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.     

Cytokeratin distribution and functional properties of growth hormone-producing pituitary adenomas

In addition to its structural function, cytokeratin may have other important roles within cells. We have reported that in growth hormone-producing adenomas (GH cell adenomas), two distinct types can be recognized by their cytokeratin distribution patterns (dot-like or perinuclear pattern) and that each type has different clinicopathological and endocrinological properties. To confirm these phenomena in a larger series and to clarify the significance of different cytokeratin distribution patterns, we studied cytokeratin localization in 70 GH cell adenomas from acromegalic patients. Type I adenomas ( 15) almost exclusively (>98%) composed of cells with a prominent, dot-like distribution; type 2 adenomas (36) comprised of cells with perinuclear cytokeratin; and type 3 adenomas (11) comprised of both cell types were separated. The remaining 8 did not exhibit a distinct distribution pattern. By electron microscopic immunocytochemistry for cytokeratin, dot-like distribution corresponded to fibrous bodies, whereas perinuclear distribution represented immune deposition in the perinuclear zone. Immunohistochemistry for GH, prolactin, β-thyrotropin, and α-subunit of glycoprotein hormones revealed a reduced expression of these hormones in type 1 adenomas, compared with types 2 and 3 adenomas. In normal pituitary glands, almost all GH cells showed a perinuclear cytokeratin distribution, and only a few GH cells exhibited a dot-like pattern. These findings suggest that a dot-like cytokeratin distribution in GH cells may be pathological (a change from physiological perinuclear distribution) and that adenomas with such a distribution may reduce endocrine activities as a result of unknown factors.     

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.     

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.     

Animal models of human pituitary tumors

Three animal models of pituitary tumors were compared: the spontaneous prolactinoma in Wistar/Furth (W/Fu) rats, an estrogen-induced transplantable tumor in the Fischer rat (MtTF4), and a spontaneous prolactin transplantable tumor in the W/Fu rat (SMtTW1). The spontaneous prolactinoma in W/Fu rats is interesting in that it resembles the human prolactinoma by its morphological characteristics, its benign nature, and its secretion of prolactin alone. It may be useful to study the initial factors of tumorigenesis but it is very expensive and the variability of tumor growth makes it difficult to plan experiments. The MtTF4 tumor is an easy model to study because it is transplantable but this tumor differs from most human pituitary tumors by its induction by estrogen, its malignancy, its undifferentiated aspect and its secretion of ACTH, GH, and prolactin. The SMtTW1 tumor, a new model of transplantable tumor, is close to the human pituitary tumor because the initial tumor is spontaneous, the transplanted tumors are benign and well differentiated. They secrete prolactin only. These transplantable tumors are valuable for studying the factors of growth. Since no single tumor system is a perfect model, researchers have to work on different models each of which is appropriate for investigating specific problems.     

The immunohistochemical identification of hormone-producing cells in formalin-fixed, paraffin-embedded human pituitary tissue.

A method is described, combining immunohistochemistry and histological staining methods in the same section, for the identification of ACTH- and STH-producing cells in formalin-fixed, routine processed human pituitary tissue. The results indicate that this method is suitable to employ in practice.     

Expression of glycoprotein hormones and intracytoplasmic distribution of cytokeratin in growth hormone-producing pituitary adenomas.

Sixteen growth hormone (GH)-producing pituitary adenomas were studied for the expression of glycoprotein hormone subunits and cytokeratin by light microscopic immunohistochemistry. Cytokeratin immunoreactivity was demonstrated in all adenomas, but its intracytoplasmic distribution showed two distinct patterns; a prominent, dot-like pattern and a diffuse, perinuclear pattern. Seven adenomas (type 1) were exclusively composed of cells with cytokeratin in a dot-like pattern, whereas 9 adenomas (type 2) comprised of cells with cytokeratin of perinuclear distribution. The expression of alpha-subunit of glycoprotein hormone was significantly different between the two types of adenomas; 8 of 9 adenomas of type 2 contained many alpha-subunit immunoreactive cells but none of type 1 adenomas showed any immunoreactivity. Only a small number of adenoma cells were positive for beta-subunit of thyrotropin stimulating hormone in 3 adenomas of type 2. beta-subunits of follicle stimulation hormone and luteinizing hormone were negative in all adenomas. These findings suggest that the expression of glycoprotein hormone subunits in GH-producing adenomas may be closely linked to their types distinguishable by the cytokeratin distribution pattern.     

Prenatal development of growth hormone-producing cells in the rat anterior pituitary as studied by immunogold electron microscopy

The existence of three morphologically different types of GH cells in the rat anterior Pituitary glands has been reported previously. Among these three types, Type III which contains the smallest granules has been considered to be immature, because this type occurs more frequently in neonatal rats than in adults. In this study, the prenatal development of GH cells in the rat fetus was observed by immunoelectron microscopy. In the rat fetus at 18.5 days of gestation, Type III cells were most numerous (48.5%), followed by Type II cells (45.5%). Type I cells were almost absent from these fetal pituitaries. At 20.5 days Type II cells exceeded Type III in frequency, and Type I cells also increased to about 35%. These results indicate that Type III cells are the most immature type of GH cells, and might transform into Type II and, in turn, to Type I, as the rats mature. Images indicating active secretory functions such as granule formation in the Golgi apparatus and/or GERL, exocytosis and crinophagy were observed in the GH cells even in the fetal stage.     

Analysis of human pituitary tumors by in situ hybridization

A procedure for performing in situ hybridization histochemistry (ISH) on frozen and paraffin sections of human pituitary tissues is described. The use of oligonucleotide probes for hPRL and hGH labeled with 35S allowed detection of a specific messenger RNA in frozen and paraffin sections. This technique can be combined with immunochemistry to localize both the gene product and the hormone(s) produced by specific cells and should be very helpful in the characterization of normal and neoplastic human pituitary cells.     

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.     

Presence of growth factors in human pituitary.

Recent reports indicate that fibroblast growth factors known to be present in the pituitary in high levels regulate the action of growth hormone and prolactin. New data also suggest a regulatory role in the pituitary for other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Since in most systems cooperation of several growth factors is required for their optimal function, we sought to demonstrate the presence of certain growth factors in the pituitary. Acid ethanol extracts from approximately 50 autopsy-derived human pituitaries were subjected to molecular sieve chromatography and were tested for growth factors. Low molecular weight protein (10 micrograms) eluted from the molecular sieve column contained 10-20 ng material binding to the EGF/TGF-alpha receptor as determined by the EGF/TGF alpha radioreceptor binding assay which represents 11 ng EGF/TGF alpha per pituitary. By Western blotting we found EGF but could not document the presence of TGF-alpha in this material. Radioimmunoassay for insulin-like growth factor I detected 0.4-0.8 ng insulin-like growth factor-I/100 micrograms extract. TGF-beta eluted between 14,000 and 20,000 M(r) at levels of 3-4 ng/pituitary. Its ability to inhibit growth of CC164 mink lung cells was abolished by antibody to TGF-beta 1 but not by antibody raised against TGF-beta 2. The detection of platelet derived growth factor was equivocal and not fully reproducible. We have partially purified TGFe from the pituitary; it stimulated soft agar growth of carcinoma SW-13 cells, and it followed an elution pattern identical to bovine kidney TGFe on molecular sieve column and high pressure liquid and high performance electrophoretic chromatography. Our data show that in addition to fibroblast growth factors, the human pituitary contains other growth factors, such as EGF/TGF-alpha, TGF-beta, insulin-like growth factor I, and TGFe.     

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.