Immunocytochemical localization of adrenodoxin in bovine adrenal cortex by protein A-gold technique

Summary To investigate the intracellular localization of the enzymes that are involved in steroid hormone synthesis, an immunocytochemical study of the distribution of adrenodoxin in cells of the bovine adrenal cortex was carried out by the post-embedding immunostaining method and by immuno-cryoultramicrotomy in combination with the protein A-gold technique. Gold particles were seen on the matrix and the inner membrane of all the mitochondria examined, which have typical vesicular or tubulo-vesicular cristae, in parenchymal cells of the zona fasciculata and the zona reticularis. Gold particles were distributed homogeneously among the mitochondria. The density of gold particles on mitochondria in the parenchymal cells of the zona glomerulosa was lower than that of the zona fasciculata, which was similar to that of the zona reticularis. Gold particles were also seen on round, electron-dense intramitochondrial bodies in the parenchymal cells. The intramitochondrial bodies were abundant in the zona glomerulosa and the outer region of the zona fasciculata.Supported by a Grant-in-Aid for Scientific Research on Priority Areas (no. 63635505) from the Ministry of Education, Science and Culture of Japan     

Intramitochondrial bodies in sheep adrenal zona glomerulosa cells

Summary Relatively large, mostly rounded, very electron dense intramitochondrial bodies in adrenal zona glomerulosa cells of sheep are described and their nature and connection to protein in the mitochondria discussed. The so called azocarmine granules seen in the light microscope may be identical with the intramitochondrial bodies in the zona glomerulosa cells.     

Effect of acute heat stress on rat adrenal cortex — a morphological and ultrastructural study

The stereological structure of rat adrenal gland was analysed by light and electron microscopy after an acute (60 min) exposure to high ambient temperature (38°C). Under these conditions a significant increase in plasma corticotrophin (ACTH), serum corticosterone and aldosterone levels were observed. Histological and stereological investigation at light microscopy showed significant decrease in volume density of capsule and zona glomerulosa, increase in volume of fasciculata cells, and decrease of numerical density of zona fasciculata cells and mean diameter of blood vessels. At the ultrastructural level, volume density of nuclei and mitochondria of zona glomerulosa cells were significantly increased and that of lipid droplets decreased. Volume density of mitochondria of fasciculata cells was significantly increased, while number of lipid droplets per μm² of cell was reduced. In the cells of zona reticularis significant increase in the number of lipid droplets was found. The response of zona glomerulosa may be interpreted as immediate reaction to dehydration, while alterations detected in zona fasciculata, which were less extensive, were related to purely stressogenic effects of high ambiental temperature.     

Adrenal cortical foci and nodules in Sprague-Dawley rats treated with N-nitrosomorpholine. Light and electron microscopic findings

Treatment of male Sprague-Dawley rats with N-nitrosomorpholine (NNM) in the drinking water at a dose of 120 mg/l for 7 weeks resulted in a subsequent enhanced development of focal and nodular lesions in the adrenal cortex. Sequential observation revealed that focal lesions in the zona reticularis/fasciculata or the zona glomerulosa developed both earlier and at a significantly higher incidence in animals treated with carcinogen than in untreated controls. Foci observed within or adjacent to the zona glomerulosa were all of pale cell appearance and contained large numbers of electron-dense cytoplasmic granules similar to those observed in normal granulosa cells. The foci and nodules which arose in the zona reticularis/fasciculata were, in contrast, characterized by a reduction or loss of the dense osmiophilic droplets normally seen in the cells of this region of the adrenal cortex, a pronounced increase in pleomorphic mitochondria of atypical appearance and the development of vacuoles.     

Immunohistochemical localization of adrenal ferredoxin and distribution of adrenal ferredoxin and cytochrome P-450 in the rat adrenal

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P-450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P-450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P-450 are not distributed uniformly throughout the rat adrenal cortex.     

Cytochrome P-45011 beta and P-450scc in adrenal cortex: zonal distribution and intramitochondrial localization by the horseradish peroxidase-labeled antibody method

In an attempt to elucidate the regulation mechanism(s) of adrenocortical steroidogenesis, cytochrome P-450scc and cytochrome P-45011 beta were localized in bovine adrenal glands by the direct peroxidase-labeled antibody method. At the light microscopic level, parenchymal cells of the zona fasciculata and the zona reticularis stained heavily for both cytochromes, while the parenchymal cells of zona glomerulosa stained lightly for both. At the electron microscopic level, these two cytochromes were associated with the matrix side of the inner mitochondrial membranes of parenchymal cells from all three zones of the adrenal cortex. The association of cytochrome P-450 with the inner mitochondrial membrane, in a manner similar to that previously reported for adrenodoxin and adrenodoxin reductase (F Mitani, Y Ishimura, S Izumi, K Watanabe, Acta Endocrinol 90:317, 1979), establishes that the steroid monooxygenase systems exist at this site. The degree of immunocytochemical staining within a single cell varied from one mitochondrion to another: some stained intensely along the entire inner membrane, including the cristae, some stained only along segments of the inner membrane, and some did not stain at all. This heterogeneity in staining was observed in mitochondria stained in situ as well as in isolated mitochondria. These findings suggest that there is a heterogeneity in steroidogenesis among mitochondria contained within a single cell of the adrenal cortex.     

