The Pituitary in Isolated ACTH Deficiency: A Histologic,Immunocytochemical, and In Situ Hybridization Study

A 74-year-old man presented in a near terminal state with progressive generalized muscular weakness, gastrointestinal disturbances, and lethargy. Investigations revealed hypotension, hyponatremia, hypoglycemia, and low plasma cortisol concentration accompanied by undetectable plasma adrenocorticotropic hormone (ACTH) level. The patient died shortly after admission to hospital, with adrenocortical failure being the provisional cause of death. Autopsy disclosed profound bilateral atrophy of adrenal cortices with evidence of a mild focal inflammatory reaction. The pituitary gland appeared normal on both gross and histologic examinations. There was no histologic evidence of inflammation, fibrosis, or adenohypophysial cell hyperplasia. By immunocytochemistry, no ACTH and β-endorphin immunoreactive cells were identified in the adenohypophysis.In situ hybridization (ISH) for pro-opiomelanocortin (POMC) mRNA yielded conclusively negative results. The case presented here was regarded as isolated ACTH deficiency. Although the remaining pituitary functions were not assessed, clinical and morphologic findings strongly support the supposition that aside from ACTH deficiency, secretory function of other pituitary hormones was preserved. This is the first case in which the pituitary was studied by immunocytochemistry and ISH. The possible pathogenetic mechanisms accounting for the isolated ACTH deficiency are discussed.     

Amyloid Deposits in Pituitaries and Pituitary Adenomas: Immunohistochemistry and In Situ Hybridization

The patterns of deposition and immunoreactivity of interstitial amyloid were studied in 11 pituitary glands obtained at autopsy and 9 surgically resected pituitary adenomas using Congo red staining and a panel of antisera directed against 5 major amyloid fibril proteins and all pituitary hormones. The deposition pattern of amyloid in pituitary glands differed from that in adenomas but all amyloid deposits showed an immunostaining with anti-amyloid λ-light chain. The remaining antisera were immunonegative.In situ hybridization using an oligodeoxyribonucleotide-probe complementary to the mRNA coding for the constant region of human λ-light chain yielded no hybridization signals in the pituitaries or pituitary adenomas, excluding local synthesis and secretion of immunoglobulins. Since no case studied suffered from generalized Aλ-amyloidosis and adsorption of immunoglobulins to the unknown amyloid fribril protein of the pituitary seems to be unlikely, crossreaction of the polyclonal antisera with an undefined antigen is probable. The similar immunostaining properties of amyloid deposits in “normal” pituitaries and pituitary adenomas suggest they both originate from the same precursor protein.     

In Situ Hybridization Method Reveals (Pro)renin Receptor Expressing Cells in the Pituitary Gland of Rats: Correlation with Anterior Pituitary Hormones

Expression of (pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, was studied in rat pituitary gland. In situ hybridization showed that cells expressing (P)RR mRNA were widely distributed in the anterior lobe and intermediate lobe of the pituitary gland. Double-staining using in situ hybridization for (P)RR mRNA and immunohistochemistry for the pituitary hormones showed that (P)RR mRNA was expressed in most of the GH cells and ACTH cells in the anterior lobe. (P)RR mRNA was also expressed in a few prolactin cells and TSH cells, but not in LH cells. The present study has shown for the first time the distribution of (P)RR mRNA expressing cells in the rat pituitary gland. These findings suggest that (P)RR plays physiological roles in the pituitary gland, such as the modulation of the pituitary hormone secretion.     

CARD In Situ Hybridization: Sights and Signals

During the last decade, several strategies have been developed to improve the detection sensitivity ofin situ hybridization (ISH) by amplification of either target nucleic acid sequences prior to ISH (e.g.,in situ PCR), or the detection signals after the hybridization procedures (signal amplification). Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, summarize applications as well as possible limitations of CARD ISH, and discuss some future directions ofin situ nucleic acid detection using this amplification strategy.     

