Pituitary adenomas in old Sprague-Dawley rats: a histologic, ultrastructural, and immunocytochemical study.

Fifty-five adenomas were identified and characterized in the anterior pituitaries of 27 male and 39 female SD rats, over 24 months of age, by histology, ultrastructural morphology, and immunocytochemistry. Adenomas were found in 85% of male and 79% of female rats; all known adenohypophysial hormones were represented in various tumors. Prolactin (PRL)-containing adenomas were the most common (47.2%); luteinizing hormone-(LH)-containing adenomas (16.3%), immunonegative adenomas (12.7%), PRL- and growth hormone (GH)-containing adenomas (10.9%), thyroid-stimulating hormone (TSH)-containing adenomas (3.6%), adrenocorticotropin hormone (ACTH)-containing adenomas (3.6%), and GH-containing adenomas (1.8%) were also identified. Unexpected combinations were observed in 3 tumors (5.4%); a GH-LH-containing adenoma, a PRL-ACTH-containing adenoma, and a PRL-LH-TSH-containing adenoma were noted. One intermediate lobe adenoma and 1 metastatic plasmacytoma were diagnosed. It can be concluded that spontaneous pituitary adenomas in aging SD rats are potential models of the human disease because of diversity of hormone content and morphologic appearance.

Five different adenomas derived from the rat adenohypophysis: immunocytochemical and ultrastructural study.

With the use of electron microscopic morphology and immunochemistry, 5 tumors were studied: a spontaneous prolactin-producing adenoma (LEP rats); an estrogen-induced intrasellar tumor (R-Amsterdam rats); and 3 transplanted tumors, MtT.W10 and MtT.W5 (WF rats) and MtT.F4 (F344 rats). All tumors were derived from rat adenohypophysis and are known to secrete prolactin, growth hormone, or adrenocorticotropic hormone. The spontaneous tumor consisted of a uniform population of cells containing only immunoreactive prolactin. In the estrogen-induced tumor, prolactin and growth hormone were localized in separate cell types with the use of the immunoperoxidase technique. In the MtT.W10 tumor, both immunoreactive prolactin and growth hormone were observed in the same cell and in separate cell types. In the MtT.F4 and MtT.W5 tumors, one cell type was identified that was characterized by lack of morphologic differentiation, reduced secretory granule number, and inconclusive immunopositivity.

Ultrastructural differentiation in the nude mouse of transformed cells isolated from the human fetal pituitary gland.

Spontaneously transformed human fetal pituitary cells were isolated from first-passage cultures after 3 to 6 months of long-term maintenance in growth medium containing 15% fetal calf serum. The cells, when injected into nude mice, developed into large tumors in 1 to 2 weeks. Attempts to detect growth hormone, prolactin, follicle-stimulating hormone, luteinizing hormone, or thyroid-stimulating hormone from nude mouse plasma and tumor homogenate by radioimmunoassay were unsuccessful. These hormones could not be demonstrated in tumor cell cytoplasm by immunocytochemistry. Two human fetal pituitary (HFP) cell strains (HFP-T2, HFP-F4) from tissue culture examined at the electron microscopic level were undifferentiated by ultrastructural criteria. However, tumor cells in the nude mice showed signs of fine structural differentiation with well-developed rough endoplasmic reticulum profiles, secretory granules, and intercellular junctions. The tumor cells lost the features of morphological differentiation when returned to tissue culture. These changes could be repeated by alternate passage in nude mouse and tissue culture.

Chronic administration of corticotropin-releasing factor increases pituitary corticotroph number.

