Maria C. Magalhães

Microbodies (Peroxisomes) in rat adrenal cortex

Electron microscope examination of the rat adrenal cortex revealed the presence of round or ovoid small bodies measuring 0.10–0.76 μ in diameter, limited by a single unit membrane (∼ 69 A thick), in the cytoplasm of cells of all zones, particularly the zona fasciculata. These bodies contained a finely granular matrix of moderate density and were occasionally connected with the endoplasmic reticulum. When glutaraldehyde-fixed, 30-μ thick sections were incubated in DAB-containing medium prepared according to Beard and Novikoff (5) and post-fixed with osmium tetroxide; dense deposits of reaction product appeared in the matrix of those bodies. Reaction product was absent from the bodies in sections incubated in medium without DAB, and was decreased or absent after incubation in medium without H2O2. Addition of catalase completely inhibited the slight staining observed in bodies incubated in medium without H2O2. High concentrations of H2O2, aminotriazole or KCN in the incubation medium rendered the reaction negative. It is concluded that such bodies contained catalase and may therefore be considered as true peroxisomes.

PROLIFERATION OF CAPSULAR STEM CELLS INDUCED BY ACTH IN THE RAT ADRENAL CORTEX

Despite great efforts devoted to clarifying the localization of proliferative activity in the adrenal cortex, the agents that stimulate proliferation remain controversial, and the nature of the stem cells from which cortical cells differentiate is incompletely understood. We studied proliferative activity in the rat adrenal cortex using an immunohistochemical method to detect the presence of the Proliferating Cell Nuclear Antigen (PCNA) (an intranuclear enzyme whose synthesis reaches the maximum intensity during the S-phase of the cell cycle). Groups of six rats were subjected to daily intraperitoneal injection of either corticotropin (ACTH1-24—0.2 mg/kg), dexamethasone (Dexa—4 mg/kg) or 0.9% saline for three consecutive days and killed 24 h after the last injection. Adrenal weight was significantly increased by ACTH treatment and reduced by Dexa. Concentrations of endogenous ACTH in plasma were lower in the Dexa group than in controls, and curiously, this was true in the ACTH1-24 treated group as well, probably in consequence of the increased corticosterone levels providing negative feedback at the hypothalamic-pituitary level. Corticosterone levels, as expected, were increased by the ACTH stimulus and reduced by the use of Dexa. Proliferating cell nuclear antigen immunostaining was close to zero in Dexa treated animals and low in controls. In ACTH treated rats, a significantly increased number of cells were positively stained. Positive cells were identified in both in zona glomerulosa (ZG) and zona intermedia (ZI) but many were located in the capsule. Zona fasciculata (ZF) and zona reticularis (ZR) were devoid of staining in all of these cases. We conclude that pharmacological doses of ACTH induce proliferation of capsular fibroblasts. Following descriptions by early 20th century researchers it is possible that these cells may also be stem cells and differentiate into adrenal cortex cells.

Age-related changes in the inner zone of the adrenal cortex of the rat—a morphologic and biochemical study

In this work, a correlative morphologic and biochemical study on the effects of ageing on the rat adrenal Inner Zone (IZ) was made. Male Wistar rats were studied at 2, 6, 12, 18 and 24 months. Structural data of Zona Fasciculata (ZF) showed age-related increase in cell volume (P < 0.05), decrease in mitochondria (P < 0.01) and smooth endoplasmic reticulum (SER) volumes, and increase in lipid droplets (P < 0.01) and lipofuscin granules (P < 0.01) volumes. In Zona Reticularis, the main change observed was the increase in lipofuscin granules (P < 0.001). Serum corticosterone from unstimulated rats increased until 12 months but decreased thereafter (P < 0.01), to levels below those from 2-month-old rats. Similarly, plasma adrenocorticotrophic hormone (ACTH) presented a maximum at 12 months, followed by a decrease to levels higher than at 2 months (P < 0.05). In rats injected either with only ACTH or dexamethasone, before ACTH stimulation, corticosterone level had a maximum at 12 months. In aged rats, serum high density lipoprotein (HDL) and adrenal cholesterol ester increased significantly (P < 0.05 and P < 0.01, respectively), whereas adrenal corticosterone decreased. Products of lipid peroxidation, assayed with the thiobarbituric acid reaction and fluorimetry showed an age-related increase (P < 0.05). The age-related decrease in mitochondria and SER volumes is consistent with the decrease of serum corticosterone. The increase in lipid droplet and HDL and the reduction of adrenal corticosterone level correlate with the increase of adrenal cholesterol ester content. These suggest a continued uptake of steroid precursor but a reduced steroid synthesis. On the whole, the data provide evidence for an age-related reduced functional ability of IZ and particularly of ZF.

