Zona Reticularis Cells Research Articles

Adrenocortical function in 4-APP treated rats: A coupled stereological and biochemical study

Adrenocortical function in 4-APP-induced (4-aminopyrazolo[3,4- d]pyrymidine) lipoprotein-deficient rats was studied in relation to quantitative morphologic changes in the gland. 4-APP treatment results in enlargement of the adrenal cortex and its zona fasciculata and reticularis cells. In enlarged livers, cholesterol and free fatty acid concentrations were similar to that of control rats, however a marked accumulation of triglycerides with a concomitant drop in hepatic Δ 4-steroid hydrogenase activity was found. A profound drop in serum cholesterol in both, high and low density lipoproteins, as well as triglycerides and plasma corticosterone concentrations was accompanied by a marked lowering of cholesterol and corticosterone concentration in the adrenal gland. Corticosterone output by adrenal homogenates was higher in 4-APP treated rats than in control animals. Such a treatment did not change cholesterol side-chain cleavage, 11β-hydroxylase, 3β-ol dehydrogenase-isomerase, steroid 5α-reductase and neutral lipase activities when expressing results per unit weight of tissue or protein. However, when calculating per adrenocortical cell, adenine analogue applied increased 11β-hydroxylase, steroid 5α-reductase and neutral lipase activities. Thus, coupled biochemical and stereologic studies revealed a complex and multidirectional effect of 4-APP on the rat adrenal cortex. This effect may be caused by serum lipoprotein deficiency and by toxic and stressful action of the adenine analogue on the rat. Also a direct effect of 4-APP on rat adrenal cortex may not be excluded.

Immunoreactive apolipoprotein E is a widely distributed cellular protein. Immunohistochemical localization of apolipoprotein E in baboon tissues.

Apolipoprotein (apo)E is an important protein determinant in cholesterol homeostasis in man. The protein is synthesized by the liver as well as by a number of extrahepatic tissues. In the present study, immunohistochemical techniques were used to identify apoE in specific cells in various baboon organs. In the 11 tissues studied, the following cell types have been found to harbor apoE immunoreactivity: cerebral astrocytes; thyroid follicular cells; alveolar type II pneumocytes; hepatocytes, and Kupffer cells; adrenocortical cells in zona fasciculata and zona reticularis; adrenal medullary cells; some renal tubular epithelia; some pancreatic islet cells; histiocytic macrophages in lymph nodes and the spleen; some gastric mucosal epithelia; and ovarian oocytes. These observations indicate the wide distribution of apoE in many organs and suggest that the protein might perform other important functions such as regulation of local hormonal homeostasis in addition to its role in cholesterol metabolism.

Ultrastructural and immunohistochemical studies on the zona-reticularis cells of the adrenal cortex of normal and 3-methylcholanthrene-treated mice.

The drug-metabolism activity of adrenocortical cells of normal and 3-methylcholanthrene (MC)-treated ddY mice were examined ultrastructurally and immunohistochemically. Immunoblot analyses performed prior to immunohistochemistry revealed the presence of a protein - possible equivalent to cytochrome P-450 found in the liver microsomes of MC-treated male rats - in the adrenal homogenate of MC-treated female mice. Immunohistochemistry demonstrated the presence of a cytochrome-P-450-like protein in the adrenal-cortical cells (especially in the zona-reticularis cells) of MC-treated female mice; at the same time, a remarkable increase in the amount of SER in these cells was observed by electron microscopy. These findings suggest that cells of the mouse adrenal gland, particularly those of the zona-reticularis, might participate not only in steroid biosynthesis but also in some sort of drug metabolism (detoxication). In addition, there might be a sex-related difference in ddY mice.

Histochemical studies on the zona reticularis of stress-susceptible pigs

During an investigation into the role of the adrenal gland in stress susceptibility of pigs, the large lipid masses in the inner zona reticularis were seen to be particularly related to sex differences, although stress-susceptible castrates had significantly more lipid in this zone than had stress-resistant castrates. Significant histochemical differences were also found in the activity of glucose-6-phosphate dehydrogenase and succinic dehydrogenase between the sexes and between castrates of differing susceptibility to stress. The differences in histochemical activity suggest a different origin for some of the adrenocortical cells in the inner zona reticularis.

Correlation between intramitochondrial localization of glutathione-peroxidase (mitochondrial GSH-PO) and steroidogenesis in the rat adrenocortical cells.