The effect of chronic administration of CRF on rat pituitary morphology was studied. Experimental animals received CRF (10 micrograms/day) over a period of 52 days by means of sc osmotic pumps changed at 10- to 14-day intervals. The average 0800 h plasma corticosterone levels in the treated animals were significantly greater than control values [7.52 +/- 0.99 (+/- SE) vs. 1.14 +/- 0.5 micrograms/dl; P less than 0.001]. The CRF-treated animals also had a significantly greater adrenal weight (16.44 +/- 1.38 vs. 12.24 +/- 0.85 mg; P less than 0.05) and lower thymus weight (164 +/- 12 vs. 248 +/- 27 mg; P less than 0.005). There was a marked increase in the number of ACTH-producing cells in the anterior pituitaries of the rats that received CRF (13.3 +/- 0.8% vs. 4.5 +/- 0.3% ACTH-producing cells; P less than 0.001), as determined by immunocytochemical methods. Corticotrophs of rats treated with CRF manifested a significant increase in nuclear area (24.0 +/- 0.7 vs. 21.4 +/- 0.4 micron 2; P less than 0.001) and an increased diameter of forming and storage granules (191.1 +/- 1.1 vs. 158.6 +/- 3.5 nm and 196.1 +/- 1.2 vs. 170.1 +/- 3.7 nm, respectively; P less than 0.001). There was no demonstrable increase in ACTH cell area. These data indicate that long term administration of CRF is capable of increasing the number of pituitary corticotrophs. It also supports the view that the corticotroph hyperplasia occurring after adrenalectomy, in unusual cases of ectopic CRF production, and in rare instances of Cushing's disease is a proliferative response to CRF.

Adrenocortical hemorrhagic necrosis: the role of catecholamines and retrograde medullary-cell embolism.

We investigated the pathogenesis of adrenal necrosis using animal models of the disease (induced by administration of acrylonitrile, cysteamine, or pyrazole) and human cases. Results of electron-microscopic and histochemical time-response studies with rat models revealed an early, retrograde embolization of medullary cells and cell fragments in the cortical capillaries that showed prominent endothelial injury. The experimental adrenal lesions were prevented by surgical removal of the medulla one month before administration of adrenocorticolytic chemicals, or by the administration of the alpha-adrenergic antagonist phenoxybenzamine hydrochloride. Histochemical staining for medullary (argyrophil) granules in human cases of adrenal necrosis demonstrated tissue fragments that stained positively for silver in vascular cortical spaces in nine of ten autopsy specimens and in all four surgical cases we reviewed. Thus, catecholamines released from the adrenal medulla and from the retrograde medullary emboli in the cortex may have a role in the pathogenesis of adrenocortical necrosis.

Subclinical adenomas of the human pituitary. New light on old problems.

We studied 107 adenomas, found incidentally at autopsy in 100 pituitaries, by histologic and immunohistochemical techniques to elucidate their cellular composition and hormone content. No adenohypophyseal hormones were found in 54 (50%) of the adenomas, whereas prolactin was shown in 45 (42%). Of the remaining tumors, two contained prolactin and growth hormone, four contained adrenocorticotropic hormone, one contained thyroid-stimulating hormone, and one contained luteinizing hormone. These findings are consistent with the view that all adenohypophyseal cell types can give rise to neoplasms. No correlation was found between clinical history, autopsy findings, or cause of death and the presence or type of adenoma. The adenomas caused neither local symptoms nor endocrine abnormalities. The prevalence of various adenoma types differed between autopsy specimens and surgical material.

Argyrophil granules in the human pituitary.

In order to assess the value of the Grimelius silver method in adenohypophysial cell identification and adenoma diagnosis, 22 nontumorous adenohypophyses and 50 pituitary adenomas were investigated. Grimelius positivity was localized, as documented by mirror sections, predominantly in PAS and led hematoxylin positive cells; but a few negative cells contained argyrophil granules as well. The immunoperoxidase method revealed Grimelius positivity primarily in corticotrophs, thyrotrophs and gonadotrophs, and occasionally in somatotrophs and lactotrophs. Argyrophilia was readily demonstrated in 11 out of 13 corticotroph cell adenomas, in 1 out of 1 thyrotroph cell adenoma, in 5 of 11 null cell adenomas and in 4 out of 5 oncocytomas. No argyrophilia was detected in 5 growth hormone cell adenomas and 5 prolactin cell adenomas. One our of 5 mixed, growth hormone cell-prolactin cell adenomas, and 1 out of 5 acidophil stem cell adenomas showed Grimelius positivity. By electron microscopy, silver grains were localized in the secretory granules. It can be concluded that no specific hormonal products account for argyrophilia.

