Morphologic effects of hGRH gene expression on the pituitary, liver, and pancreas of MT-hGRH transgenic mice. An in situ hybridization analysis.

Morphologic changes in the pituitary, liver, and pancreas of mice with the metallothionein-human growth hormone--releasing hormone (MT-hGRH) transgene were analyzed by in situ hybridization histochemistry (ISH). There was progression from somatotroph hyperplasia to neoplasia in pituitaries of transgenic mice. Pituitary neoplasms were present between 9 to 12 months of age in some mice. Magnetic resonance imaging (MRI) readily identified enlarged pituitaries in MT-hGRH transgenic mice. Serum mouse GH and hGRH levels were marked elevated in MT-hGRH transgenic mice. In situ hybridization histochemistry showed mRNA for hGRH in liver, pituitary, pancreas, spleen, and in most other tissues examined. Combined ISH and immunohistochemistry in the pituitary gland showed that some of the GH cells also produced hGRH, and ultrastructural immunohistochemical analysis of pituitaries showed that GH and hGRH were localized in the same cell and within the same secretory granules. Liver cells of MT-hGRH transgenic mice showed evidence of hypertrophy, and the pancreatic islets were hyperplastic with significant increases in the islet cell areas. The morphologic changes in the liver were distinctive enough to separate control littermates from MT-hGRH transgenic mice in all cases. The enlarged pancreatic islets had increased numbers of insulin-producing cells. Immunoreactive hGRH and hGRH mRNA were both localized in islet cells, and an intense hybridization signal of hGRH mRNA, but only weak staining for hGRH protein, were detected in the liver of transgenic mice. These results indicate that excessive hGRH production leads to distinct morphologic changes in various organs in MT-hGRH transgenic mice and that there is temporal progression from hyperplasia to adenomatous somatotrophs in pituitaries with chronic stimulation by hGRH that involves paracrine, endocrine, and autocrine mechanisms.     

Ultrastructural immunocytochemistry of somatotrophs and mammotrophs in embryos of the dwarf mutant mouse

The embryonic development of somatotrophs and mammotrophs in the adenohypophysis in dwarf (dw) mutant mice was studied by means of ultrastructural immunocytochemistry. Embryos at 16-18 days of gestation were removed from litters consisting entirely of dwarf homozygotes obtained from homozygous matings of adult dwarfs in which reproductivity was induced by means of hormone supplementation and renal capsule implants of normal pituitaries. Litters from normal (+/+) matings served as controls. Pituitaries from adult normal and dwarf mice also were removed and processed with the embryonic material. Thin sections were exposed to anti-growth-hormone serum (anti-GH) or to antiprolactin serum (anti-PRL) and processed immunocytochemically with the colloidal gold procedure. In the normal pituitaries, a strong positive reaction to anti-GH occurred in the adult and in embryos as early as 16 days, whereas the reaction to anti-PRL was strong in the adults but relatively weak in the embryos. In dwarf embryonic and adult pituitaries, ambiguous cells showing combinations of features characteristic of somatotrophs and mammotrophs were present. However, neither these cells nor other granulated cells reacted to anti-GH or to anti-PRL, except for a questionable reaction to the latter at 18 days and in the adult. Thus, the dwarf pituitary shows functional as well as morphological abnormalities as early as 16 days of gestation.     

Muscle mechano growth factor is preferentially induced by growth hormone in growth hormone-deficient lit/lit mice

Two muscle insulin-like growth factor-I (IGF-I) mRNA splice variants (IGF-IEa and IGF-IEb) have been identified in rodents. IGF-IEb, also called mechano growth factor (MGF) has been found to be upregulated by exercise or muscle damage. Growth hormone (GH) is the principal regulator of  IGF-I expression in several tissues including skeletal muscle. Therefore, we investigated the effect of chronic GH excess or disruption of GH receptor (GHR) signalling, and the acute effect of GH administration on expression of muscle IGF-I isoforms using transgenic mice that express bovine GH (bGH), GHR gene-disrupted (GHR–/–) mice and GH-deficient lit/lit mice before and after exogenous GH administration. MGF mRNA in skeletal muscle was increased in bGH mice whereas it was decreased in GHR–/– mice compared with control animals. Exogenous GH administration to dwarf lit/lit mice significantly increased muscle MGF but not IGF-IEa mRNA 4 h after treatment. Twelve hours after GH treatment, both MGF and IGF-IEa mRNAs in muscle were increased compared with vehicle-treated lit/lit mice. In contrast in GH-sufficient lit/+ mice, both MGF and IGF-IEa mRNAs were increased 4 h after and returned to the basal level 12 h after GH treatment. Hepatic IGF-I isoforms were regulated in parallel by GH. Thus, our results demonstrated that: (1) MGF mRNA in skeletal muscle is expressed in parallel with GH action; (2) MGF mRNA in muscle is produced preferentially in the situation of GH deficiency in contrast to the pattern in the GH-sufficient state; and (3) the induction of IGF-I isoforms by GH is tissue-specific.     