Steroidogenic characteristics of the adrenal cortex of the mare studied by electron microscopy

The three concentric zones of the horse adrenal cortex (zonae glomerulosa, fasciculata and reticularis) showed marked interpenetration and exhibited a different relative development according to their position in the gland. Whereas the three cortical zones each had a specific histological structure, the ultrastructure of their cells showed a certain qualitative homogeneity. The differences corresponded essentially to the relative abundance of the constituents which are generally considered typical of steroidogeneous cells: mitochondria with vesicular cristae, smooth endoplasmic reticulum and lipid droplets. Their importance increased progressively from the zona glomerulosa to the zona reticularis. In this zone, the presence of gap and septate-like cell junctions, and mitochondria with vesicular cristae in close proximity to a smooth endoplasmic reticulum with numerous dilated tubules, suggested that steroidogenesis may be the most active. Ultrastructural findings were indicative of only quantitative differences between the steroidogenic capacities of the three zones of the mare adrenal cortex.     

Angiotensin II-induced inositol phosphate production in isolated rat zona glomerulosa and fasciculata/reticularis cells

Angiotensin II (2.5 to 250nM) induced, within 60 sec, a significant increase in [3H]inositol-labeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate in rat zona glomerulosa cells. Neither ACTH (3nM) nor K+ (8.4mM) had any effect, although aldosterone and corticosterone were significantly stimulated by all three agonists (after 30 min incubation). A similar significant dose-dependent increase in the inositol phosphates was observed with angiotensin II in zona fasciculata/reticularis cells after 30 min, but without any effect on corticosterone. In contrast ACTH significantly increased corticosterone with only a small although highly significant increase in inositol trisphosphate and inositol bisphosphate at 0.03nM ACTH. However at the higher dose (3.0nM) only inositol bisphosphate was significantly increased. These results indicate the presence on both zona glomerulosa and zona fasciculata/reticularis cells of AII receptors, which were linked to the formation of the secondary messenger, but only in the zona glomerulosa cells are associated with steroidogenesis.     

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.     

Sex differences in adrenocortical structure and function

Summary Adrenal glands of adult female rats are heavier than the glands of corresponding male rats. Postpubertal orchiectomy increases the adrenal weight, an effect restored by testosterone replacement. Under the same conditions ovariectomy of 8 weeks duration does not change the adrenal weight while estradiol replacement enhances the relative adrenal weight.Karyometric studies have shown that nuclei in the female zona fasciculata cells are larger (app. 18%) than those of the male. Similar but only slight differences (2%) were observed in the zona reticularis. Orchiectomy results in enlargement of cell nuclei within all zones of the adrenal cortex; testosterone replacement has the opposite effect. Ovariectomy of 8 weeks duration slightly enhances the volume of nuclei of the zona glomerulosa cells, has no effect on the nuclei of the zona fasciculata and reduces the volume of nuclei in the zona reticularis. Estradiol replacement reduces the volume of nuclei of the zona glomerulosa cells but increases nuclear volume in the zona fasciculata and in the zona reticularis.Thus testosterone has an inhibitory effect on the adrenal cortex of the rat while the physiologic effect of estradiol on the morphology of this gland, particularly on the zona fasciculata cells is rather dubious.The author wishes to thank Mrs. B. Westerska and Miss K. Siejak for excellent technical assistance.This research was supported in part by a grant from the Zoological Committee, 2nd Department, Polish Academy of Sciences.This paper is dedicated to the memory of late Kazimierz Mietkiewski, M.D., Ph.D. whose encouragements and suggestions were most stimulating for my work.     

Autoradiographic localization of vitamin A in the adrenal gland of rats.

Cellular distribution of vitamin A in the rat adrenal was evaluated by autoradiography. Vitamin A was concentrated in the lipid droplets of epithelial cells of the zona fasciculata and zona reticularis. A small amount of vitamin A was also present in the cytoplasm and nucleus of these cells. The zona glomerulosa contained very little vitamin A either in lipid droplets or in the remainder of the cell. The medulla had essentially no vitamin A. Such strong cellular specificity for uptake of vitamin A by the cells of the zona fasciculata and zona reticularis supports the hypothesis that vitamin A is involved in synthesis of glucocorticoid hormones.     

