Multiple pigmented adrenal cortical nodules associated with Cushing's syndrome. Histochemical, ultrastructural and quantitative studies

Multiple pigmented adrenocortical nodules were found in a 25-year-old woman associated with Cushing's syndrome, whose laboratory data indicated that the adrenal cortex had been functioning autonomously and adrenocorticotrophic hormone (ACTH) from the pituitary gland as suppressed. The surgically removed left adrenal gland disclosed multiple black nodules measuring up to 3 mm in diameter and histologically consisting of large "compact cells" which contained numerous yellow-brown pigments, but adjacent cortical cells were not atrophied. This kind of adrenal lesion is generally regarded as nodular hyperplasia of the cortex. The present case revealed scanty lipid and markedly increased activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and glucose-6-phosphate-dehydrogenase (G6PD). Ultrastructural study showed abundant cytoplasm with a large number of mitochondria, well-developed smooth-surfaced endoplasmic reticulum (SER), less rough-surfaced endoplasmic reticulum (RER), lysosomes, and numerous granules in cells of the nodules. Mitochondria varied in size and shape up to occasional giant mitochondria. SER was vesicular or tubular forming a network of anastomosing tubules. Granules varied greatly in size from 400 millimicrons to 6 microns in diameter, with diverse electron densities, mostly exhibiting the structural features of lipofuscin. The ultrastructural features resembled those in black adenoma associated with Cushing's syndrome ever reported. Concentration of cortisol was increased in the tissue where numerous black nodules were contained.     

Effects of exogenous ACTH on the rat fetal adrenal cortex: a histochemical and electron-microscopical observation.

ACTH was administered subcutaneously to rat fetus directly at the late stage of fetal development and acute reaction on the fetal adrenal cortex was observed histochemically and electron microscopically. By administration of ACTH the adrenal cortex became remarkably hyperemic and there were swollen cells in all layers, particularly in the middle and inner layers (corresponding to the zona fasciculata and reticularis in adult rat). Marked reduction of lipid, enlarged mitochondria with increased vesicular cristae and increased smooth-surfaced endoplasmic reticulum (SER) were characteristic. The alterations of mitochondria preceded the change of SER, and thereafter mitochondria showed rapid degeneration. The outer layer (corresponding to the zona glomerulosa in adult rat) also showed similar changes by ACTH to those of the other two layers. These results indicated that the fetal adrenal cortex of rats was exogenous ACTH-reactive and its reaction which was different from that of adult cortical cells, seemed to be specifically related to the development and differentiation of the cells.     

Electron microscopic studies on the effect of ACTH and flavin adenine dinucleotide on adrenocortical atrophy of hypophysectomized rat.

Electron microscopic observation was made on the outer fasciculata cells in the adrenal cortex of hypophysectomized rats receiving 10 mg of FAD and/or 0.3 mg of ACTH intraperitoneally once a day for 5 consecutive days from 24 hours after hypophysectomy. The simultaneous administration of FAD and ACTH to the hypophysectomized rat was more effective for preventing adrenocortical atrophy induced than the administration of ACTH alone. This effect appeared as clear cells with low electron density. While the characteristics induced by hypophysectomy were the decrease in number of smooth-surfaced endoplasmic reticulum (SER) and mitochondria and also crista of mitochondria being tubular. The clear cells showed a less degree of their characteristic. From this fact, it is considered that the external FAD acts against fasciculata cells in the adrenal cortex of hypophysectomized rats as a coenzyme for flavin enzyme under ACTH and decreases oxidation-reaction in mitochondria and oxidative phosphorilation reaction in SER, being induced by hypophysectomy.     