Ultrastructural morphometry of sparsely granulated prolactin cell adenomas of the human pituitary.

Fifteen sparsely granulated prolactin-producing adenomas and 10 nontumourous adenohypophyses, removed by surgical hypophysectomy, have been studied using morphometry at the electron microscopic level. Compared to non-tumourous prolactin cells, sparsely granulated adenomatous prolactin cells showed a significant decrease in diameter and volume density of secretory granules and an increased volume density of rough-surfaced endoplasmic reticulum and Golgi apparatus. The volume density of mitochondria remained unchanged. These results indicate that the cells of the adenoma are in a highly active functional state. It appears that the equilibrium between hormone synthesis, storage and release is altered in adenomatous prolactin cells.

Protein synthesis dependent and independent mechanisms of neutrophil emigration. Different mechanisms of inflammation in rabbits induced by interleukin-1, tumor necrosis factor alpha or endotoxin versus leukocyte chemoattractants.

Inflammation constitutes the body's principal mode of defense against infection and other harmful agents. Neutrophil leukocytes are the primary effector cells in this process. The role of protein synthesis in neutrophil emigration into acute inflammatory lesions was examined. Local intradermal injections of actinomycin D, cycloheximide or puromycin could inhibit in a dose- and time-dependent manner neutrophil emigration induced by interleukin-1, tumor necrosis factor alpha or endotoxin, but not by the leukocyte chemoattractants C5a des arg (zymosan-activated plasma), n-formyl-methionyl-leucyl-phenylalanine or leukotriene B4. Maximal inhibition, measured at the time of peak emigration, was greater than 90%. The onset of neutrophil emigration induced by the cytokines or by endotoxin was delayed by 30 to 60 minutes in comparison to the leukocyte chemoattractants. These results demonstrate at least two mechanisms of neutrophil emigration: one with a slower onset and dependence on local RNA transcription and translation and the other rapid in onset and independent of protein synthesis.

Neutrophil leukocyte emigration induced by endotoxin. Mediator roles of interleukin 1 and tumor necrosis factor alpha 1.

The hypothesis that cytokines mediate neutrophil emigration induced by endotoxin (LPS) was studied by examining the potency, the kinetics of neutrophil emigration, and the tachyphylaxis of intradermal sites with IL-1, TNF-alpha and LPS. Human rIL-1 alpha and IL-1 beta, synthetic lipid A, and LPS were several orders of magnitude more potent than human rTNF. The kinetic profiles of neutrophil emigration induced by IL-1 alpha, TNF, and LPS were characterized by minimal emigration in the first 30 min, followed by rapid and transient emigration. After the injection of LPS, the onset and the time at which the rate of emigration was maximal consistently appeared 30 min later than IL-alpha or TNF, suggesting that neutrophil emigration in response to LPS was mediated by a locally generated cytokine. IL-1 and TNF were then examined as potential secondary mediators of LPS-induced emigration by comparing the patterns of tachyphylaxis between LPS and IL-1 alpha or TNF; i.e., the magnitude of neutrophil emigration into inflammatory sites was compared with sites injected 6 h previously (desensitizing injections) with a cytokine or with LPS. Tachyphylaxis was dose dependent with each and also between the IL-1 species; therefore, when tachyphylaxis between the cytokines and LPS was examined, relatively higher doses were selected for the desensitizing injections than for the test injections. With this approach, desensitizing injections of IL-1 alpha diminished the neutrophil accumulation after LPS, and LPS also desensitized sites to IL-1 alpha. However, tachyphylaxis was not observed between TNF and LPS, or between TNF and IL-1 alpha. These data suggest that IL-1, but not TNF, is a potential mediator of LPS-induced neutrophil emigration.

