Ultrastructural pathology of the adrenal gland in Cushing's syndrome

The ultrastructural pathology of the adrenal glands was studied in fifteen cases of Cushing's syndrome. Some specific features correlated with the pathological aspects of adrenals were found. In the hyperplastic adrenal cortex the cytoplasms contained a rich smooth endoplasmic reticulum and many mitochondria. Increased lipid-pigment complexes were found especially in the compact cells. In adenomas, the clear cells showed large lipid vacuoles; the compact cells presented anisomorphous mitochondria, a well developed smooth endoplasmic reticulum and many pigment bodies. The nuclei of adenomatous cells were irregular, with deep invaginations. In adrenal carcinomas, the pleomorphism of nuclei, nucleoli, mitochondria, and smooth endoplasmic reticulum was more obvious. Absolutely reliable characteristics proving malignancy at ultrastructural level do not, however, exist. The steroidogenic activity of both hyperplastic and tumoral adrenal glands can be assessed using the agranular endoplasmic reticulum and the mitochondria as functional parameters.     

Ultrastructural cytochemistry of the secretory granules of the hamster submandibular gland

Summary The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied. After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver staining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.     

Ultrastructural and histochemical observations on secretory phenomena in the resting human mammary gland

Ultrastructure and histochemistry of clinically normal appearing tissue and secretions of non-lactating human mammary glands have been investigated in order to document and analyse secretory phenomena in the resting gland. The material studied originated from women of different ages (18-74 years) who underwent plastic surgery or surgery for various disorders of the breast. The epithelia of small ducts and of alveolar enlargements as well as acini contained moderate amounts of mitochondria and of cisterns of the rough endoplasmic reticulum; the transcisterns of the Golgi apparatus which were surrounded by smooth and coated vesicles, exhibited modest dilatations, the number of lysosomes increased with age; regularly glycogen particles and bundles of intracellular filaments (phi 5 nm) were to be observed. Typical casein vesicles and stages of apocrine secretion of milk fat globules were not seen. The following features indicated secretory activity: differently sized vesicles and granules with flocculent, dense, or light contents were regularly to be seen in the apical cytoplasm often immediately below the apical plasma membrane of the epithelia of the small ducts and even more frequently in the alveolar enlargements. Secretory products of fine granular or filamentous structure, probably containing proteins, were frequently found within the lumen. Different types of lipid and liposome-like particles were detected both in intracellular localization as well as in ductal lumina. As demonstrated by lectin histochemistry the secretory products also contained a considerable amount of carbohydrate components. The composition of the secretory products of the resting gland is of clinical interest since the chronical deposition of secretions, which among others possibly contain enzymes producing oxygen radicals, may lead to pathological changes of mammary gland tissue.     

Ultrastructural evidence for the presence of secretory cells in the pineal parenchyma ofHeteropneustes fossilis

There is an extensive literature dealing with the study of indoles, especially serotonin and melatonin, but with considerably less emphasis on the cells and cell types involved in the synthetic process. In the present electron microscopical investigation of the pineal end vesicle ofHeteropneustes fossilis, pinealocytes have been revealed in the pineal parenchyma characterized with extensive synthetic apparatus viz., rough endoplasmic reticulum, free ribosomes, lipid droplets, mitochondria and Golgi bodies. Two sub-populations of the pinealocytes are easily distinguishable on the basis of electron opacity and the preponderance of one or other morphological profile: light cells and dark cells. Light cells represent the active phase of secretion while dark cells represent the storage and release phase of secretion. A neuroendocrine role for the pineal body inHeteropneustes fossilis is suggested which may be significant in view of the nocturnal habit of the fish. Dedicated to my father, Prof. C B L Srivastava     

Chromatographic evidence for high-molecular-mass galanin immunoreactivity in pig and cat adrenal glands

Galanin was measured by radioimmunoassay in extracts of pig, cat and rat adrenals using non-C- and mid to C-terminally directed antibodies. The extracts were fractioned by gel chromatography and HPLC. The non-C-terminal galanin immunoreactivity in pig was 92.8 +/- 11.7 pmol/g, in cat 9.1 +/- 0.9 pmol/g and in rat less than 1 pmol/g. Two higher molecular forms of galanin have been identified in both pig and cat adrenal. One major large form behaves as if it was N-terminally extended (Kav pig 0.58, cat 0.48) and the other, a very high-molecular-mass form (Kav pig 0.10, 0.24, cat 0.10), as if it had both N- and C-terminal extensions.     