EFFECTS OF EXOGENOUS ACTH ON THE RAT FETAL ADRENAL CORTEX: A HISTOCHEMICAL AND ELECTRON-MICROSCOPICAL OBSERVATION

ACTH was administered subcutaneously to rat fetus directly at the late stage of fetal development and acute reaction on the fetal adrenal cortex was observed histochemically and electron microscopically. By administration of ACTH the adrenal cortex became remarkably hyperemic and there were swollen cells in all layers, particularly in the middle and inner layers (corresponding to the zona fasciculata and reticularis in adult rat). Marked reduction of lipid, enlarged mitochondria with increased vesicular cristae and increased smooth-surfaced endoplasmic reticulum (SER) were characteristic. The alterations of mitochondria preceded the change of SER, and thereafter mitochondria showed rapid degeneration. The outer layer (corresponding to the zona glomerulosa in adult rat) also showed similar changes by ACTH to those of the other two layers. These results indicated that the fetal adrenal cortex of rats was exogenous ACTH-reactive and its reaction which was different from that of adult cortical cells, seemed to be speciflcally related to the development and differentiation of the cells. ACTA PATH. JAP. 27 : 477–484, 1977.     

ELECTRON MICROSCOPIC STUDIES ON THE EFFECT OF ACTH AND FLAVIN ADENINE DINUCLEOTIDE ON ADRENOCORTICAL ATROPHY OF HYPOPHYSECTOMIZED RAT

Electron microscopic observation was made on the outer fasciculata cells in the adrenal cortex of hypophysectomized rats receiving 10 nag of FAD and/ or 0.3 mg of ACTH intraperitoneally once a day for 5 consecutive days from 24 hours after hypophysectomy.
The simultaneous administration of FAD and ACTH to the hypophysectomized rat was more effective for preventing adrenocortical atrophy induced than the administration of ACTH alone. This effect appeared as clear cells with low electron density. While the characteristics induced by hypophysectomy were the decrease in number of smooth-surfaced endoplasmic reticulum (SER) and mitochondria and also crista of mitochondria being tubular. The clear cells showed a less degree of their characteristic. From this fact, it is considered that the external FAD acts against fasciculata cells in the adrenal cortex of hypophysectomized rats as a coenzyme for flavin enzyme under ACTH and decreases oxidation-reaction in mitochondria and oxidative phosphorilation reaction in SER, being induced by hypophysectomy. ACTA PATH. JAP. 27: 637˜645, 1977.     

Observations on the fine structure of propylthiouracil-induced "brown degeneration" in the zona reticularis of mouse adrenal cortex.

Propylthiouracil (6-propyl-2-thiouracil), an anti-thyroid agent, was fed to mice in a concentration equal to 0.1% of their diet for periods of 10 and 15 weeks. The cells of the inner zone of the adrenal cortex were examined with the electron microscope. In animals receiving propylthiouracil for ten weeks mitochondria were altered and the smooth endoplasmic reticulum (SER) showed a marked focal proliferation. In contrast to control animals rough endoplasmic reticulum was abundant and was frequently associated with the hyperplastic SER. After 15 weeks these alterations were no longer present but had been replaced by a spectrum of "brown degeneration." The less affected cells were characterized by increased numbers of liposomes and lysosomes and the more affected cells by liposomal and mitochondrial degeneration. These observations emphasize that "brown degeneration" is a true degenerative process and not a spontaneous proliferation of ceroid pigment. It is suggested that the changes described may be directly related to an alteration in cholesterol metabolism.     

American association of anatomists. Eighty eighth annual session,University of California Schools of Medicine and Dentistry,Los Angeles,California

Propylthiouracil (6-propyl-2-thiouracil), an anti-thyroid agent, was fed to mice in a concentration equal to 0.1% of their diet for periods of 10 and 15 weeks. The cells of the inner zone of the adrenal cortex were examined with the electron microscope. In animals receiving propylthiouracil for ten weeks mitochondria were altered and the smooth endoplasmic reticulum (SER) showed a marked focal proliferation. In contrast to control animals rough endoplasmic reticulum was abundant and was frequently associated with the hyperplastic SER. After 15 weeks these alterations were no longer present but had been replaced by a spectrum of “brown degeneration.” The less affected cells were characterized by increased numbers of liposomes and lysosomes and the more affected cells by liposomal and mitochondrial degeneration. These observations emphasize that “brown degeneration” is a true degenerative process and not a spontaneous proliferation of ceroid pigment. It is suggested that the changes described may be directly related to an alteration in cholesterol metabolism.     