Electron Microscopic and Confocal Laser Scanning Microscopic Observation of Subcellular Organelles and Pituitary Hormone mRNA: Application of Ultrastructural In Situ Hybridization and Immunohistochemistry to the Pathophysiological Studies of Pituitary Cells

Nonradioisotopic electron microscopic (EM)in situ hybridization (ISH) (EM-ISH) with biotinylated oligonucleotide probes is utilized for the ultrastructural visualization of pituitary hormone mRNA in rat pituitary cells. EM-ISH is an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cell using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex can provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein. Another focus of this review is the utilization of a recently developed imaging system of confocal laser scanning microscopy (CLSM). The combination of CLSM and image analysis system (IAS) enables us to visualize an individual dimensional image of the intracellular distribution of mRNA and subcellular organelles successfully at any optional cross sections of light microscopic ISH studies, and can be another useful tool for the ultrastructural ISH study of mRNA.     

Molecular Pathology of the Pituitary Adenomas

Molecular techniques have been applied to the study of pituitary adenomas by many investigators. We have used nonisotopicin situ hybridization (ISH) to analyze pituitary adenomas in our laboratory. These studies have provided insight into mRNA production by various adenomas, and have contributed toward our new proposed clinical and cytofunctional classification of pitutiary adenomas. Presented in part at the Molecular Endocrine Pathology Symposium at the International Academy of Pathology XXI International Congress, Budapest, Hungary, October 20–25, 1996.     

Automated Molecular Pathology : One Hour In Situ DNA Hybridization

Abstract

We describe a rapid automated technique for colorimetric in situ DNA hybridization using the Code-On Immuno-DNA Slide Stainer. We report recent advances in positively charged glass slide technology, rapid limolene/xylene dewaxing, stable ribonuclease and pepsin predigestion, heat cycling during hybridization, and alkaline phosphatase enhancement. These amplify the signal to noise ratio of colorimetric molecular hybridization to the point where complete one hour assays can be performed from dewaxing to counterstaining by automation. Both biotin labeled oligonucleotide and nick translated DNA probes can localize their viral targets in situ with this single, mechanized protocol at the rate of one slide per minute. (The J Histotechnol 14:219, 1991)     

Changes in Laminin Chain Expression in Pre- and Postnatal Rat Pituitary Gland

Cell–matrix interaction is required for tissue development. Laminin, a major constituent of the basement membrane, is important for structural support and as a ligand in tissue development. Laminin has 19 isoforms, which are determined by combinational assembly of five α, three β, and three γ chains (eg, laminin 121 is α1, β2, and γ1). However, no report has identified the laminin isoforms expressed during pituitary development. We used in situ hybridization to investigate all laminin chains expressed during rat anterior pituitary development. The α5 chain was expressed during early pituitary development (embryonic day 12.5–15.5). Expression of α1 and α4 chains was noted in vasculature cells at embryonic day 19.5, but later diminished. The α1 chain was re-expressed in parenchymal cells of anterior lobe from postnatal day 10 (P10), while the α4 chain was present in vasculature cells from P30. The α2 and α3 chains were transiently expressed in vasculature cells and anterior lobe, respectively, only at P30. Widespread distribution of β and γ chains was also observed during development. These findings suggest that numerous laminin isoforms are involved in anterior pituitary gland development and that alteration of the expression pattern is required for proper development of the gland.     

Localization of Activin A/EDF in the Normal and Diabetic Rat Pancreas: Immunohistochemical and In Situ Hybridization Studies

We examined the immunohistochemical localization of activin A/erythroid differentiation factor (EDF) in the pancreases of normal and diabetic rats with insulin-dependent diabetes mellitus or noninsulin-dependent diabetes mellitus to elucidate how activin A/EDF modulates insulin secretion. In both the normal and the diabetic pancreas, all of the cells staining with antirecombinant human (rh) activin A/EDF antiserum were insulin-producing B-cells, and they gradually decreased in number with the development of diabetic changes in both types of diabetic rats. No rh activin A/EDF immunoreactivity was detected in A-, D-, or pancreatic polypeptide (PP) cells in the islets, or in the exocrine cells. Thein situ hybridization (ISH) method showed that activin A/EDF mRNA is localized in activin A/EDF-positive cells. These data indicated that activin A/EDF localizes in insulin-producing B-cells of normal and diabetic pancreases, and may act as an autocrine modulator of insulin secretion.     

