Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization

Acidophilic pituitary adenomas commonly produce growth hormone (GH) or prolactin (PRL), according to studies employing immunohistochemical and ultrastructural methods. To examine this question, in situ hybridization with oligonucleotide probes was done on routinely processed tissues received in the pathology laboratory to analyze for the presence of GH and PRL messenger RNA (mRNA) in 4 normal pituitaries, 10 prolactinomas, and 16 GH-secreting adenomas. Most acidophilic cells in normal pituitaries expressed either GH or PRL hormone and the respective mRNAs, but GH mRNA and PRL hormone were also detected in some of the same cells. Patients with a clinical diagnosis of prolactinoma had cells with only PRL mRNA in their tumors, while most (14 of 16) patients with a clinical diagnosis of acromegaly or gigantism had both GH and PRL mRNAs in their tumors. The GH adenomas varied in these studies. In situ hybridization was helpful in characterizing the adenoma from a patient with acromegaly who had immunoreactive PRL, but no immunoreactive GH in the resected tumor; in situ hybridization analysis revealed mRNAs for both GH and PRL in the same tumor cells. Our findings indicate that pituitary adenomas from patients with acromegaly commonly express PRL mRNA. It is concluded that in situ hybridization provides new information about the clinical biology and the histopathologic classification of pituitary adenomas.     

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.     

垂体ACTH细胞腺瘤的免疫电镜研究

5 cases of pituitary adenomas associated with Cushing's disease or Nelson's syndrome were studied with electron microscopy and immunoelectron microscopy by using protein A--gold complex. Diversified ultrastructure was displayed in these tumors, among which 4 revealed presence of ACTH positive secretory granules. These granules were round or polyhedric in shape, varied in number, size and electronic density. Bundles of microfilaments could be seen in the tumor cells frequently, which were of the highest diagnostic value. There was no significant difference found in ultrastructure and immunocytochemical reaction of adenomas in Cushing's disease and Nelson's syndrome.     

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     

Pergolide for the treatment of pituitary tumors secreting prolactin or growth hormone

We gave pergolide mesylate, a new long-acting ergot derivative with dopaminergic properties, to 47 patients with hypersecretion of prolactin or growth hormone. Single doses produced long-lasting reductions of serum prolactin levels; after 24 hours, the values remained depressed at a mean of 28.8 per cent of the base-line value. Among 41 patients (22 women and 19 men) with hyperprolactinemia who took pergolide for three months or more, prolactin levels fell to normal in 37 and remained slightly elevated in 2. In the two patients in whom the levels fell to only 38 to 52 per cent of base line, treatment was regarded as a failure. The level of growth hormone fell to a mean of 52.8 per cent of base line in patients with acromegaly who were taking 100 micrograms of pergolide per day. Among patients for whom adequate CT scans were available, definite tumor shrinkage occurred in 10 of 13 with macroadenomas and definite or probable shrinkage in 5 of 9 with microadenomas. Menses returned in 76 per cent of treated women and testosterone levels rose in 10 of 14 men. We conclude that pergolide reduces hypersecretion and shrinks most prolactin-secreting macroadenomas. In some patients long-term pergolide therapy may be superior to surgery and x-ray treatment.     

Expression of vascular endothelial growth factor in normal pituitary cells and pituitary adenomas producing adrenocorticotropic hormone

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation and an increase in capillary permeability. Because the anterior pituitary gland and pituitary adenomas are highly vascular, expression of VEGF was examined immunohistochemically. Some normal pituitary cells stained positively for VEGF, and restaining for ACTH, prolactin, TSH, LH, FSH, and S-100 protein after VEGF staining revealed that almost all cells staining positively for ACTH also stained for VEGF. Only adenomas staining positively for ACTH stained for VEGF. These results suggest that VEGF is produced by normal pituitary cells and adenomas producing ACTH.     

Immunoelectron microscopic demonstration of glycoprotein subunits in the FSH producing pituitary adenomas

Immunoelectron microscopic observations disclosed the presence of FSH alpha, beta subunits in secretory granules and rough endoplasmic reticulum (RER). Golgi saccules were consistently negative. The presence of these subunits in secretory granules which were located in the vicinity of RER or perinuclear spaces (PNS) suggested the possible formation of secretory granules in RER or PNS. The tumor cells showed heterogeneity in size and immunoreactivity of the secretory granules but non-neoplastic cells showed uniformity in these aspects.     