Immunohistochemical study of p53 protein in human and animal pituitary tumors

In many human cancers, p53 gene mutations are frequently occurring genetic abnormalities, which may be detected by immunohistochemical staining for p53 protein. In the present study, p53 immunoreactivity was investigated in formalin-fixed, paraffin-embedded tissues from human and animal pituitary tumors, using the avidin-biotin-peroxidase complex technique. No p53 was detected in 3 nontumorous human adenohypophyses or in 40 human pituitary tumors including 5 GH cell adenomas, 10 PRL cell adenomas, 2 mixed GH cell-PRL cell adenomas, 2 acidophil stem cell adenomas, 8 ACTH cell adenomas, 1 TSH cell adenoma, 1 FSH/LH cell adenoma, 5 null cell adenomas, 5 oncocytomas, and 1 plurihormonal adenoma. Twenty nontumorous and hyperplastic pituitaries of hGRH transgenic mice and 8 tumors in these transgenic animals were immunonegative for p53. All pituitary tumors found in AVP/SV40 transgenic mice contained p53 immunoreactivity in the nuclei, while the nontumorous adenohypophysis of one such transgenic mouse was negative. It can be concluded that p53 mutations are apparently not involved in the pathogenesis of human pituitary adenomas or of the pituitary tumors which develop in hGRH transgenic mice. However, pituitary tumors in AVP/ SV40 transgenic mice are accompanied by p53 expression.     

Local expression of GH and IGF-1 in the hippocampus of GH-deficient long-lived mice

Beneficial effects of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) on the development and function of the central nervous system are well documented. In spite of primary deficiency of GH and secondary IGF-1 deficiency, Ames dwarf mice live considerably longer than normal animals, exhibit apparently normal cognitive functions and maintain them into advanced age. In an attempt to reconcile these findings, we have examined local expression of GH and IGF-1 in the hippocampus of normal and Ames dwarf mice. We found that both hippocampal GH and IGF-1 protein levels are increased and the corresponding mRNAs are normal in Ames dwarf as compared with normal mice. Increased phosphorylation of Akt and cyclic AMP responsive element-binding protein (CREB) were detected in the hippocampus of Ames dwarf mice. Our results suggest that increase in hippocampal GH and IGF-1 protein expression and subsequent activation of PI3K/Akt-CREB signal transduction cascade might contribute to the maintenance of cognitive function and is likely to be responsible for the integrity of neuronal structure, and maintenance of youthful levels of cognitive function in these long-lived mice during aging.     

Effect of intravenous infusion of growth hormone-releasing hormone on the morphology of rat pituitary somatotrophs

The effect of GRH infusion on rat adenohypophysial morphology was studied by light microscopy, immunocytochemistry, in situ hybridization, and electron microscopy. Synthetic rat GRH was intravenously administered by osmotic minipumps at 14.4, 72, 360 and 720 μg/ day/rat for 1 week. In one group treated for 1 week with a daily dose of 720 μg GRH, the rats were killed 7 days after withdrawal of GRH. Control rats in which GRH was replaced by excipient, or those that received no treatment, were included as well. GRH infusion with daily doses of 360 and 720 μg resulted in a significant increase in pituitary weight and weaker GH immunoreactivity compared with other groups. Ultrastructurally, the somatotrophs were increased in size and became sparsely granulated, and the organelles involved in hormone sythesis were very prominent. The intensity of the GH mRNA signal did not differ from control animals, suggesting the desensitization of somatotrophs to GRH. The highest GRH dose induced an increased number of nuclei immunoreactive for proliferation cell nuclear antigen (PCNA). One week after GRH withdrawal, shrinkage of cytoplasm, involution of RER and Golgi complex, and a decrease of cell attachment sites indicated the reversibility of changes induced by GRH. In conclusion, GRH infusion induced, within days, hypertrophy and proliferation of somatotrophs with ultrastructural features of highly stimulated, sparsely granulated cells. Morphological changes were reversible.Endocr Pathol 4:131–139, 1993.     