Interphase Analysis of Chromosome 11 in Human Pituitary Somatotroph Adenomas by Direct Fluorescence In Situ Hybridization

Chromosome 11 numerical abnormalities were detected by fluorescencein situ hybridization (FISH) technique in four surgically removed pituitary somatotroph adenomas from patients clinically associated with acromegaly. The tumors were diagnosed by histology, immunocytochemistry, and electron microscopy, and included two densely granulated somatotroph (DG-SM) and two sparsely granulated somatotroph (SG-SM) adenomas. For demonstration of chromosome 11, the direct FISH technique was applied on imprints from fresh adenoma tissue fixed in acetone using an α-satellite specific centromeric probe. The slides were studied with a fluorescence microscope and the percentages of positive nuclei with aberrant fluorescent signals were counted. All tumors exhibited numerical chromosomal abnormalities in 8–23% of their cell population and included nuclei containing 1–3 extra chromosome 11 copies. The SG-SM adenomas exhibited more prominent abnormalities compared with the DG-SM adenomas. We conclude that numerical chromosome 11 abnormalities represent a frequent event among somatotroph adenomas with a tendency for higher frequency in SG-SM adenomas.     

Pituitary Corticotroph Adenoma in a Woman with Long-Standing Addison's Disease: A Histologic,immunocytochemical, Electron Microscopic,and In Situ Hybridization Study

A 64-year-old woman with long-standing Addison’s disease owing to destructive immune adrenalitis presented with hyperpigmentation and progressively increasing blood adrenocorticotrophic hormone (ACTH) levels. Magnetic resonance imaging demonstrated a pituitary microadenoma, which was removed by transsphenoidal surgery and investigated by histology, immunocytochemistry, transmission electron microscopy, andin situ hybridization (ISH). The morphologic studies revealed a basophilic, periodic acid-Schiff (PAS)-positive pituitary adenoma immunoreactive for ACTh and β-endorphin and in several cells for α-subunit. By transmission electron microscopy, the tumor was a densely granulated corticotroph adenoma, which, by ISH, expressed pro-opiomelanocortin (POMC) mRNA. The lack of corticotroph hyperplasia in the nontumorous adenohypophysis was an intriguing finding. Corticotroph adenomas in patients with long-standing Addison’s disease were very rarely examined by morphology. Our report includes a detailed morphologic analysis and is the first demonstration of POMC mRNA in the tumor cells using ISH. The question of whether the adenoma was related to increased secretory activity secondary to protracted hypocorticism or developed independently unrelated to deranged endocrine homeostasis remains unresolved. The lack of corticotroph hyperplasia in the nontumorous adenohypophysis favors the interpretation that hypothalamic stimulation played no major role in adenoma formation in our case.     

Detection of inhibin α and βA subunits (inhibin A, B, activin A,AB) in the human pituitary gland and in pituitary adenomas by immunohistochemistry and nonradioisotopicin situ hybridization