垂体生长激素细胞腺瘤的电镜及免疫电镜观察

24 cases of growth hormone(GH)-producing pituitary adenomas were studied with electron microscopy and immunoelectron microscopy by protein A-gold complex, 6 cases were identified as densely granulated GH adenoma and 15 cases as sparsely granulated GH adenoma, among which 4 cases were proved by immunoelectron microscopy to be containing granules with prolactin(PRL) activity simultaneously. Intracytoplasmic fibrous bodies were often seen in the sparsely granulated cells anyhow, not all those cells with fibrous bodies possess the secretory granules with GH activity, and fibrous bodies were also detected in some PRL cells of certain mixed type adenoma. This suggests that fibrous bodies might not be the specific morphological feature of pituitary growth hormone cell adenomas.     

Immunoelectron microscopic studies of GH and alpha subunit in GH secreting pituitary adenomas

9 of 15 cases of GH secreting adenomas showed the localization of GH and a subunit in the adenoma cells. GH and a subunit were frequently colocalized in the same adenomas. Immunoelectron microscopically, GH and a subunit were localized in secretory granules which were packed in the cytoplasm. Immunoelectron microscopic double staining (preembedding method and post-embedding method) showed colocalization of GH and a subunit in the same secretory granules. These data suggested corelease of GH and a subunit by the same secretory granules.     

Sparsely granulated prolactin cell adenomas of the pituitary gland

Summary The possible relationship between the preoperative plasma prolactin levels of patients having a sparsely granulated prolactin cell adenoma of the pituitary gland and the morphology of the tumors was studied by means of quantitative electron microscopy. To this end, a number of ultrastructural variables were chosen which are generally regarded to be indicative of cellular activity and which could be determined in a quantitative or semiquantitative way. These variables were determined in 19 adenomas from 17 patients and plotted against the corresponding prolactin levels. It appeared that marked endocrine activity was associated with a small number of granules per cell, a high frequency of exocytosis, and a marked development of the rough endoplasmic reticulum. Granule size and development of Golgi apparatus and lysosomes were not at all, or only poorly correlated with the plasma hormone levels. Finally, the number of mitochondria per cell showed a totally unexpected inverse correlation with endocrine activity. Due to the close mutual correlation existing between several of the variables investigated, combining them in a multivariate analysis did not significantly improve the correlation with the hormone level.     

Pathology of prolactin cell adenomas of the human pituitary

Prolactin (PRL) cell adenoma is the most common tumor type in the human pituitary. It accounts for 30% of surgically removed adenomas, while its prevalence is even higher (45%) among incidental pituitary tumors observed at autopsy. Most PRL cell adenomas are highly differentiated with a characteristic ultrastructure. Administration of bromocriptine, a dopaminergic agonist, evokes profound morphologic changes in responsive PRL cell adenomas, while it leaves the fine structure of unresponsive tumors unchanged. The importance of immunocytochemical and electron microscopic investigation of pituitary biopsies is emphasized as tumors with different cell derivation, biological behavior, and therapeutic responsiveness may mimic PRL cell adenomas clinically.     

Mixed growth hormone cell- prolactin cell pituitary adenoma with acromegaly: α-subunit most growth hormone cells

A 51 -year-old woman with mixed growth hormone (GH) cell-prolactin (PRL) cell pituitary adenoma is presented. She had clinical signs due to hypersecretion of GH and PRL. Resected tissue was studied immunohistochemically and morphologically. The serial sections revealed that GH and α-subunit were co-localized in most cells, while GH and PRL were localized in different cells.     

The pituitary in cirrhosis: ultrastructure, growth hormone, and prolactin concentrations.