Bromocriptine suppression of dispersed pituitary lactotrophs from estrogen-pretreated rats: a quantitative electron microscopic study.

Dispersed rat lactotrophs were treated with bromocriptine (10(-10) M) for either 30 min or 3 h to investigate its effect on cell morphology using light and electron microscopy and ultrastructural morphometry. After 30 min, lactotrophs treated with bromocriptine exhibited an increase in storage granule size (p less than 0.05). Crinophagy was also evident and a significant increase in lysosome volume density was observed (p less than 0.001). However, no significant change in prolactin content in the media was detected until 3 h of incubation in bromocriptine (p less than 0.05). At this time a nonsignificant decrease in Golgi region volume density was also observed.

Monomorphous plurihormonal adenoma of the human pituitary. A histologic, immunocytologic and ultrastructural study.

A well-developed 23-year-old man, complaining of blurred vision but with no endocrine symptoms, was found to have a large pituitary adenoma spreading outside the sella. Endocrine investigations disclosed growth hormone deficiency, hyperprolactinemia (responsive to thyrotropin-releasing hormone), and very high blood alpha-subunit (72 ng/ml) level. Histology showed a chromophobic, slightly acidophilic pituitary adenoma with focal fibrosis and calcification. The immunoperoxidase technique revealed prolactin and alpha-subunit in the cytoplasm of a single-cell type, at the light and electron microscopic level, indicating that monomorphous, plurihormonal adenomas exist in the human pituitary. Immunostaining with antibodies raised against beta-thyroid stimulating hormone, beta-follicle stimulating hormone and alpha-endorphin were observed in scattered cells. Those cells that contained immunoreactive alpha-endorphin did not appear to contain alpha-subunit. The ultrastructural features of adenoma cells showed no resemblance to any known cells in nontumorous or tumorous pituitaries. It can be postulated that adenohypophysial cells, after neoplastic transformation, may have the ability to secrete a number of biochemically unrelated hormones, suggesting that during embryologic development they pass through a common progenitor cell stage, capable of plurihormonal activity.

Enhanced responsiveness of lactotrophs in ectopic pituitaries to bromocryptine and estrone acetate.

Intrasellar and transplanted pituitaries removed from the same rats were studied using light microscopy, immunocytology, electron microscopy and ultrastructural morphometry following treatment with bromocryptine and/or estrone acetate. Ectopic lactotrophs of rats treated with bromocryptine showed an increase in storage granule size and number, which was not observed in the lactotrophs of corresponding intrasellar pituitaries. In lactotrophs of intrasellar and ectopic pituitaries, treatment with estrone acetate caused a decrease of storage granule size, while rough endoplasmic reticulum (RER) and Golgi region volume density increased. When rats were treated with a combination of bromocryptine and estrone acetate, intrasellar lactotrophs exhibited increased volume density and diameter size in forming granules as well as increased RER volume density. In ectopic lactotrophs, combined treatment led to a decrease in RER, volume densities of Golgi region and forming granules, as well as increased storage granule size. It can be concluded that ectopic lactotrophs were more sensitive to the inhibiting effects of bromocryptine treatment than intrasellar lactotrophs. Intrasellar lactotrophs were more susceptible to estrone acetate than bromocryptine treatment. As a result, the increased responsiveness of ectopic lactotrophs to estrone acetate was less conspicuous.

Some functional and morphological characteristics of an acutely dispersed purified cell suspension of rat lactotrophs prepared with Percoll.