Ultrastructural evidence for endogenous testosterone immunoreactivity in the pituitary gland of the rat

Summary Several attempts have been made to localize steroids by means of immunocytological techniques. However, these methods were found inadequate for detecting steroids bound to their receptors. To localize endogenous testosterone (T) in its target cells at the ultrastructural level, an immunocytological technique was performed on ultrathin sections obtained by cryo-ultramicrotomy. T was detected in the pituitary glands obtained from intact male or female rats and castrated rats, but not in castrated + adrenalectomized rats. Animals were also injected either with testosterone, with other steroids (estradiol, progesterone, corticosterone) or with an androgen antagonist (cyproterone acetate). In addition, some ultrathin sections were preincubated either with phosphate buffers of various pH, corticosterone, cyproterone acetate solution, or with T solution. The content of T in the pituitary before and after fixation was measured by radioimmunoassay; it decreased after fixation. T immunoreactivity was localized in the gonadotropic cells only, both in the male and female rats. At the subcellular level, the immunoreactivity was detected in the cytoplasmic matrix and in the nucleus. Immunoreactive T disappeared 1) in rats after castration+adrenalectomy; by means of radioimmunoassay no T was measured in these pituitary glands; 2) in rats injected with 25 (g/rat of cyproterone acetate; 3) after preincubation of pituitary sections on a drop of cyproterone acetate (1 × 10^-6 M). The immunocytological reaction was not modified when the rats were injected with estradiol, progesterone or corticosterone (1 mg/rat), or after preincubation of the sections with corticosterone (1 × 10^-3 M), or a buffer solution at pH 7.6. Lower or higher pH values led to a strong decrease in the immunoreactivity. After injection of T (15 g/rat) the immunocytological reaction was more abundant in the nucleus and less in the cytoplasm. The immunoreactivity was again observed when the sections were preincubated with cyproterone acetate solution and then with T solution. These data suggest that T can be detected by means of immunocytochemistry. It is probably bound to a specific binding site.This work was presented in part at the VI^th International Congress on Hormonal Steroids, Jerusalem, 1982     

Mass-spectrometric evidence for spontaneous and angiotensin-induced generation of prostacyclin by perfused cat lungs

Isolated perfused cat lungs secreted spontaneously a PGI₂-like substance that relaxed a strip of bovine coronary artery. The presence of PGI₂ was confirmed by the identification of 6-oxo-PGF_α by GC-MS. Both bioassay and mass fragmentography showed that PGI₂ was released at a rate of 4 – 12 ng/ml. Generation of PGI₂ by the perfused cat lungs was stimulated by angiotensin II (3 – 10 ng/ml). The above results along with our $\text{in vivo}$ experiments point to the lung as an important source of endogenous PGI₂ in the body.     

Morphological and functional changes in cell junctions during secretory stimulation in the perfused rat submandibular gland

To examine the influence of cholinergic and beta-adrenergic agents on paracellular transport, we applied confocal microscopy and freeze-fracture to the isolated, perfused submandibular gland of the rat. By confocal microscopy, perfusion of lucifer yellow through an arterial catheter, revealed a bright fluorescence in the basolateral spaces of acini, but not in the intercellular canaliculi. However, addition of isoproterenol on carbachol stimulation, induced lucifer yellow fluorescence in intercellular canaliculi. This finding indicates that isoproterenol is capable of opening the paracellular route. The tight junction strands surrounding intercellular canaliculi were visualized using freeze replicas. Fixation was carried out both by vascular perfusion with Karnovsky's solution and by metal contact rapid freezing with liquid helium. In the chemically-fixed specimens, the strand particles of tight junctions formed 2-5 lines at the P-face along most of the apical portion at rest. With carbachol/isoproterenol stimulation, the strand particles rearranged with free ends and terminal loops. In the rapidly frozen specimens, the strand particles were arranged more irregularly even in the resting state. The meshwork of strands became more disheveled and interrupted during carbachol/ isoproterenol stimulation. The present findings led us to conclude that: 1) the beta-adrenergic agent, isoproterenol, can open the paracellular transport. 2) in the rapidly frozen specimen, the tight junction strand particles are arranged roughly and become disheveled and interrupted during stimulation by carbachol/isoproterenol. These findings may be related to rearrangement of subcellular structures, especially of the actin filament network.     

Ultrastructural evidence for a possible secretory function of Merkel cells in the barbels of a teleost fish,Cyprinus carpio

Summary Examination of barbels of the carp (Cyprinus carpio) revealed cells showing the characteristics of Merkel cells. Some ultrastructural features of these cells suggest a secretory function.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

High-intensity exercise training produces morphological and biochemical changes in adrenal gland of mice