In order to confirm the relationship between mitochondrial GSH-PO and steroidogenesis in the rat adrenal cortex, we have studied the immunocyto-chemical localization of GSH-PO in the rat adrenocortical cells of Metopiron administration, which blocks the conversion of deoxycorticosterone to corticosterone. In the control rat adrenocortical cells, mitochondrial GSH-PO was mainly localized in lipid-depleted compact cells, of inner fasciculata cells, but not zona glomerulosa and zona reticularis cells. By Metopiron administration, the adrenal cortex was markedly hypertrophic. Immunohistochemically, GSH-PO was observed in outer fasciculata cells and intracellular localization of GSH-PO was mainly observed in cytosol (cytosol GSH-PO). However, mitochondrial GSH-PO was less than that in the control rat adrenocortical cells.Based on our findings, it is strongly suggested that mitochondrial GSH-PO may be a very important enzyme for steridogenesis, especially, corticosterone biosynthesis.

Steroid biosynthesis by zona fasciculata and zona reticularis cells purified from the mammalian adrenal cortex

Steroidogenesis by zona reticularis (ZR) and zona fasciculata (ZF) cells from guinea-pig, macaque and human adrenals has been examined to establish the relative importance of the two cell types in the production of adrenal androgens. Microdissection and equilibrium density sedimentation of enzymically dispersed cells on Percoll gradients have been used to separate the two cell types with varying degrees of purity. Outputs of androstenedione and cortisol were measured for all the species studied and dehydroepiandrosterone sulphate (DHAS) for human adrenal cells. In all three species, the ZF cells produced more cortisol than the ZR cells. This production was stimulated to a greater extent in the ZF than the ZR cells in the guinea-pig, but to a similar extent in the macaque and human cells. Outputs of androstenedione were greater for ZF cells than ZR cells in the guinea-pig both basally and in response to an ACTH stimulus but basal production of this androgen was slightly greater from the ZR than the ZF in tissue incubations (1.6-fold), although cortisol output was still higher for ZF tissue by a factor of 2. Examination of androstenedione/cortisol ratios for all the species, both tissue and cells and in the presence or absence of ACTH, demonstrated clearly the inherent bias of ZR cells towards androgen synthesis by the fact that this ratio was always higher for ZR than for ZF cells or tissue. The most marked difference in this ratio was found in the human cells comparing the purest ZR and the purest ZF cell preparations even though the highest estimated purity of either cell type was only 80%. The difference in the ratio DHAS/cortisol was even greater comparing these human ZR and ZF cell preparations, ranging from 18- to 44-fold basally and in response to ACTH. The preliminary experiment on human adrenal cells indicates that the ZR cells are much more important than the ZF cells in the production of androgens even in the presence of ACTH, in contrast to the other species studied.

Changes in Blood Cortisol and Aldosterone Levels and Ultrastructure of the Adrenal Cortex During Hemorrhagic Shock

Changes of secretory activity and structure of the adrenal cortex were observed in dogs subjected to hemorrhagic shock. Blood cortisol and aldosterone levels were rapidly increased after hemorrhage. The cortisol level reached a maximum value about 5 times that of normal 1 h after hemorrhage, while aldosterone levels attained maximum levels showing a 10-fold increase over normal, considerably later (at the end of the impending stage) than the cortisol. Cortisol secretion was quickly depleted at the end of critical stage and terminal stage of normovolemic shock, while the aldosterone secretion remained relatively high. Morphologically, sticking of leukocytes to the sinusoidal wall and accumulation of SER around lipid droplets in the fasciculata and reticularis cells became conspicuous 1 h after hemorrhage. At the end of the impending stage, infiltration of leukocytes occurred in the zona fasciculata and reticularis. Irregular masses of fibrin strands appeared in the sinusoidal space and at large gaps of the sinusoidal wall. Degenerative changes such as aggregation of SER, dilatation of endoplasmic reticulum and edema were observed in the cells of zona fasciculata and reticularis, especially of inner half of the former. At the end of the critical stage, small necrotic foci and hemorrhage with inflammatory infiltration were scattered in the inner half of the zona fasciculata. Degeneration of the cells of zona fasciculata and reticularis became more severe in this period, while the cells of zona glomerulosa remained unchanged. At the terminal stage of normovolemic shock after reinfusion, degeneration and necrosis were remarkable in the cells of zona fasciculata and reticularis, especially of the inner half of the zona fasciculata. Moreover, destruction of cordal structure, hemorrhage and inflammatory infiltration were distinct in the zona fasciculata and reticularis, while the zona glomerulosa was relatively well preserved.

