Morphology and ultrastructure of the adrenal gland in Bactrian camels (Camelus bactrianus)

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.     

Morphometric and histological studies on the adrenal glands of the camel Camelus dromedarius

The adrenal gland of the camel consists of an outer cortex and an inner medulla. The general disposition of the cortex and medulla, however, differs occasionally from that of other mammals. Extensions of medulla could reach as far as the periphery of the cortex. Islet of medullary tissue may be found in sections of the cortex and cortical tissue consisting of all zones of the cortex may occur around arteries or nerves in the medulla. The medulla may be separated from the cortex by connective tissue especially in old camels. The arrangement of noradrenaline-secreting cells is different from that in other ruminants; they are found in groups scattered between the adrenaline-secreting cells. Bundles of smooth muscle occur in venules at the corticomedullary interface. Accessory adrenal glands are found embedded in the renal fat. They are similar in structure to the adrenal gland. The adrenal cortex forms 74% of the volume of the gland and the ratio of the cortex to medulla is 4:1. The zona glomerulosa, fasciculata and reticularis constitute about 13%, 53%, and 29% by volume of the cortex, respectively.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

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.     

Monoamine Oxidase in Rat and Bovine Endocrine

Monoamine oxidase (MAO) was characterized in tissue homogenates from rat pancreatic islets, rat neurohypophysis and adenohypophysis, and rat and bovine adrenal medulla and adrenal cortex. Phenylethylamine was preferentially deaminated by rat pancreatic islet and bovine adrenal medulla MAO and with slight preference by rat neurohypophysis MAO, whereas 5-hydroxytryptamine was preferentially deaminated by MAO from all other endocrine tissues. Tyramine was a good substrate for all tissues. Clorgyline, a selective inhibitor of MAO-A, preferentially inhibited deamination of 5-hydroxytryptamine by all tissue homogenates, whereas deprenyl, a selective inhibitor of MAO-B, preferentially inhibited deamination of phenylethylamine. Km values for 5-hydroxytryptamine and tyramine were higher by one to two decimal powers than for phenylethylamine in homogenates from all endocrine tissues. Km values were significantly lower for 5-hydroxytryptamine and significantly higher for phenylethylamine in rat and bovine adrenal cortex than in adrenal medulla. According to these results, the contributions of MAO-B to total enzyme activity were 70% for rat pancreatic islets, 45% for rat neurohypophysis, 15% for rat adenohypophysis, 20% for rat adrenal medulla, 10% for rat adrenal cortex, 60% for bovine adrenal medulla, and 20% for bovine adrenal cortex. PC 12 cells also contained predominantly MAO-A (90%); however, an increased Km for phenylethylamine and a sensitivity of deamination of this MAO-B substrate to inhibition by clorgyline are indicators of abnormal behavior of MAO in this clonal rat pheochromocytoma cell line.     

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.     

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.     

Characterization and immunohistochemical localization of nucleoside triphosphate diphosphohydrolase (NTPDase) in pig adrenal glands (presence of a non-sedimentable isoform)

Considering that adrenal glands possess a variety of purinoceptors associated with various cell types and that some of these cells (chromaffin cells) secrete large amounts of adenine nucleotides, it was of interest to localize nucleoside triphosphate diphosphohydrolase (NTPDase) in these glands and to define the biochemical characteristics of this ectonucleotidase. Immunolocalization produced a moderate reaction in capsula and medulla, with no signal in zona glomerulosa and zona reticularis. In contrast, a very strong reaction was found in zona fasciculata. Biochemical analysis of particulate fractions isolated from whole glands revealed high levels of ATPase and ADPase activities. This appeared to be attributable to the NTPDase since the level of ADPase was as high as ATPase. Both ATPase and ADPase activities were similarly inhibited by sodium azide. Additionally electrophoretograms with these two substrates showed comparable patterns. Western blots with 'Ringo', an antibody that recognizes the different isoforms of mammalian NTPDases, showed the presence of isoforms of NTPDases at 54 and 78 kDa, respectively. Interestingly, the 54 kDa isoform remains in the supernatant of a chromaffin granule lysate after ultracentrifugation. Up until now little interest has been given to the relationship between adrenal medulla and cortex. Presence of purinoceptors and ectonucleotidases in both these regions together with the effects of ATP in vivo and in vitro in different species indicate that purines play a significant role in adrenal glands.     