The effects of training are dependent on complex, adaptive changes which are induced by acute physical exercise at different levels. In particular, evidence shows that the hypothalamus-pituitary-adrenocortical axis, as well as the sympatho-adrenomedullary system, is mainly involved in mediating the physiological effects of physical exercise. The aim of the present study was to investigate, through a morphological and biochemical approach, the effects of training on the adrenal gland of mice, following two different protocols consisting of either low- or high-intensity training. Mice were run daily on a motorised treadmill for 8 weeks, at a velocity corresponding to 60% (low-intensity exercise) or 90% (high-intensity exercise) of the maximal running velocity previously determined by an incremental exercise test. We found that physical exercise produced an increase in the adrenal gland size compared with the control (sedentary) mice. The increase was 31.04% for mice that underwent high-intensity exercise and 10.08% for mice that underwent low intensity exercise, and this appeared to be the result of an increase in the area of both the adrenal cortex and adrenal medulla. Morphological analysis of the adrenal cortex showed that both types of exercise produced an increase in cytoplasmic vacuoles in steroidogenic cells, appearing more abundant after high-intensity exercise. No change was found in the reticulate zone. In the adrenal medulla, despite the absence of morphological changes, immunohistochemistry for tyrosine hydroxylase, dopamine β-hydroxylase and phenyl-ethanolamine-N-methyltransferase demonstrated an increased immunopositivity for these cathecolamine-synthesizing enzymes after intense exercise. These results were confirmed by immunoblot accompanied by densitometric analysis.     

Sex-dependent effect of melatonin on the secretory activity of rat and hamster adrenal gland in vitro

Adrenal quarters from adult male or female hamsters were incubated in the presence of melatonin (10(-7) or 10(-4)M), and cortisol concentration in the incubation medium was assayed by RIA. Melatonin did not change cortisol output by adrenals obtained from the male hamsters, while a slight stimulatory effect was observed in female glands, the lower concentration of melatonin being more effective than the higher one. At both concentrations tested, melatonin notably stimulated corticosterone output by isolated rat adrenocortical cells derived from the males, and lowered corticosterone secretion by the cells obtained from the female glands only at a concentration of 10(-7) M. The lower concentration of melatonin increased ACTH (0.1 mU.ml-1)-stimulated corticosterone output by the cells of male and female rat adrenals. The pineal hormone was ineffective at a concentration of 10(-4) M, as well as in the presence of a higher dose of ACTH (1.0 mU.ml-1). These findings indicate a distinct sex-dependent effect of melatonin on in vitro cortisol and corticosterone production, and demonstrate that the modulatory effect of melatonin of the secretion of steroid hormones is more effective at lower concentrations.     

Ultrastructural evidence for a sensory-motor neuron in Ctenophora

A survey of the nerve-net of four species of ctenophores, using electron microscopy, reveals two types of highly differentiated sensory neurons. These two kinds of receptors are widely distributed all over the ectodermal epithelia. These sensory neurons form synapse-like junctions with epithelial effectors, for example mucous secretory cells. The evolutionary and behavioural significance of such relationships is discussed.     

Control of aldosterone secretion in zona glomerulosa cell suspensions and in the perfused adrenal gland of the rat

The secretion of aldosterone and its responses to stimulation have been studied in rat adrenal zona glomerulosa tissue incubated as intact capsules or as collagenase-dispersed cell suspensions, and in intact perfused rat adrenal glands. Several differences are apparent in the functions of the various preparations. Aldosterone secretion rates are similar in incubated intact capsules and in the perfused gland. Relative to corticosterone, lower yields of aldosterone are obtained in dispersed glomerulosa cell in vitro. This may be related to the loss in the dispersed cells of a pool of tissue steroid (aldosterone or a precursor) which is revealed only in intact tissue incubations by trypsin stimulation of aldosterone secretion. Trypsin-released aldosterone is increased by prior dietary sodium restriction. In addition, differences occur in the responses of dispersed cells and perfused glands to stimulation. Perfused glands from animals on a normal diet are less sensitive to stimulation by ACTH or alpha-MSH, but more sensitive than dispersed cells to angiotensin II amide. In the perfused gland, sensitivity of response (lowest effective concentration) to all three stimulants is increased by prior dietary sodium restriction, in contrast to dispersed cells in which increased sensitivity has been reported only to alpha-MSH. The perfused gland is particularly sensitive to angiotensin II amide, and a bolus administration of 1 amol gives significant stimulation in glands from animals on low sodium intake. Electrical (field) stimulation or dopamine administration at 10(-6) mol/l (which is ineffective in dispersed cells) both depress aldosterone secretion by the perfused gland. The data suggest that the sequestered pool of steroid is utilized in the perfused gland for aldosterone secretion. They furthermore suggest that in the intact gland there are mechanisms, which involve neural components, for intraglandular regulation of aldosterone secretion, which are lost in dispersed cells in vitro. Such mechanisms may be involved in sensitivity increases in sodium depletion.     

Identification of a biochemical marker for the secretory pathway in Tetrahymena thermophila

Tetrahymena thermophila is a ciliated protozoan studied by investigators from a wide range of disciplines as a model system for a variety of specialized eukaryotic cell functions. The proteinaceous secretory products of T thermophila have been isolated and characterized and in this study we identify the major secretory product, a 34,000 Mr polypeptide, and use an antiserum prepared against this secretory protein to (1) demonstrate that this 34,000 Mr polypeptide is truly a secreted protein in Tetrahymena and (2) monitor the synthesis and transport of this protein by indirect immunofluorescence and light microscopy during mucocyst biogenesis.