The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum

The normal fine structure of the zona glomerulosa and the zona fasciculata of the opossum is described, with emphasis on the structure of the smooth surfaced endoplasmic reticulum (SER) and mitochondria. The cells of the zona glomerulosa are characterized by the presence of numerous rod-shaped mitochondria with shelf-like cristae, small amounts of tubular SER and moderate amounts of rough surfaced endoplasmic reticulum. The cells of the zona fasciculata possess large quantities of SER, spherical mitochondria with tubular cristae and a few profiles of rough surfaced reticulum. A Golgi apparatus is present in cells of both zones as are coated vesicles, coated invaginations of the plasmalemma, lysosomes and lipid droplets. It is concluded that the tubular SER seen in electron micrographs corresponds closely to the form of this membrane system found in the living cell. The functional significance of the associations observed among lipid droplets, mitochondria and SER are discussed.     

Adrenocorticotropin-independent unilateral adrenocortical hyperplasia with Cushing's syndrome: Immunohistochemical studies of steroidogenic enzymes, ultrastructural examination and a review of the literature

A 60-year-old woman presented with a history of palpitations, headaches and severe hypertension, which was resistant to hypotensive agents. She had a 2-year history of obesity and a moon face. Her plasma adrenocorticotropic hormone level was below the limits of detection and did not respond to corticotropin-releasing hormone. Urinary-free cortisol was elevated and the circadian rhythm of serum cortisol level had completely disappeared. Imaging analysis demonstrated a unilaterally functioning mass in the left adrenal gland. Serum cortisol level in the left adrenal vein was elevated. The resected adrenal mass measured 4 x 3.5 x 2.5 cm, and ranged from yellow to tan in color. The adrenal cortex adjacent to the nodule did not demonstrate cortical atrophy. The mass was well circumscribed but not encapsulated, and consisted of multiple cortical nodules. These nodules were composed predominantly of clear cortical cells, and partly of compact cortical cells. Immunoreactivity of steroidogenic enzymes including cholesterol side-chain-cleavage P450, 3beta-hydroxysteroid dehydrogenase, 21-hydroxylase cytochrome P450, 11beta-hydroxylase cytochrome P450 and 17alpha-hydroxylase cytochrome P450 was marked in cortical nodules, but minimal in non-nodular cortex. Ultrastructural examination of nodular cortical cells also demonstrated well-developed mitochondria and smooth endoplasmic reticulum, consistent with elevated steroidogenesis in these cells.     

Ultrastructure of the fetal rat adrenal gland at full-term and during prolonged gestation

The ultrastructure of the fetal rat adrenal gland is described at full-term (day 22) and during artificially prolonged gestation (days 23, 24, 25). The latter was achieved by daily subcutaneous injections of seven mg progesterone to gravid females from the twentieth through the twenty-fourth day. Cortical and medullary cells contained well developed organelles at all stages. Nonetheless, zonation of the gland was not yet distinct. Whereas the zona glomerulosa and zona fasciculata were fairly well delineated, the zona reticularis and medulla were as yet poorly defined. The latter regions were intermingled, and thus collectively referred to as the “zona reticulo-medullaris.” During prolonged gestation (days 24 and 25 only), many cells of the zona fasciculata and zona reticulo-medullaris contained dilated smooth endoplasmic reticulum and numerous enlarged mitochondria. Enlarged mitochondria were observed also in medullary cells concurrent with a paucity of catecholamine storage granules. Moreover, meconium staining, which is indicative of fetal stress, was also observed on days 24 and 25. The ultrastructural changes observed were interpreted to be an expression of response by the fetal adrenal gland to fetal stress produced by as yet undetermined factors arising during prolonged gestation.     