Inhibin and activin are gonadal hormones produced in human ovaries. They are known to act on anterior pituitary cells to regulate the synthesis and secretion of follicle-stimulating hormone (FSH). The purpose of the present study was to determine the localization of inhibin and activin subunits α and βA as endocrine markers in the human normal pituitary gland and pituitary adenomas, using immunohistochemistry andin situ hybridization (ISH) methods. Pituitary tissues from surgical and autopsy materials were fixed in 10% formalin and embedded in paraffin. Five normal pituitary glands and 79 pituitary adenomas were immunostained with the avidin-biotin peroxidase complex (ABC) method using polyclonal antibodies against inhibin and activin subunits α and βA. The other antibodies against anterior pituitary hormones used in this study were as follows: antigrowth hormone (anti-GH), antiprolactin (anti-PRL), antiadrenocorticotropic hormone (anti-ACTH), anti-FSHβ, antilutenizing hormone (anti-LH) β, antithyroid-stimulating hormone (anti-TSH) β, and antiglycoprotein α-subunit (anti-α-SU). We analyzed gene expressions of subunits α and βA by nonradioisotopic ISH in pituitary adenomas. In the normal human pituitary glands, inhibin and activin subunits α and βA immunoreactivities were found diffusely in the cytoplasm of anterior pituitary cells. The percentage of subunit α-immunopositive cells was 40% of the anterior pituitary cells. Subunit βA immunoreactivities were observed in about 15% of the anterior pituitary cells. By the double-staining method, subunit α immunoreactivity was detected in all types of anterior pituitary cells, and it was colocalized most frequently with GH and α-SU-positive cells. Subunit βA immunoreactivity was colocalized predominantly with PRL, FSH-β, LH-β, and α-SU. Among the 79 adenomas, 75 cases (94.9%) were positive for subunit α, and 50 cases (63.3%) were positive for subunit βA. Subunit βA was positive in tumor cells with the following incidences: GH adenomas, 3 of 14 (21.4%); PRL adenomas, 5 of 8 (62.5%); ACTH adenomas, 6 of 6 (100%); TSH adenomas, 7 of 7 (100%); nonfunctioning adenomas, 29 of 44 (65.9%), including gonadotropin-positive, 16 of 22 (80.0%). The ISH signals for subunits α and βA were strongly expressed in gonadotropin-positive adenomas among the nonfunctioning adenomas. The mRNA signals were low and infrequent in the GH-producing adenomas. Inhibin and activin subunit α localization did not demonstrate cell-type specificity in pituitary adenomas. In contrast, subunit βA demonstrated predominant positivity in the functioning pituitary adenomas (ACTH- and TSH-secreting) and nonfunctioning adenomas (including gonadotropin-positive adenomas). The present results suggest that the functional role of inhibin and activin in the differentiation of cells in normal human pituitary glands and adenomas is present in subunit βA.     

Insulin-like growth factor I in human thyroid tissue: Specific localization by immunohistochemistry andIn Situ hybridization

Insulin-like growth factor I and II (IGF-I and IGF-II) have been implicated in the replication of normal thyroid follicular cells in vitro. This study evaluates the distribution and abundance of immunoreactive IGF-I by histochemical analysis in human thyroid tissue with different histopathologic characteristics. We used two types of highly specific and sensitive polyclonal rabbit anti-IGF-I antibodies and one monoclonal antibody (MAb) with the immunoperoxidase technique on sections of 25 glands harboring adenomatous goiter; 11 glands with follicular adenoma (FA); 45 glands with thyroid carcinoma of papillary, follicular, and undifferentiated types; and 18 glands with Graves' disease. Immunoreactive IGF-I was present in some thyroid follicular cells of all thyroid tissues examined. The percentage of cells staining positively varies among the different processes, being lowest in normal thyroid tissues and highest in all thyroid carcinomas. The cytoplasmic pattern of IGF-I immunoreactivity also varied among the different thyroid conditions. Furthermore, using nonradioactivein situ hybridization (ISH) we detected IGF-I mRNA in the thyroid cells of adenomatous goiter. The expression was higher in the histologically hyperplastic areas. These findings provide further support for an autocrine and/or paracrine role of IGF-I in the function and/or growth of normal thyroid follicular cells and suggest that IGF-I may play a role in the dysfunctional growth of thyroid follicular cells in adenomatous goiter, thyroid carcinoma, and Graves' hyperthyroidism.     