Ultrastructural Characteristics of Adult Rat Adrenocortical Cells Maintained in vitro With and Without ACTH

Bhattacharyya, T. K., Butler, D. G., Price, C. S. 1980. Ultrastructural characteristics of adult rat adrenocortical cells maintained in vitro with and without ACTH. (Ramsay Wright Zoological Laboratories, Department of Zoology, University of Toronto, and Division of Endocrinology, Toronto Western Hospital, and Department of Medicine, University of Toronto, Canada.) — Acta zool. (Stockh.) 61(1): 9–21. The ultrastructural morphology of adrenocortical cells of adult rats maintained in culture for four days with and without corticotropin (ACTH) was studied in comparison to freshly dissociated cells. Identification of cells belonging to zona glomerulosa, zona fasciculata, and zona reticularis was made on the characteristics of the mitochondria and smooth endoplasmic reticulum (SER), and was confirmed by comparison with cellular layers from intact adrenals. Without ACTH, glomerulosa and fasciculata cells showed disappearance of SER, atrophied Golgi apparatus (GA), and a striking proliferation of granular reticulum. Fascicular cells had a loss in mitochondrial matrix density and the mitochondrial cristae showed a tendency to convert to lamellar glomerulosa-type cristae. Zona reticularis cells were not strikingly altered. Maintenance with ACTH led to increased cell size and islet formation of cortical cells. Glomerulosa cells had normal appearance and fasciculata cells manifested a pronounced development of GA and SER, and a normal configuration of mitochondria. The effects of ACTH on glomerulosa cells suggest a trophic influence of ACTH on these cells in vitro. The alterations observed in fascicular cell mitochondria and SER can be explained in terms of known concepts of steroid biosynthesis and basically agree with the patterns of steroid synthesis observed in these cells previously (Price et al. 1975).     

Leukotrienes as common intermediates in the cyclic AMP dependent and independent pathways in adrenal steroidogenesis

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and adrenocorticotropin (ACTH). Since the mitochondria can recognize factors generated by AII (cyclic-AMP-independent) and ACTH (cyclic AMP dependent), it is reasonable to postulate the existence of a common intermediate in spite of a different signal transduction mechanism. We have evaluated this hypothesis by stimulation of mitochondria from glomerulosa gland with fractions isolated from glomerulosa gland stimulated with AII or from fasciculata gland stimulated with ACTH; the same fractions were tested using mitochondria from fasciculata cells. Postmitochondrial fractions (PMTS) obtained after incubation of adrenal zona glomerulosa with or without AII (10(-7) M) or ACTH (10(-10) M), were able to increase net progesterone synthesis 5-fold in mitochondria isolated from non-stimulated rat zona glomerulosa. In addition, AII in zona glomerulosa produced in vitro steroidogenic fractions that were able to stimulate mitochondria from zona fasciculata cells. Inhibitors of arachidonic acid release and metabolism blocked corticosterone production in fasciculata cells stimulated with ACTH. This concept is supported by the experiment in which bromophenacylbromide and nordihydroguaiaretic acid also blocked the formation of an activated PMTS. In fact, non-activated PMTS, in the presence of exogenous arachidonic acid AA, behaved as an activated PMTS from ACTH stimulated cells. We suggest that the mechanisms of action of ACTH and AII involve an increase in the release of AA and an activation of the enzyme system which converts AA in leukotriene products.     

Stimulation of the adrenal gland of the mongolian gerbil after ovariectomy

The effect of ovariectomy on the adrenal gland was studied in the Mongolian gerbil. Castration stimulated cells in the zona fasciculata as well as those in the region between the zona fasciculata and zona reticularis. No alterations occurred in the zona reticularis. The width of the intermediate region was enlarged, the cells were hypertrophic and contained an increased number of concentric whorls of rough endoplasmic reticulum. Smooth endoplasmic reticulum was hypertropic in cells of the zona fasciculata and intermediate region. Many mitochondria in cells of the intermediate region were larger than those in controls. Stimulation of cellular ultrastructure is directly correlated with a significant elevation in plasma cortisol in ovariectomized gerbils.     

Effects of atrial natriuretic peptide AP II on the morphofunctional state of the adrenal cortex of white rats]

The effect of atrial natriuretic peptides synthetic analog AP II on adrenal zona glomerulosa and zona fasciculata physiological regeneration have been studied on male rats. The 3H-thymidine incorporation into DNA in adrenal cortical cells was evaluated in 4 and 24 h after intraperitoneal injection of 10 or 100 mcg/kg AP II. Besides, we have investigated the influence of AP II on adrenal cortical cells karyometric parameter in 4 and 24 h and aldosterone plasma concentration in 1 h after injection. 10 mcg/kg AP II increased the fraction of labelled nuclei in zona glomerulosa and decreased the aldosterone plasma level. No significant changes were seen in zona fasciculata cells proliferation. 100 mcg/kg AP II inhibited the DNA synthesis process in adrenal zona fasciculata, but had no significant influence on zona glomerulosa physiological regeneration and aldosterone plasma concentration. No nuclear morphometric parameter changes were observed in adrenal glomerulosa and fasciculata cells of AP II--treated animals.     