免疫组化电镜示垂体LH细胞

应用免疫组化电镜显示垂体黄体生成素(LH)细胞的形态特徵、超微结构以及反应阳性颗粒的分布状况等,并按此将LH细胞分为四型。一、二型细胞内大小分泌粒均含有LH;三型细胞长(?)的150 nm及200 nm颗粒显免疫反应阳性,且部分胞膜和微绒毛顶部界膜内外附着致密细粒;四型细胞出现反应阴性颗粒,仅在rER膜囊外显有直径30～40 nm黑色细粒。LH细胞的分泌可能以外排、分子渗透和局部分泌三种方式排出LH。     

Estrogen-producing adrenocortical carcinoma. A light and electron microscopic study

A case of adrenocortical carcinoma with feminization seen in a man aged 35, is reported. The levels of estron (E1) and estradiol (E2) in the venous blood draining the tumor were high, and it was confirmed by in vitro assay of tumor cells taken from the primary tumor of the left adrenal gland that the tumor produced estrone. The light microscopic examination demonstrated that the primary tumor was composed of mixture of large cells with pleomorphic nuclei and vacuolated cytoplasm and uniform cells with ovoid nuclei and eosinophilic cytoplasm. The electron microscopic examination on the latter cells revealed numerous large and irregularly shaped mitochondria with mostly tubular or lamellar and occasionally vesicular cristae and electron-dense matrix, well-developed smooth-surfaced endoplasmic reticulum in the cytoplasm. However, lipid droplets and lysosomes or lipofuscin granules were scanty. From these findings, it is suggested that cells of the present tumor have characteristics of those in the zona reticularis of the adrenal cortex as well as in the fetal cortex, and the functional property of this tumor is well correlated with its morphological features.     

Ultrastructural evidence for steroid-hormone production in the adrenal of the marsupial,Isoodon macrourus,at birth

To determine whether the adrenal gland of the bandicoot is actively producing steroid hormones at birth, the cellular structure was examined by electron microscopy, and the concentration of cortisol in the gland was determined by radioimmunoassay. Two distinct cell populations were seen in the adrenal gland. One group of cells possessed dark staining granules, normally observed in catecholamine-secreting cells, and the second group contained large amounts of smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae. Both features are characteristic of steroid-secreting cells. A concentration of 0.094 ng of cortisol per adrenal was determined by pooling glands from nine individual newborn bandicoots. This evidence of cortisol secretion from the bandicoot adrenal at birth indicates that, as in many eutherians, the marsupial adrenal may play an important role in determining the length of gestation.     

ULTRASTRUCTURAL ZONATION OF THE HUMAN ADRENAL CORTEX

Electron microscopic observations of sections from adrenal cortex obtained from a twenty-four-year-old male with hypertension were made. In the zona glomerulosa the cells showed a large nuclear-cytoplasmic ratio. The mitochondria had a dark matrix and plate-like inner structure. The agranular endoplasmic reticulum revealed a close association with lipid granules. The Golgi apparatus was prominent and frequently accompanied by centrioles. Free ribosomes were abundant. The zona fasciculata can be subdivided into two layers. In the outer layer the cells are characterized by numerous large lipid granules. Two kinds of mitochondria are discernible; one with a pale matrix and tubulo-vesicular or vesicular inner structure and the other with a dark matrix and tubulo-vesicular or lamelllform cristae. In the inner zona fasciculata and reticularis there are two types of cells besides the dark cells. The type 1 cells have a compact cytoplasm, elliptic or irregularly shaped mitochondria with a dark matrix and tubulo-vesicular or vesicular internal structure. The type 2 cells are large and ovoid, having a clear transparent cytoplasm. The mitochondria are numerous having a clear matrix and almost exclusively vesicular inner elements. Abundance in agranular endoplasmic reticulum, decreased lipid granules, and cytoplasmic incisions are common in both the inner fasciculata and reticularis. In addition the zona reticularis is characterized by increased number of dark cells, numerous pigment bodies, and the appearance of huge mitochondria.
On the basis of these observation were discussed the significance of the organelles in steroid biosynthesis, mode of hormone secretion, and cyto-genesls in the adrenal cortex.     