Micronodular cirrhosis was induced in male SUAH substrain Wistar rats by combined phenobarbitone and carbon tetrachloride treatment. Both pituitary and serum concentrations of growth hormone were significantly reduced in cirrhotic rats compared with age-related untreated rats or those treated only with phenobarbitone. Ultrastructurally growth hormone-secreting cells (somatotrophs) of pituitaries of cirrhotic rats appeared relatively inactive, having few hormone-containing granules, sparse rough endoplasmic reticulum, and small nuclei with areas of condensed chromatin. The cells themselves were smaller than similar cells of untreated rats with a reduced cytoplasmic area. In addition immunocytochemistry of pituitaries at light microscope level, using sheep anti-rat growth hormone antibody, showed that somatotrophs of cirrhotic rats were more heteromorphic and disorganized than those in controls. There was marked development of the folliculo-stellate cell system in pituitaries of cirrhotic rats, the cells were enlarged with distinct golgi, and numerous microvilli were projecting into dilated follicular lumena.     

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.     

Effects of propylthiouracil on growth hormone and prolactin messenger ribonucleic acids in the rat pituitary

The effects of hypothyroidism induced by propylthiouracil (PTU) treatment on growth hormone (GH) and prolactin (PRL) messenger ribonucleic acid (mRNA) levels were analyzed in adult female rat adenohypophyses by in situ hybridization histochemistry and Northern hybridization analyses. Twenty-eight days of PTU treatment produced a significant decrease in GH mRNA levels and a smaller decrease in PRL mRNA determined by both in situ hybridization histochemistry and Northern hybridization analyses. A combined procedure of in situ hybridization histochemistry followed by immunochemistry on the same sections revealed mammosomatotropic cells expressing GH mRNA and PRL protein in the same pituitary cells from all treatment groups. Cells expressing GH mRNA and thyroid-stimulating hormone protein were not detected by this method. Immunochemical staining revealed a decrease in GH cells and an increase in thyroid-stimulating hormone cells in hypothyroid rats. Cells expressing both GH and thyroid-stimulating hormone protein were not detected by immunostaining. These results indicate that hypothyroidism produces significant decreases in GH mRNA and also decreases PRL mRNA and that mammosomatotropic cells can be detected in pituitaries from normal and hypothyroid rats.     

Growth hormone (GH) and prolactin (PRL) gene expression and immunoreactivity in GH- and PRL-producing human pituitary adenomas

Summary Growth hormone(GH)-producing pituitary adenomas are morphologically heterogeneous and frequently contain not only GH immunoreactivity but also variable numbers of prolactin (PRL) immunopositive cells. Paraffin sections of 59 surgically removed GH- and/or PRL-producing adenomas classified by histology, immunocytochemistry (ICC) and electron microscopy were studied using in situ hybridization (ISH) for GH and PRL mRNA and combined with ICC for the coded hormones. Somatotroph adenomas (10 densely and 10 sparsely granulated tumours) and mammosomatotroph adenomas (10 cases) contained both GH mRNA and GH immunoreactivity. In 4 densely and 4 sparsely granulated somatotroph adenomas and 4 mammosomatotroph adenomas, only GH mRNA and its product were found. In 28 cases (6 densely and 6 sparsely granulated somatotroph adenomas, 10 mixed somatotrophlactotroph adenomas and 6 mammosomatotroph adenomas) both GH and PRL mRNA were present, although no PRL immunoreactivity was not in 2 densely granulated somatotroph adenomas. In these cases, ISH for PRL mRNA combined with GH immunostaining revealed the presence of variable numbers of mammosomatotrophs. In 9 acidophil stem cell adenomas only PRL mRNA and its product were found; one tumour expressed both GH and PRL mRNA and their products. Nine lactotroph adenomas contained only PRL mRNA and PRL immunoreactivity. The results show that GH and/or PRL mRNA content could not be correlated with ICC for coded proteins and ultrastructural features. The mammosomatotrophs were more numerous using ISH when compared with ICC. Somatotroph, mammosomatotroph and mixed adenomas are closely related and they can be considered to represent one basic tumour type originating in a cell committed to GH production. This may undergo clonal differentiation towards a mammosomatotroph and further to the lactotroph line. The results also indicate that lactotroph adenomas arise in a cell committed to PRL production. Acidophil stem cell adenomas seem to be more closely related to lactotroph cells than somatotroph.     

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.