An ultrastructural, morphometric and autoradiographic study of the effects of 7,12-dimethylbenzanthracene on the rat adrenal cortex.

The effects of chronic treatment (up to 9 consecutive days) with 7,12-dimethylbenzathracene (DMBA) on the adrenal glands of adult male Wistar rats were investigated. Morphometry showed that DMBA provokes atrophy of the zona reticularis which was due to the decrease in both cell volume and number. The zona fasciculata showed only a decrease in the cell volume, whereas the zona glomerulosa did not display any significant changes. Autoradiography demonstrated that DMBA induces a significant increase in the number of mitoses and "S" phase cells in the zona glomerulosa and outer zona fasciculata, which may be interpreted as a repair mechanism of the DMBA-provoked slight necrosis in the inner adrenocortical layers. The mechanism(s) underlying the cytotoxic effect of DMBA is discussed in the light of our ultrastructural observations showing that the chemical causes a decrease in the volume of the mitochondrial and nuclear compartments and in the surface of smooth endoplasmic reticulum as well as an increase in the volume of the lipid compartment.     

The suprarenal glands of a prosimian primate,the lesser bushbaby (Galago senegalensis)

The gross relationships and light microscopic anatomy of the suprarenal gland of a prosimian primate, Galago senegalensis, is described. The left gland is located medial to the pole of the left kidney in a fascial compartment of its own. The right suprarenal is located medial to the pole of the right kidney in intimate apposition to the liver and inferior vena cava. The capsule of the right gland blends with the capsule of the right lobe of the liver and is also contiguous with the adventitia of the inferior vena cava. The histologic appearance of the gland is similar to that of other primate genera. The zona glomerulosa is poorly developed; the zona fasciculata is composed of cell cords and is relatively well developed and the zona reticularis shows no unusual characteristics. The organization of the lipid content of the various cortical zones show a considerably different pattern than previously reported. The zona glomerulosa contains numerous large lipid droplets. In contrast to the bi-laminar pattern of lipid deposition seen in other primates, the Galago shows three distinct layers of lipid droplets in the zona fasciculata. The zona reticularis has a moderate population of lipid droplets essentially similar to that reported in most other forms. The medulla, except for a sparse number of centrally displaced zona reticularis cells, is completely devoid of lipid deposits. The junction of the zona reticularis and medulla is distinct, although a connective tissue capsule is not present.     

Carbonic anhydrase isoenzymes II and I are present in the zona glomerulosa cells of the human adrenal gland

Human carbonic anhydrase isoenzymes I and II (HCA I and II) were purified from human erythrocytes by inhibitor affinity chromatography and ion-exchange chromatography. These isoenzymes were then located in the human adrenal gland using specific polyclonal antisera raised in rabbits and specific detection by immunohistochemical techniques. Both HCA II and I were located in the zona glomerulosa cells, although the staining for HCA I was faint. The cells of the zona fasciculata and the zona reticularis failed to stain with either antiserum. Control stainings with preimmune or anti-HCA VI sera were negative. The presence of HCA II and I in the zona glomerulosa cells may be linked to regulation of the biosynthesis or secretion of mineralocorticoids.     

Proliferation and distribution of adrenocortical cells in the gland of ACTH- or dexamethasone-treated rats

The effects of prolonged (7-day) ACTH and dexamethasone administrations on rat adrenocortical-cell turnover have been investigated by combined stereological and metaphase-arrest techniques. ACTH was found to increase the number of parenchymal cells in each adrenal zone; however, ACTH altered the cell distribution in the cortex, lowering their percentage in the zona glomerulosa (ZG) and zona fasciculata (ZF) and enhancing it in the zona reticularis (ZR). The cell birth-rate was markedly raised by ACTH exclusively in ZG and ZF. Dexamethasone notably decreased the number of ZF and ZR cells, without altering that of ZG cells. Moreover, dexamethasone increased the percentage of parenchymal cells in ZG and ZF, and lowered it in ZR. In the adrenal cortices of dexamethasone-administered animals, metaphases were virtually absent. These data indicate that ACTH increases the cell birthrate in ZG and possibly ZF, and enhances the centripetal migration of newly-formed cells and their accumulation in ZR. Dexamethasone inhibits both proliferation of adrenocortical cells in the outer cortical layers and their centripetal migration into ZR. Moreover, it appears to cause parenchymal-cell loss in the inner adrenocortical layers.