A cell suspension containing more than 90% lactotrophs can be prepared from enzymically dispersed adenohypophyses obtained from male rats pretreated with estradiol. The lactotrophs are separated from the mixed cell population by centrifugation on a discontinuous density gradient prepared from a commercial preparation of colloidal silica (Percoll, Pharmacia). The method allows isopycnic separation of these delicate cells under very mild conditions; normal ionic strength and normal pH were maintained throughout the gradient, centrifugal acceleration did not exceed 1600 X g, and all procedures were done at room temperature. Histological verification that at least 90% of the cells were lactotrophs was done using specific immunoperoxidase staining. The functional capability of the lactotrophs was established by measuring the dose--response to the dopamine agonist bromocriptine and to thyrotropin-releasing hormone (TRH). Bromocriptine decreased spontaneous release in a dose-related way over the concentration range of 10(-10) to 10(-8) M. TRH, which causes an in vivo release of prolactin (PRL) in estrogen-primed rats, produced a dose-related increase in the release of PRL over the concentration range of 3 X 10(-10) to 3 X 10(-8) M after the high spontaneous release had been previously reduced by bromocriptine (3 X 10(-8) M).

The effect of the duodenal ulcerogen cysteamine on somatostatin and gastrin cells in the rat.

Previous studies showed a rapid decrease of somatostatin concentration in the gut and an increase in serum gastrin levels after a single dose of the duodenal ulcerogen cysteamine. An attempt was made to identify morphologic changes that would correlate with these functional changes. Rats were killed 1, 4, 8, or 24 hr after a single dose of cysteamine and sections of gastric mucosa and pancreas were processed for electron and light microscopy. Subtle ultrastructural alterations were seen in D cells of the stomach (e.g., dilation of mitochondrial cristae and endoplasmic reticulum, and apparent increase in electron density of secretory granules) after cysteamine administration. The number of somatostatin-positive cells visualized by the immunoperoxidase technique using light microscopy was decreased in 1-4 hr but returned to normal by 24 hr. The alterations observed in the G cells after cysteamine administration are consistent with release of gastrin from mature granules and increased synthesis of the hormone. The lack of major morphologic changes in the D cells suggests that cysteamine affects somatostatin without causing cell necrosis or alteration in lysosome formation. The effect of the drug may thus be mediated at the biochemical level without marked morphologic alterations.

Aminoglutethimide-stimulated corticotrophs. An immunocytologic, ultrastructural and immunoelectron microscopic study of the rat adenohypophysis.

Thirty-six rats of both sexes in two groups were treated with aminoglutethimide (AG), a steroid synthesis inhibitor, for 1 and 8 weeks respectively. The anterior pituitaries were investigated by light and electron microscopy, the techniques used including immunocytochemical and morphometric methods. Corticotrophs were identified by the avidinbiotin-peroxidase complex technique at the light and electron microscopic levels. AG administration resulted in hyperplasia of ACTH-containing cells. The increase in the volume density of corticotrophs was prevented by simultaneous medication with 3 mg corticosterone in male rats, whereas in female rats a larger dose of corticosterone was required to suppress pituitary corticotroph hyperplasia. Electron microscopy revealed that in AG-treated rats, corticotrophs were larger and contained more secretory granules than those in controls, the mean secretory granule diameter increasing from 100 nm to 165 nm. Under AG stimulation, corticotrophs showed considerable variations in structural features probably reflecting differences in their functional state. It was apparent that only one ACTH-producing cell type existed in the pars distalis of the rat adenohypophysis, although this cell type may undergo substantial morphologic alterations due to changes in endocrine activity. Gonadotrophs and thyrotrophs showed morphologic signs of stimulation and exhibited hyperplasia following AG treatment indicating that the effect of the drug was not restricted to corticotrophs.

Effects of cysteamine on the hypothalamic-pituitary axis in the rat.