Adrenal gland: structure, function, and mechanisms of toxicity

The adrenal gland is one of the most common endocrine organs affected by chemically induced lesions. In the adrenal cortex, lesions are more frequent in the zona fasciculata and reticularis than in the zona glomerulosa. The adrenal cortex produces steroid hormones with a 17-carbon nucleus following a series of hydroxylation reactions that occur in the mitochondria and endoplasmic reticulum. Toxic agents for the adrenal cortex include short-chain aliphatic compounds, lipidosis inducers, amphiphilic compounds, natural and synthetic steroids, and chemicals that affect hydroxylation. Morphologic evaluation of cortical lesions provides insight into the sites of inhibition of steroidogenesis. The adrenal cortex response to injury is varied. Degeneration (vacuolar and granular), necrosis, and hemorrhage are common findings of acute injury. In contrast, chronic reparative processes are typically atrophy, fibrosis, and nodular hyperplasia. Chemically induced proliferative lesions are uncommon in the adrenal cortex. The adrenal medulla contains chromaffin cells (that produce epinephrine, norepinephrine, chromogranin, and neuropeptides) and ganglion cells. Proliferative lesions of the medulla are common in the rat and include diffuse or nodular hyperplasia and benign and malignant pheochromocytoma. Mechanisms of chromaffin cell proliferation in rats include excess growth hormone or prolactin, stimulation of cholinergic nerves, and diet-induced hypercalcemia. There often are species specificity and age dependence in the development of chemically induced adrenal lesions that should be considered when interpreting toxicity data.     

Spironolactone bodies in an adrenal adenoma.

Spironolactone bodies were observed in an adrenal cortical adenoma that was removed from a patient with primary hyperaldosteronism (Conn syndrome) treated preoperatively with spironolactone. The electron microscopical evaluation of this adrenal cortical adenoma shows origin of spironolactone bodies from whorls of endoplasmic reticulum in cells with the cytoplasmic features of those from the zona glomerulosa of the adrenal gland. There was no evidence that the bodies were derived from mitochondria, which confirms recent ultrastructural findings in patients treated with spironolactone. These bodies have been described in the adrenal cortex only in patients who have received spironolactone, and the pharmacologic specificity of the bodies strongly suggests a direct mode of action by spironolactone on aldosterone production by cells of the adrenal zona glomerulosa.     

Bilateral primary pigmented nodular adrenocortical disease--a case of report describing a rare cause of Cushing's syndrome.

A case of Cushing''s syndrome due to bilateral pigmented nodular adrenal disease in a 35-year-old male is presented. The adrenals showed multiple, black, variable sized nodules. Histologically the cells contained lipofuscin and either had a clear cytoplasm or an eosinophilic cytoplasm with a prominent nucleus. Lymphocytic infiltration and fatty metaplasia within the nodules are two of the prominent histological features. There is extreme internodular atrophy which suggests that primary pigmented nodular adrenocortical disease is a non-adrenocorticotropic hormone dependent condition. Since the disorder appears to involve primarily the cortex of both adrenals, the treatment of choice is bilateral adrenalectomy followed by steroid replacement. The characteristic clinicopathological manifestations that separate this diagnosis from other types of adrenal disease are also discussed. This is the first reported case in Korea to be documented with the pertinent clinicopathological findings.     

The adrenal cortex in spontaneously hypertensive rats. A quantitative ultrastructural study.