Effects of age and adrenocorticotropin on microsomal enzymes in guinea pig adrenal inner and outer cortices.

Guinea pig adrenals possess a distinctive zona reticularis which increases in volume as the animal matures, coming to occupy over 50% of the gland in mature 800-g animals. The adrenocortical cells of this zone possess abundant smooth-surfaced endoplasmic reticulum. In this paper we demonstrate that microsomes obtained from guinea pig inner cortices of both young and mature animals, consisting predominantly of zona reticularis tissue, possess higher activities of 21-hydroxylase, an enzyme that hydroxylates steroids, and ethylmorphine demethylase, a drug-metabolizing enzyme, as well as higher contents of cytochromes b5 and P450, than microsomes derived from outer cortices, which contain predominantly zona fasciculata tissue. In addition, we show that while the inner cortex contains 90% of the total adrenal activity for ethylmorphine demethylase, ACTH suppresses this activity by 55% and decreases activity in the outer cortex to negligible levels. ACTH does not affect 21-hydroxylase activity or cytochrome b5 or P450 content in either cortical region. These observations suggest that differential regulation of microsomal functions for steroid hydroxylation and drug metabolism occurs largely in the zona reticularis. Furthermore, they suggest that other activities for xenobiotic metabolism, previously reported as a property of whole guinea pig adrenals, may reside in the inner cortex and be unique functions of the abundant smooth endoplasmic reticulum of the zona reticularis cells.

Preparation and steroidogenic properties of purified zona fasciculata and zona reticularis cells from the guinea-pig adrenal gland.

Mixtures of zona fasciculata (ZF) and zona reticularis (ZR) cells, obtained by enzyme dispersion of decapsulated guinea-pig adrenal glands, were separated either by unit gravity sedimentation or by equilibrium density sedimentation. There was no evidence of deleterious effects on ultrastructural integrity or the ability of cells to respond to (1-24)ACTH (Synacthen) after either separation technique. Unit gravity sedimentation gave one fraction in which 90% of the cells were from the ZR and another fraction in which 70% of the cells were from the ZF. Equilibrium density sedimentation of cell mixtures on Percoll gradients gave fractions containing either 90% pure ZR or 95% pure ZF cells. Cortisol, 11-deoxycortisol, corticosterone, deoxycorticosterone, 11 beta-hydroxyandrostenedione and androstenedione were all formed from [14C]pregnenolone on incubation with purified preparations of both types of cell. No product was seen to be unique to either cell type although ZR cells appeared deficient in 11 beta-hydroxylase activity relative to ZF cells. The ratio of androstenedione to cortisol (formed either from labelled pregnenolone or from endogenous precursors) was higher for ZR cells than for ZF cells. When the purer cells obtained by equilibrium density sedimentation were studied, it was found that (1-24)ACTH stimulated greater steroid production (both androstenedione and cortisol) by the ZF cells compared with the ZR cells.

Delta 4-Hydrogenase in guinea pig adrenal: evidence of localization in zona reticularis and age-related change.

The guinea pig adrenal increases in the size with age, yet cortisol secretion per gram adrenal declines. It has been suggested that this could be due to catabolism of cortisol by increased adrenal delta 4-hydrogenase. In this paper we show that delta 4-hydrogenase is localized in the zona reticularis and increases coincidently with the large increase in the volume of this zone with age. Glands were microdissected to give inner vs. outer cortices, and the postmitochondrial supernatants of these two portions were assayed for delta 4-hydrogenase activity. The enzyme was found almost solely in preparations from inner cortices, which were predominantly zona reticularis. Cells were isolated from each portion and assayed for delta 4-hydrogenase activity. Again, only those which contained zona reticularis cells, that is, those from the inner cortices, had activity. The activity was shown to be present in both microsomes and cytosol, although the latter was preferentially expressed in a 15,000 X g supernatant. When the metabolites were separated by thin layer chromatography, the products of the microsomal enzyme were 5 alpha- and 5 beta-dihydrocortisol (1:1) and that of the cytosolic enzyme was 5 beta-dihydrocortisol, whereas in the 15,000 X g supernatant the 5 beta-isomer was the predominant product. Its identity as 5 beta-dihydrocortisol was confirmed by mass spectroscopy. Tetrahydrocortisol comprised a minor portion of the products.

The influence of the potassium ions on the adrenal cortex secretory activity in the common vole ( Microtus arvalis pall.)