The effects of cysteamine (CSH) on hypothalamic concentrations of neuropeptides were reviewed and correlated with available information on changes in pituitary hormone content and circulating pituitary hormone levels. In our study, we found notable changes in the morphology of lactotropes from female Long-Evans rats treated for 7 days with CSH (300 mg/(kg X day) per os). Forming granules increased in number, and crinophagy, which is the augmented incorporation of these granules into lysosomes, was evident. Storage granules were reduced in number. These changes were not suppressed by simultaneous administration of 17 beta-estradiol (50 micrograms/day s.c.) for 7 days. CSH administration failed to prevent estrogen-induced lactotrope hyperplasia. Serum prolactin levels were unaffected by CSH treatment. The morphological changes in the adenohypophysis did not resemble those observed when rats were treated with bromocriptine. The rough endoplasmic reticulum luminal density was reduced in gonadotropes from intact CSH-treated rats after 1 wk. CSH treatment suppressed the development of castration cells and significantly reduced serum luteinizing hormone levels in ovariectomized rats. The morphological effects of CSH appeared to be confined to lactotropes and gonadotropes.

The effect of bromocriptine on the mammary glands of hyperprolactinemic rats. A histologic, ultrastructural and morphometric study.

Old female Long-Evans rats with elevated serum prolactin levels were used to study the effect of bromocriptine on the stimulated mammary glands by histology, electron microscopy, and morphometry. Untreated rats were divided into normoprolactinemic (below 30 ng prolactin/ml serum) and hyperprolactinemic control groups (above 30 ng/ml). Treated groups were injected subcutaneously with 1 mg/kg of bromocriptine once daily for 1 day, 30 days and 44 days. One group was treated with bromocriptine for 30 days, and then the drug was withdrawn for an additional 14 days. The light and electron microscopic features of breasts of hyperprolactinemic control, 1 day treatment and withdrawal groups resembled those of postpartum lactating breasts. A unique feature was the presence of large membrane-bound bodies, possibly lysosomes. The breasts of normoprolactinemic control, 30 and 44-day treatment groups were similar by histology and ultrastructure to involuted breasts after cessation of lactation. However, many large lysosome-like bodies and microvilli were found in the alveolar cells which remained tightly apposed. Hyperprolactinemic control rats showed breast stimulation which could be correlated with serum prolactin levels. Serum prolactin concentrations fell within 1 day of bromocriptine treatment, whereas breast tissue responded only after a longer interval. Pituitary weights could be correlated with serum prolactin levels. Withdrawal of bromocriptine resulted in an elevation of serum prolactin levels and breasts returned to the stimulated stage. It can be concluded that bromocriptine reversibly suppresses the stimulation of mammary glands in hyperprolactinemic rats.

An immunohistochemical and ultrastructural comparison of the effects of 2-bromo-alpha-ergocryptine on intrasellar and transplanted rat pituitaries.

Following 2 weeks of administration of 2-bromo-alpha-ergocryptine, a marked decrease was observed in prolactin immunoreactivity of the grafted pituitaries, whereas no reduction was noted in the intrasellar pituitaries. No evidence of crinophagy was revealed by electron microscopy in prolactin cells of 2-bromo-alpha-ergocryptine-treated rats.

Morphometric evidence of sexual dimorphism in the ultrastructure of gonadotroph adenomas of the human pituitary.

Gonadotroph adenomas resected from the pituitaries of 8 males and 8 females were studied by transmission electron microscopy and morphometry in order to establish whether they exhibit sexual dimorphism. Random photographs were taken and the following parameters compared: (1) cellular volume, (2) nuclear volume, (3) cytoplasmic to nuclear ratio, (4) volume density of endoplasmic reticulum, (5) volume density of Golgi bodies, (6) volume density of mitochondria, (7) volume density of secretory granules, (8) volume density of lysosomes, (9) diameter of secretory granules, (10) surface to volume ratio of secretory granules, (11) surface to volume ratio of Golgi bodies, (12) surface to volume ratio of nuclei, (13) surface to volume ratio of cells. The results showed that gonadotroph adenomas of males differed significantly from those of females in cell size, cell shape, and electron microscopic features of Golgi apparatus. It remains to be established whether differences in the morphology of non-neoplastic gonadotrophs of males and females can explain the development of morphologically distinct adenoma cells between the sexes.