Immunocytochemistry of ambiguous cells in adult and embryonic dwarf (dw) mouse pituitaries

In the pars distalis of the pituitary gland in adult and embryonic dwarf (dw/dw) mutant mice, ambiguous cells exhibiting ultrastructural features common to growth hormone (GH) cells and prolactin (Prl) cells were analyzed by means of colloidal gold ultrastructural immunocytochemistry in order to define the functional nature of these peculiar cells. Adult and 18-day embryonic pituitaries from normal (+/+; dw/+) and dwarf (dw/dw) mice were processed with antibodies to GH, Prl, TSH (thyroid-stimulating hormone), ACTH (adrenocorticotropic hormone), LH (luteinizing hormone), FSH (follicle-stimulating hormone), and HCG (chorionic gonadotropic hormone). In the adult and embryonic dwarf pituitaries, the ambiguous cells reacted negatively to all of the antibodies except for anti-ACTH, which labeled them well. In addition, the ACTH-positive cells showed a much wider variety of shapes and granule size and distribution, as compared with normal adults. In the embryos, this variability in ACTH cell morphology occurred not only in dwarf embryos, but in their normal counterparts as well. The results thus suggest that adult dwarf pituitaries may retain an embryonic or incompletely differentiated form of ACTH cells. © 1993 Wiley-Liss, Inc.     

Hormonal and morphological study of the pituitaries in reeler mice

Reelin is a neuronal glycoprotein that plays a crucial role in brain layer formation during prenatal development. The reeler mutant mouse lacks Reelin, leading to abnormalities in the neuronal layering of cerebral cortex and cerebellum, producing ataxia, tremor and abnormal locomotion. Reeler mice are reported to have growth retardation and most of them are sterile or unable to bring up their newborns. Since the brain is one of the main regulator of pituitary hormone secretion and no information was reported regarding pituitary function and structure in these mutant mice, we studied pituitary endocrine activity and morphology in reeler mice. Mice were classified in three groups as reeler homozygote (RHM), reeler heterozygote (RHT) or control (CO). Pituitary hormone blood levels were assessed by enzyme immunoassay (EIA) and immunoradiometric assay (IRMA). Animals and their pituitaries were weighted and pituitaries were studied by histology, immunohistochemistry and electron microscopy. Results showed statistically significant differences in body weight and in adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) blood levels between the three groups. In contrast, growth hormone (GH) blood levels showed a high individual variation and no decrease in reeler groups compared with CO. Morphological studies revealed no differences in pituitary cell types except that somatotrophs appeared to be slightly smaller in RHM and RHT. Although it seems that pituitary hypofunction is not responsible for growth retardation, more studies are needed to obtain a deeper insight into the endocrine status of these mutant mice to elucidate the cause of their low body weight and reproductive behaviour.     

Oncogene-Induced Senescence in Pituitary Adenomas—an Immunohistochemical Study

Oncogene-induced senescence (OIS) serves as an initial barrier to cancer development, being proposed as a possible explanation for the usually benign behavior of the pituitary adenomas. We aimed to explore the immunohistochemical expression of the OIS markers, senescence-associated lysosomal β-galactosidase (SA-β-GAL), p16, and p21 in different types of 345 pituitary adenomas and compared it with the expression in the normal pituitary and in the specimens from the repeated surgeries. SA-β-GAL was overexpressed in the pituitary adenomas, compared to the normal pituitaries. Growth hormone (GH) producing adenomas showed the strongest SA-β-GAL, with densely granulated (DG)-GH adenomas more reactive than the sparsely granulated (SG). Nuclear p21 was decreased in the adenomas, except for the SG-GH adenomas that had higher p21 than the normal pituitaries and the other adenomas. p16 was significantly lower in the adenomas, without type-related differences. SA-β-GAL was slightly lower and p16 slightly higher in the recurrences. Our findings indicate alterations of the senescence program in the different types of pituitary adenomas. Activation of senescence in the pituitary adenomas presents one possible explanation for their usually benign behavior, at least in the GH adenomas that show a synchronous increase of two OIS markers. However, subdivision into GH adenoma subtypes reveals differences that reflect complex regulatory mechanisms influenced by the interplay between the granularity pattern and the hormonal factors, with possible impact on the different clinical behavior of the SG- and DG-GH adenoma subtypes. p16 seems to have a more prominent role in the pituitary tumorigenesis than in the senescence. Recurrent growth in a subset of the pituitary adenomas is not associated with consistent changes in the senescence pattern.     