1. 1. The experiment was carried out on 56 common vole males. The animals were fed with 10 g of alfalfa, parsley and cabbage containing 230, 1000 and 500mg of potassium per day respectively. 2. 2. The KCl solutions (14.3% KCl solution and 25% of this solution in 0.4ml per day) were administered per os to the animals. 3. 3. The histophotometric determinations of DNA level in cells of zona glomerulosa, fasciculata externa and reticularis of the adrenal cortex were made. 4. 4. The DNA level was the highest in the cells of zona fasciculata, while the lowest in zona reticularis cells.

Cyclic AMP levels in purified rat adrenal zonae fasciculata and reticularis cells and the effect of adrenocorticotrophic hormone.

Cyclic AMP levels were measured in combined cells and supernatant fraction from incubations of dispersed rat adrenal zona fasciculata and zona reticularis cell preparations purified by unit gravity sedimentation. These measurements were correlated with deoxycorticosterone (DOC) and corticosterone outputs from the cells in the presence or absence of ACTH. Similar measurements of cyclic AMP outputs were made for unpurified dispersed, decapsulated rat adrenal cell preparations and they were found to correspond to previously reported measurements made by other workers on such preparations. The response of the purest zona reticularis cells to ACTH in terms of cyclic AMP output was 28-fold lower than that of the purest zona fasciculata cells (compared with a fivefold lower DOC output and a 20-fold lower corticosterone output) and the response to ACTH of the mixed-cell preparations was related to the number of zona fasciculata cells in the preparation, i.e. the greater the proportion of zona fasciculata cells in the preparation the greater the response in terms of both outputs of cyclic AMP and of either of the two steroids measured. This correlation is in accordance with the theory that cyclic AMP may be the secondary messenger for both zona fasciculata and zona reticularis cells of the rat adrenal cortex in mediating the response to an ACTH stimulus.

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).

Effect of cyproterone acetate on structure of adrenal cortex in hypophysectomized, ACTH treated mice.

Hypophysectomized adult male C57BL/6J mice injected with 0.08 mg/g of cyproterone acetate showed large aggregates of cellular lipid as well as nodular formations in the zona fasciculata of the adrenal cortex. Daily ACTH injections (10 IU) reversed the atrophic changes noted above. At the ultrastructural level, the zona fasciculata and zona reticularis cells exhibited decreased cellular lipid content, proliferated profiles of rough endoplasmic reticulum, an increase in polyribosomes, and an active Golgi apparatus. It is believed that cyproterone acetate inhibited adrenocortical steroidogenesis by interfering with pituitary release of ACTH.

PROPERTIES OF RAT ADRENAL ZONA RETICULARIS CELLS: PRODUCTION AND STIMULATION OF CERTAIN STEROIDS

The outputs of corticosterone, deoxycorticosterone and androstenedione from dispersed, purified rat adrenal zona reticularis and zona fasciculata cells have been measured by radioimmunoassay. Preferential production of deoxycorticosterone by zona reticularis cells was demonstrated by their higher basal deoxycorticosterone : corticosterone ratio when compared with zona fasciculata cells. Adrencorticotrophin (ACTH) stimulated corticosterone output by all cell pools prepared by unit gravity (1 g) sedimentation, zona fasciculata cells being stimulated 130-fold compared with 20-fold for the zona reticularis cells in relation to their basal corticosterone output. In every cell pool, ACTH stimulated the output corticosterone more than it stimulated the output of deoxycorticosterone. In parallel cell preparations, it was shown that ACTH increased the conversion of tracer amounts of radioactive deoxycorticosterone to corticosterone and decreased the conversion of radioactive corticosterone to 11-dehydrocorticosterone. Adrenocorticotrophin did not increase the conversion of radioactive deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone). It is unlikely therefore that ACTH stimulates 11 beta-hydroxylation. Data indicate that the ratio of deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone) is characteristic for each cell type, and that this ratio will be relatively independent of ACTH stimulation or the amount of pregnenolone substrate available. Basal androstenedione outputs were similar for both types of cell, and ACTH stimulation was very small, being slightly greater for zona fasciculata than for zona reticularis cells. The contribution of the zona reticularis cells to the basal output of any steroid by the cells of the inner two zones of the adrenal cortex of the rat was relatively small (20% for deoxycorticosterone and 10% for corticosterone) and was even less after stimulation by ACTH. Unless a specific stimulus can be found, therefore, a significant role for the zona reticularis cannot yet be established.