The adrenal cortex of spontaneously hypertensive rats (SHR) has been examined by quantitative morphologic techniques for electron microscopy. The volume and surface area of smooth endoplasmic reticulum and the volume of Golgi apparatuses in zona glomerulosa cells of SHR was significantly greater than those of Wistar-Kyoto strain (W/KY) normotensive controls; the volume of lipid droplets and nucleus was significantly less in SHR than in W/KY animals. A stimulation of the zona glomerulosa in SHR may well be attributable to the elevation in systolic blood pressure. A distinct lipid-free subglomerulosa was observed in the adrenal gland of W/KY rats; the cell volume was similar to that of the zona glomerulosa although the cells showed a significantly greater volume of mitochondria and surface area of mitochondrial membranes and greater volume of smooth endoplasmic reticulum and lysosomes. In the zona fasciculata, cell volume, volumes and surface area of mitochondria and smooth endoplasmic reticulum, and volume of lipid droplets were significantly lower in SHR than in W/KY rats. The volume of the Golgi apparatus was greater in SHR than in W/KY rats. Glycogen particles were observed in focal areas of some zona fasciculata cells. The adrenal cortex of another strain of normotensive Wistar rat (W/CFN) was compared with that of the W/KY and SHR. Although the relative adrenal weights of SHR and W/KY animals were identical, the weight of that in W/CFN was significantly smaller. The volume of the zona glomerulosa of SHR was significantly greater than that of W/KY although the volume of the zona glomerulosa in W/CFN was significantly greater than the other two groups. The volume of nucleus and lipid droplets of zona glomerulosa in W/KY was significantly greater than that in the S/CFN; the volume of the cell, mitochondria, smooth endoplasmic reticulum, lipid droplets, and lysosomes, and the surface area of smooth endoplasmic reticulum and mitochondrial membranes of W/KY animals was significantly greater than those of W/CFN animals. It is concluded that the W/CFN rat is not an appropriate control for spontaneously hypertensive rats.     

The development of adrenal homolog of rainbow trout<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>: an immunohistochemical and ultrastructural study

In this work we describe the adrenal homolog of the rainbow trout Oncorhynchus mykiss during development. At the histological level, the interrenal primordium is clearly evident in larvae 25 days after fertilization (dpf), and the immunohistochemical reactions for tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT), which mark the chromaffin cells, appear as early as 27 dpf. Both reactions are evident in cells localized in the head kidney and in some, probably migrating, cells close to the notochord. In 27-dpf larvae, the ultrastructural analysis shows the presence of the interrenal cells with mitochondria with tubulovesicular cristae, typical of steroidogenic cells, sometimes surrounded by smooth endoplasmic reticulum (SER) cisternae, indicating that in this stage the cells have the capacity for steroid synthesis and secretion. In the same stage the chromaffin cells are characterized by few and small membrane-bound granules containing cores of heterogeneous electron density. Both types of cells show large nuclei, numerous free or clumped ribosomes, developed rough endoplasmic reticulum (RER), and scarce SER. Rare nerve endings contacting chromaffin cells are present. In the subsequent developmental stages, a further differentiation of both types of cells is evidenced by modifications of cell organelles as mitochondria, chromaffin granules, RER, SER, and so on. A clear discrimination of the two types of catecholamine-containing cells, adrenaline and noradrenaline cells, is evident only 5 days after hatching. The presence of different interrenal cell types in larvae at 5 and 10 days after hatching probably indicates the activation of a physiological cellular cycle. The immunohistochemical and ultrastructural results are compared with those obtained by other authors in the same and other vertebrate species.     

Fine structure and morphogenesis of spironolactone bodies in the zona glomerulosa of the human adrenal cortex

Numerous spironolactone bodies have been detected in the zona glomerulosa cells of the adrenal cortex of a 36-year-old spironolactone-treated woman whose non-tumorous right adrenal gland was removed surgically because of primary hyperaldosteronism. Electron microscopy revealed spherical laminated whorls which consisted of a central core composed of an amorphous electron-dense material surrounded by numerous smooth-walled concentric membranes. Continuous with and deriving from the endoplasmic reticulum, they were present in viable cells and were not associated with ultrastructural features indicating cellular injury. Cytoplasmic inclusions similar to spironolactone bodies can be detected in other organs after the administration of various compounds. Thus, they can be regarded as neither specific to spironolactone treatment nor exclusively inducible in the zona glomerulosa of the adrenal cortex.