Coated Glass Slides TACAS Are Applicable to Heat-Assisted Immunostaining and In Situ Hybridization at the Electron Microscopy Level

We performed pre-embedding electron microscopic study for visualizing the antigen and genome of severe fever with thrombocytopenia syndrome (SFTS) virus in the cytoplasm of macrophages of the human splenic red pulp, both requesting preheating treatment of sections. To pursue this, coated glass slides with unique characteristics are needed. Namely, during staining they must prevent detaching off sections, but after staining the sections must be transferred to epoxy resin. Aminopropyltriexoxysilane-coated glass slides, widely used for immunostaining, were resistant to transfer to epoxy resin. In contrast, coated glass slides designated as Thinlayer Advanced Cytology Assay System (TACAS) were suitable for this purpose. The technique is also applicable to the coated glass slide-requiring cytology practice, in which immunocytochemical evaluation is needed after cell transfer to another glass slide.     

Organization and chromosomal location of repetitive DNA sequences in three species of squamate reptiles

Repetitive DNA sequences were isolated from the genomes of species representing three major clades of squamate reptiles. A repetitive sequence (Cn4C7) was isolated from the New Mexican whiptail lizard,Cnemidophorus neomexicanus. This sequence is distributed throughout the chromosomes, but is more concentrated in the telomeric region. Cn4C7 also hybridizes to the chromosomes of otherCnemidophorus. Some evidence was found for concerted evolution of this repeat in hybrid unisexual lineages. In the lesser earless lizard,Holbrookia maculata, the predominant repeat in the genome is represented by a sequence (Hm1E11) which is restricted to the area flanking the centromere in all species ofHolbrookia. Two families of repetitive sequences (one dispersed, and the other telomeric) were isolated from the western diamondback rattlesnake,Crotalus atrox. The type and distribution of repetitive sequences in squamates is often taxon-specific, and may be useful as characters for elucidating taxonomic relationships.     

Autosomal location of a new subtype of 1.688 satellite DNA ofDrosophila melanogaster

During the screening of aDrosophila melanogaster YAC library with DNA from the minichromosomeDp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.accepted for publication by D. Ward     

Vascular Endothelial Growth Factor (VEGF) Expression in Human Pituitary Adenomas and Carcinomas

Vascular endothelial growth factor (VEGF) is a key mediator of endothelial cell proliferation, angiogenesis, and vascular permeability. Little is known about its expression in human pituitary adenomas. We examined 148 human pituitary adenomas for VEGF protein expression by immunohistochemistry. The strongest immunoreactivity was present in GH adenomas, corticotroph, silent corticotroph, silent subtype 3, and nononcocytic null cell adenomas. GH adenomas treated with octreotide strained less intensely than did untreated tumors. Relatively weak staining was present in PRL, gonadotroph, thyrotroph, and oncocytic null cell adenomas in the same sections showed evidence of down-regulation of VEGF protein expression in adenomas. Pituitary carcinomas usually had stronger staining than adenomas. In situ hybridization studies with oligonucleotide probes showed positive staining in all groups with stronger staining in GH, ACTH, TSH, and gonadotroph adenomas and in pituitary carcinomas. These results indicate that VEGF expression is more prominent in certain adenoma subtypes, that decreased expression occurs in adenomas as compared to nontumorous pituitary and that carcinomas show increased VEGF expression relative to adenomas suggesting up-regulation of VEGF during pituitary tumor progression.     

Physical relationship between satellite I and II DNA in centromeric regions of sheep chromosomes

Fluorescence in situ hybridization (FISH) with probes representing sheep satellite I and satellite II DNAs shows a different distribution of the two repetitive DNA families in the centromeric region of most chromosomes. The single signal per chromosome produced by the satellite I probe suggests close proximity of this DNA family to the primary constriction. Satellite II produces two separate signals on the sister chromatids, and large blocks of satellite II DNA constitute most of the short arm of all acrocentric chromosomes. We have isolated and sequenced a phage clone containing a junction between discrete blocks of satellite I and satellite II sequences. The junction is characterized by an abrupt juxtaposition of arrays of the two satellites. The possibility that the peculiar structural features of this junction could have a functional significance is discussed.This revised version was published online in November 2005 with corrections to the Cover Date.