Perturbation of differentiated functions in vivo during persistent viral infection. III. Decreased growth hormone mRNA

Lymphocytic choriomeningitis virus (LCMV) persistent infection that results from the inoculation of C3H/St newborn mice causes growth hormone (GH) deficiency and associated disease characterized by both reduced weight and serum glucose levels. Molecular analysis of pituitary nucleic acids shows GH deficient mice have, on average, fivefold reduced levels of GH mRNA although the histopathology of such GH producing cells is normal. Northern blots indicate that the length of GH mRNA is comparable in the GH deficient, virus infected mice and the GH normal, uninfected age-matched controls. Hence, truncated GH mRNA cannot account for hormonal defect. Mice infected congenitally through mating of persistently infected parents have normal growth and blood glucose levels. GH mRNA levels in pituitaries of these mice are equivalent to those of uninfected age-matched controls but significantly greater than those seen in neonatally infected GH deficient mice. Although infectious virus titers in the sera are equivalent in congenitally and neonatally infected age- and sex-matched mice, virus titers are significantly lower in pituitaries and brains of the congenitally infected mice when compared to neonatally inoculated mice. Additionally, the number of GH-producing pituitary cells expressing viral proteins is less in congenitally infected mice relative to those in neonatally inoculated mice. Hence there is a direct association between viral replication in GH-producing cells, lowered GH mRNA, and GH deficiency.     

Immune growth hormone (GH): localization of GH and GH mRNA in the bursa of Fabricius

Expression of growth hormone (GH) and GH receptor (GHR) genes in the bursa of Fabricius of chickens suggests that it is an autocrine/paracrine site of GH production and action. The cellular localization of GH and GH mRNA within the bursa was the focus of this study. GH mRNA was expressed mainly in the cortex, comprised of lymphocyte progenitor cells, but was lacking in the medulla where lymphocytes mature. In contrast, more GH immunoreactivity (GH-IR) was present in the medulla than in the cortex. In non-stromal tissues, GH-IR and GH mRNA were primarily in lymphocytes, and also in macrophage-like cells and secretory dendritic cells. In stromal tissues, GH mRNA, GH and GHR were expressed in cells near the connective tissue (CT) between follicles and below the outer serosa. In contrast, GH (but not GH mRNA or GHR), was present in cells of the interfollicular epithelium (IFE), the follicle-associated epithelium (FAE) and the interstitial corticoepithelium. This mismatch may reflect dynamic temporal changes in GH translation. Co-expression of GHR-IR, GH-IR, GH mRNA and IgG was found in immature lymphoid cells near the cortex and in IgG-IR CT cells, suggesting an autocrine/paracrine role for bursal GH in B-cell differentiation.     

Pituitary Changes in Ataxia-Telangiectasia Syndrome: An Immunocytochemical,In Situ Hybridization,and DNA Cytometric Study of Three Cases