Zonal distribution of cytochromes P-450 and related enzymes of bovine adrenal cortex--quantitative assay of concentrations and total contents

The zona glomerulosa, zona fasciculata, zona reticularis, and medulla were separated from bovine adrenal glands and cytochromes P-450 and related enzymes in each zone were investigated immunochemically by Western blotting using antisera from chickens or rabbits against cytochromes P-450scc, P-450(11)beta, P-450s21, and b5, NADH-cytochrome b5 reductase, NADPH-cytochrome P-450 reductase, NADPH-adrenodoxin reductase, and adrenodoxin. Concentrations of cytochrome P-450(11)beta, NADPH-cytochrome P-450 reductase, and cytochrome b5 per milligram of protein of homogenate were higher in the zona glomerulosa than in the other zones; the levels of the other components were higher in the zona fasciculata. The total enzyme content of all components was the highest in the zona fasciculata. The amount of adrenodoxin was about 10 times that of NADPH-adrenodoxin reductase in each zone.     

Strain differences in rabbit adrenocortical structure and morphological evidence of corticomedullary communication

Interstrain differences in the structure of the mouse and rat adrenal cortex are well known, but related data on rabbits are not available. This study was planned to demonstrate possible strain differences in rabbit adrenal morphology and morphological evidence of a communication between cortex and medulla. For this purpose, the zonation of the adrenal cortex of intact mature male and female rabbits of different strains (New Zealand, Chinchilla and California) were compared using morphometry and the corticomedullary junction was evaluated for close relationship. Marked intersex and interstrain differences were found in the adrenocortical zonation. Female rabbits had larger adrenocortical zones than corresponding males. The rabbits of Chinchilla and California strains had the largest cortical sizes. This difference depended largely upon the higher thickness of the zona fasciculata. Whereas, there were no significant strain differences in the thickness of zona glomerulosa and zona reticularis. Furthermore, examination of corticomedullary junction showed that cortex and medulla were interwoven. Single and small accumulations of cortical cells were spread throughout the medulla. Our morphological data showed the presence of significant interstrain differences in the adrenocortical zonation in the rabbits and provide evidence for a possible paracrine interaction between medullary and cortical cells.     

Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex

An assumption that the aldosterone-synthesizing enzyme exists only in zona glomerulosa cells apparently contradicts our recent findings that a purified bovine adrenocortical cytochrome P-45011 beta catalyzes the aldosterone formation and the enzyme exists in both zones of the adrenal cortex. To gain more insight into the zone specificity of aldosterone production, the aldosterone-synthesizing activity of mitochondria prepared from the isolated zones of adrenal cortex of various animal species was investigated. The intact mitochondria from the bovine or porcine zonae fasciculata-reticularis could not produce aldosterone whereas those from the zona glomerulosa produced it at a significant rate. When the mitochondria from the zonae fasciculata-reticularis were solubilized by the addition of cholate, they produced aldosterone from corticosterone at a rate comparable to that of those from the zona glomerulosa. The presence of specific factor(s) in the zonae fasciculata-reticularis mitochondria inhibiting expression of the aldosterone synthetic activity is discussed. The mitochondria of the rat zonae fasciculata-reticularis could hardly catalyze aldosterone synthesis under the detergent-solubilized conditions, whereas those of the zona glomerulosa could. Immunoblot analysis revealed that the mitochondria of the zonae fasciculata-reticularis contained a protein of Mr 51,000 which was immunocrossreactive with a monoclonal antibody directed against P-45011 beta, whereas those of the zona glomerulosa contained two immunocrossreactive proteins of Mr 51,000 and 49,000. These results suggest that in the case of rat adrenal cortex, a specific aldosterone-synthesizing enzyme exists in the zona glomerulosa.     

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.     

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.     

Human adrenal cells that express both 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) contribute to adrenal androstenedione production

