D1, cyclin D3, and Topoisomerase IIα was studied in a collection of 60 clinically inactive surgically removed pituitary adenomas of the follicle-stimulating hormone/luteinizing hormone (FSH/LH) cell complex (20 null cell adenomas, 20 oncocytomas, and 20 FSH/LH cell adenomas) for correlation with other proliferation markers (Ki-67, PCNA) and with clinical data. Whereas cyclin D1 was positive only in one invasive null cell adenoma (1.7%) with some p53-positive nuclei, cyclin D3 was overexpressed in the nuclei of 41 tumors (68%). Topoisomerase IIα was demonstrated in the nuclei of 42 adenomas (70%) with no significant differences discernible between the three adenoma subtypes. There was no significant correlation to the time of development of tumor symptoms, but a correlation of Topoisomerase IIα with cyclin D3 and the proliferation marker Ki-67 (Mib1). From these data we conclude that cyclin D3 and Toposomerase IIα appear to be additional markers for proliferation which can be used for prognosis index in surgical pathology of the pituitary.     

Growth factor receptor expression in anal squamous lesions: modifications associated with oncogenic human papillomavirus and human immunodeficiency virus

High prevalence of squamous anal lesions is linked to oncogenic human papillomavirus (HPV). Human immunodeficiency virus (HIV) promotes anal carcinogenesis. Epidermal growth factor receptor (EGFR), HER2/neu, c-Met, and vascular endothelial growth factor receptor-1 (VEGFR1) (tyrosine kinase growth factor receptors) are implicated in tumor progression, but little is known about their role in anal lesions. We investigated their expression and distribution in normal, dysplastic, and carcinomatous anal epithelium and then tried to analyze the effects on these variables of HPV and the HIV-positive status. Seventy-one HIV-positive and 47 HIV-negative patients were selected. We studied growth factor receptors, p16 and Ki67 expression, by in situ hybridization, fluorescent in situ hybridization (FISH) and chromogen in situ hybridization (CISH), immunocytochemistry, and morphological quantification in 226 lesions, either infected by HPV6 and 11 (31 condylomas acuminata) or infected with oncogenic HPVs (48 invasive cancers, 147 anal intraepithelial neoplasias). No HER2/neu was detected. Strong EGFR immunolabeling was not accompanied by gene amplification. The number and intensity of EGFR- and c-Met–immunoreactive cells increased significantly during lesion progression, highlighting the effects of oncogenic HPVs. EGFR, c-Met, VEGFR1, and p16 were coexpressed in 96% of invasive cancers. HIV-modified c-Met expression in condyloma acuminata (P < .008) and invasive cancers (P < .02). Strong HIV-related immunodeficiency and an absence of antiretroviral therapy increased c-Met and/or EGFR expression. HIV-positive anal cancers showed correlated c-Met and VEGFR1 (P < .003), strong p16 labeling, and an increased Ki67 proliferation. The finding that EGFR, c-Met, and VEGFR1 involved in carcinogenesis are well-represented and coexpressed in anal cancers, especially in HIV-positive population, suggests possible novel targeted treatments for anal diseases.