Fine structure of the rabbit adrenal cortex and the effects of short-term ACTH administration

The fine structure of the rabbit adrenal cortex was investigated. The parenchymal cells display the ultrastructural features of steroid-producing cells, and also contain numerous electron-dense bodies frequently located near intercellular canaliculi, when open into the subendothelial space. Short-term ACTH-administration induced a noticeable decrease in the volume of the lipid compartment in the cells of all three cortical zones and a significant increase in the volume of dense bodies in the cells of zona fasciculata and zona reticularis. The hypothesis that these dense bodies are secretory granules is discussed in the light of biochemical evidence showing that ACTH increases the concentration of both corticosterone and cortisol in the decapsulated-enucleated adrenal homogenate and does not affect the activity of two lysosome-marker enzymes (i.e., acid phosphatase and beta-glucuronidase).

Properties of dispersed cells from rat adrenals

Using the physical methods of unit gravity (1 g) sedimentation and column filtration (Sephadex) on the rat collagenase-dispersed adrenal gland (either capsule or decapsulated gland), purified preparations of all three cell types, zonae glomerulosa, fasciculata and reticularis, were obtained. Morphological studies have determined the modal diameters of the cells to be: zona glomerulosa, 10.5 μm; zona fasciculata, 18 μmm; zona reticularis, Type I, 9 μm, and Type II, 13 μm. All types of adrenal cell produced at least one corticoid in common, corticosterone. Zona glomerulosa cells responded to small changes in potassium ion (K +) concentration (×2.3 corticosterone output), angiotensin II (× 1.7 corticosterone output), ACTH ( × 3 corticosterone output) and in vitro, serotonin ( × 2.1 corticosterone output). Aldosterone was produced exclusively by the zona glomerulosa cells. Zona reticularis cells had a different quantitative pattern of steroid output compared with zona fasciculata cells such that the ratio of total 11-oxygenated steroids/deoxycorticosterone may be used to characterise the different cells. ACTH stimulated steroidogenesis (corticosterone output) in all three types of adrenal cell (zona reticularis, × 19.5; zona fasciculata, × 130; zona glomerulosa, × 3). The effect of ACTH on the corticosterone output of the zona reticularis and fasciculata cells was, in part, due to an alteration in the biosynthesis favouring corticosterone rather than 11-dehydrocorticosterone after stimulation. There was a minor effect on total 11-oxygenation in zona fasciculata cells

Effect of thyroparathyroidectomy (TPX) on the zona reticularis: a quantitative ultrastructural study.

Atrophy of zona reticularis cells was observed two weeks after surgical thyroparathyroidectomy (TPX). Quantitative morphological techniques for electron microscopy showed significant decreases in the volume of cytoplasm, nucleus, mitochondria, smooth endoplasmic reticulum and lipid droplets in the zona reticularis of TPX rats. In addition, many mitochnodria contained lipid droplets, some of which occupied virtually the entire matrix of mitochondria. The volume per cell of mitochondria with these inclusions increased significantly after TPX. The lipid droplets may well arise from cytoplasmic droplets by increased transport or reduced metabolism of cholesterol, or by direct incorporation of droplets into the mitochondrial matrix. The serum corticosterone level of TPX rats sacrificed under quiescent conditions did not differ significantly from that of controls. Hypothyroidism induced a significant increase in the volume of peroxisomes per cell in TPX animals which may be related to changes in lipid metabolism or transport.

Adrenal gland involvement in mice with hereditary obesity and Diabetes mellitus

The contribution of the adrenal gland to the development of the spontaneous syndrome of obesity and diabetes in Yellow-KK (Y-KK) mice was studied. Six-month old Y-KK mice exhibited hyperadrenocorticism and adrenal cortex enlargement. Light microscopic morphometric studies of Y-KK adrenals revealed an expanded volume of the adrenal cortex resulting from hyperplasia of zona fasciculata and reticularis cells. Ultrastructural studies revealed fewer lipid droplets, increased numbers of mitochondria and a more extensively developed Golgi system with zona fasciculata and reticularis cells. This cytological evidence of enhanced steroid biosynthetic and secretory activity was consistent with increased levels of plasma immunoreactive corticosterone. Structural and functional abnormalities of Y-KK adrenals were preceded by the development of obesity, hyperglycaemia and hyperinsulinaemia. It is unlikely, therefore, that the adrenal plays a casual role in the syndrome's pathogenesis, although, hyperadrenocorticism may be in part responsible for an exacerbation of the observed phenomena.

Adrenal Luteinizing Hormone Releasing Hormone Receptors

BERNARDO, LINDA A.; PETRALI, JOHN P.; WEISS, LEON P.; STERNBERGER, LUDWIG A. Author Information