Androstenedione is one of several weak androgens produced in the human adrenal gland. 3β-Hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) are both required for androstenedione production. However, previous studies demonstrated the expression of HSD3B2 within the zona glomerulosa (ZG) and fasciculata (ZF) but low levels in the zona reticularis. In contrast, CYB5A expression increases in the zona reticularis (ZR) in human adrenal glands. Although their colocalization has been reported in gonadal theca and Leydig cells this has not been studied in the human adrenal. Therefore, we immonolocalized HSD3B2 and CYB5A in normal human adrenal glands and first demonstrated their co-expression in the cortical cells located at the border between the ZF and ZR in normal human adrenal. Results of in vitro studies using the human adrenal H295R cells treated with the HSD3B2 inhibitor, trilostane, also demonstrated a markedly decreased androstenedione production. Decreasing CYB5A mRNA using its corresponding siRNA also resulted in significant inhibition of androstenedione production in the H295R cells. These findings together indicate that there are a group of cells co-expressing HSD3B2 and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex, and these hybrid cortical cells may play an important role in androstenedione production in human adrenal gland.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in beef adrenal cortex.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity (mevalonate:NADP+ oxidoreductase )CoA-acylating) EC 1.1.1.34) was demonstrated in beef adrenal cortex. Most of the HMG-CoA reductase activity is in the microsomal fraction while a small percentage of the activity is associated with the mitochondria, Mitochondria purified on a linear sucrose gradient are enriched in HMG-CoA reductase and cytochrome c oxidase activities. The reductase present in microsomal preparations from the whole adrenal cortex has an apparent Km of 5.6 X 10(-5) M for (R,S)-HMG-CoA. Reductase activities found in the microsomal fractions from the zona glomerulosa, the zona fasciculata, and the zona reticularis were 1.32, 7.37, and 9.74 nmol mevalonate formed per milligram protein in 30 min respectively.     

An immuno-electron-microscopic study of the localization of VIP-like immunoreactivity in the adrenal gland of the rat

Summary VIP-like immunoreactivity was revealed in a few chromaffin cells, medullary ganglion cells and a plexus of varicose nerve fibers in the superficial cortex and single varicose fibers in the juxtamedullary cortex and the medulla of the rat adrenal gland. VIP-like immunoreactive chromaffin cells were polygonal in shape without any distinct cytoplasmic processes and they appeared solitarily. Their cytoplasm contained abundant granular vesicles having a round core and the immunoreactive material was localized to the granular core. VIP-immunoreactive ganglion cells were multipolar and had large intracytoplasmic vacuoles. The immunoreactive material was localized not only in a few granular vesicles but also diffusely throughout the axoplasm. VIP-immunoreactive varicose nerve fibers in the superficial cortex were characterized by abundant small clear vesicles and some large granular vesicles, while those in the juxtamedullary cortex and medulla and the ganglionic processes were characterized by abundant large clear vesicles, as well as the same vesicular elements as contained in the nerves in the superficial cortex. The immunoreactive material was localized on the granular cores and diffusely in the axoplasm in both nerves. Based on the similarity and difference in the composition of the vesicles contained in individual nerves, it is likely that the VIP-immunoreactive nerve fibers in the medulla and the juxtamedullary cortex are derived from the medullary VIP-ganglion cells, while those in the superficial cortex are of extrinsic origin. The immunoreactive nerve fibers in both the cortex and the medulla were often in direct contact with cortical cells and chromaffin cells, where no membrane specializations were formed. The immunoreactive nerve fibers were sometimes associated with the smooth muscle cells and pericytes of small blood vessels in the superficial cortex. In addition they were often seen in close apposition to the fenestrated endothelial cells in the cortex and the medulla, only a common basal lamina intervening. Several possible mechanisms by which VIP may exert its effect in the adrenal gland are discussed.     

Steroid 5α-reductase type 1 immunolocalized in the adrenal gland of normal, gonadectomized, and sex hormone-supplemented rats

 Steroid 5α-reductase in the rat adrenal gland is supposed to play a role in the catabolism of adrenal steroids. We showed immunohistochemically the cellular and subcellular localization of 5α-reductase in the rat adrenal gland, using a polyclonal antibody against 5α-reductase rat type 1. In the adrenal cortex, positive immunoreaction was found in the cells of the zonae fasciculata and reticularis but was absent in those of the zona glomerulosa. The positive staining was restricted to the cytoplasm but not to the nucleus, Golgi complexes or mitochondria. The staining intensity showed a marked change depending on the steroidal milieu. Gonadectomy for 6 weeks increased the immunoreaction, regardless of the sex. Testosterone replacement for the last 2 weeks considerably reduced the immunoreaction in 6-week-castrated males, and estradiol supplement for the last 2 weeks also resulted in the marked reduction of immunostaining in 6-week-gonadectomized females. In the adrenal medulla, the immunoreaction was localized in the supporting cells and the Schwann cells but not in the chromaffin cells. In these cells, the immunoreaction was not affected by steroidal treatments. These findings suggest that the expression of 5α-reductase in the rat adrenal cortex is regulated by sex hormones from the gonads, and the enzyme may participate in the conversion of adrenal steroids depending on the steroidal environment, although the functional significance of the enzyme in the adrenal medulla remains unclarified. Accepted: 27 June 1997     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.