Ataxia-telangiectasia (AT) syndrome (cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency, susceptibility to infections, and neoplasia) is associated with cyto- and nucleomegaly in several organ systems. Our aim was to determine (1) whether such cellular abnormalities in the pituitary selectively involve specific cell types, and (2) the proliferation and DNA ploidy status of such cells. Three AT autopsy pituitaries were studied by histology, immunohistochemistry (pituitary hormones, MIB-1, p53 protein),in situ hybridization (pituitary hormones), and Feulgen stain image analysis for ploidy. Results indicated that, in adenohypophyses the scattered pleomorphic, bizarre nuclei were mainly those of somatotrophs and corticotrophs, growth hormone (GH), or adrenocorticotropic hormone (ACTH) immunoreactive and expressing the GH or ACTH gene, respectively. Cyto-and nucleomegaly were less frequent in other secretory cells but were also noted in pituicytes of the posterior lobe. Affected cells were immunonegative for MIB-1 and for p53 protein. Image morphometric DNA analysis showed the bizarre cells to be aneuploid with complex histogram patterns, including many nuclei with DNA contents >8 n. No adenomas were found. We conclude that in AT adenohypophyseal cells with cyto- and nucleomegaly, as well as pleomorphism, synthesize and store adenohypophyseal hormones, mainly GH or ACTH. They and affected pituicytes are nonproliferative and are aneuploid.     

Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas.

Galanin is a neuropeptide that regulates the secretion of several pituitary hormones, including prolactin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in the rat anterior pituitary is cell lineage specific, with predominant expression in lactotrophs and somatotrophs. The authors examined the cellular distribution of human Gal-IR in seven normal postmortem pituitaries and 62 pituitary tumors by immunoperoxidase staining. In contrast to the rat, Gal-IR in human anterior pituitaries was present in corticotrophs scattered throughout the gland, but not in lactotrophs, somatotrophs, thyrotrophs, or gonadotrophs. Distinct Gal-IR also was present in hyperplastic and neoplastic corticotrophs in 19 of 22 patients with Cushing's disease. In noncorticotroph cell tumors, unequivocal Gal-IR was present in 5 of 11 GH-secreting tumors associated with clinical acromegaly, 9 of 18 nonfunctioning pituitary adenomas, and 2 of 14 prolactinomas. Of these galanin-positive tumors, four of the five GH-secreting adenomas, six of the nine nonfunctioning adenomas, and both of the prolactinomas also contained adrenocorticotropic hormone immunoreactivity (ACTH-IR). Immunostaining and in situ hybridization on adjacent sections using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant galanin expression in normal corticotrophs. Immunoelectron microscopy confirmed the presence of Gal-IR in pituitary cells characteristic of corticotrophs in both normal and neoplastic pituitaries. Thus, as in the rat, galanin gene expression in the human pituitary is cell-type specific. Unlike the rat, however, human galanin gene expression is restricted to the corticotroph lineage. Studies of tumors confirmed the observed coexpression of galanin and adrenocorticotropic hormone. The divergent cell type specificity of galanin production in human and rat pituitaries reflects different patterns of gene activation in these two species. In addition, these results suggest that galanin in the human pituitary may participate locally in the regulation of the hypothalamic-pituitary-adrenal axis.     

A morphological analysis endocrine tumour genesis in pancreas and anterior pituitary of AVP/SV40 transgenic mice

Summary Insertion into the mouse genome of the hybrid oncogene made up of bovine vasopressin gene derived 5 upstream sequences and the coding sequences of SV40 large T-antigen promoted tumours in anterior pituitary and endocrine pancreas of mice bearing this transgene. In order to investigate the morphology of the steps in the neoplastic process, we used light and electron microscopy to study these organs in 42 animals belonging to the 3rd, 4th and 5th generations, subdivided into 4 age groups from 20 days to 100 days of life. Antibodies to large T-antigen were used to identify sites of expression of the hybrid oncogene, thus monitoring the steps in neoplastic transformation. Large T-antigen immunoreactivity was identified in dysplastic lesions of younger animals and in both dysplastic lesions and tumours of older mice. Insulin (100% of cases) and pancreatic polypeptide (25% of cases) immunoreactivities were revealed in pancreatic lesions but no hormonal immunoreactivity was detected in the pituitary lesions. The ultrastructural study confirmed that the majority cell population of the pancreatic neoplasms was B-type and that the anterior pituitary tumours were poorly granulated. The subcellular localization of large T-antigen immunoreactivity was investigated by the immunogold method and was confined to the heterochromatin of tumour cell nuclei. These findings provide evidence for the dysplasia-neoplasia sequence in the genesis of endocrine tumours of pituitary and pancreas of transgenic mice. The vasopressin-SV40 large T-antigen transgenic mice may therefore be an useful model for the study of endocrine cell oncogenesis,