Peroxisomes in adrenal steroidogenesis

Peroxisomes, cytoplasmic organelles limited by a single membrane and with a matrix of moderate electron density, are present in a great number of cells, namely in adrenal cortex and other steroid‐secreting organs. Presently peroxisomes are considered to be involved in important metabolic processes. They intervene in: (1) the production and degradation of H2O2; (2) biosynthesis of ether‐phospholipids, cholesterol, dolichol, and bile acids; (3) oxidation of very long chain fatty acids, purines, polyamines, and prostaglandins; (4) catabolism of pipecolic, phythanic and glyoxylic acids; and (5) gluconeogenesis. Recent studies demonstrated that the experimental alterations in the normal steroidogenesis, produce significant morphological and biochemical changes in peroxisomes. Besides this, the presence of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (the key enzyme in the de novo cholesterol synthesis from acetate) and of sterol carrier protein‐2 (SCP2), which is involved in the cholesterol metabolism and steroid metabolic pathways, are located in peroxisomes of steroid‐secreting cells. In addition, patients with peroxisome diseases present deficiency in steroidogenesis, as well as reduced levels of SCP2. These data pointed out the important role of peroxisomes in steroid biosynthesis. Microsc. Res. Tech. 36:493–502, 1997.

Effects of ovariectomy and estradiol administration on the adrenal macrophage system of the rat.

SummaryMacrophages of the adrenal cortex were studied in normal male and female, ovariectomized and estradiol-injected rats. In normal male rats few macrophages with numerous granules were observed in the zona fasciculatazona reticularis border, and in the zona reticularis. Granules, identified as lysosomes, were limited by a single membrane with a heterogeneous matrix; they exhibited acid phosphatase- and aminotriazole-resistant peroxidatic activities. A larger number of macrophages had identical distributions in normal female rats. In ovariectomized and estradiol-injected rats the number and distribution of adrenal macrophages were similar to those in normal females; however, in spayed animals the number of these cells in the zona reticularis was higher than in the other experimental groups. Lysosomes in macrophages of treated animals were more numerous and their contents more complex than in normal male animals. These results indicate that the adrenal macrophage system is stimulated in experimental conditions involving high levels of circulating estrogens.

Age-related changes in lipid peroxidation products in rat adrenal gland

Chloroform-methanol extracts from rat adrenals at five different ages (2, 6, 12, 18 and 24 months), were studied by fluorescence. After obtaining excitation and emission spectra, fluorescence intensity was measured at 365 nm excitation and 455 emission for all time points of aging. An additional study of lipid peroxidation employing a thiobarbituric acid reaction was made.Fluorescence intensity increased during aging from 16.39 × 103 arbitrary units of fluorescence per gram of tissue at 2 months, to 34.33 × 103 units at 24 months. Thiobarbituric acid reaction products expressed in nmol of malondialdehyde per gram of adrenal increased from 172.97 at 2 months to 640.83 at 24 months. One way analysis of variance revealed a statistically significant difference (p<0.05 and p<0.01 respectively).The results show an age-related steady increase in lipid peroxidation products in rat adrenals and suggest their accumulation in lipofuscin granules.

Postnatal development of the rat adrenal cortex: an ultrastructural morphometric study

Adrenals from 12-hr, 4 1/2, 7 1/2, 10-, and 14-day-old rats were used in this study. At 12 hr the cells of the zonae glomerulosa and fasciculata were similar to those of the adult adrenal cortex. The zona juxtamedullaris cells presented round mitochondria with vesicular cristae and very dense matrix and a large endoplasmic reticulum. After this age and until the 14th day, changes were slight and mainly regarding the size of cytoplasmic organelles. Variations in volumetric and surface densities were determined by stereological methods. These showed between 12 hr and 4 1/2 days of life in the three cortex zones a significant decrease of the volumetric densities of mitochondria (except the zona juxtamedullaris) and the endoplasmic reticulum (except in the zona glomerulosa) and a significant increase in the relative volume of the lipid droplets. The surface density of mitochondria decreased in the three zones, and the same parameter for the lipid droplets significantly increased in the zonae glomerulosa and fasciculata. After 4 1/2 days there was, in the three zones, an increase in the volumetric density of the mitochondria and endoplasmic reticulum and an increase in the mitochondrial surface density which were significant in most cases; the volumetric and surface densities of the lipid droplets increased except in the zona juxtamedullaris. These data suggest a decrease in the adrenal metabolic activity during the first days of life, which increases thereafter until reaching normal values.

Ontogeny of 3beta-hydroxysteroid dehydrogenase expression in the rat adrenal gland as studied by immunohistochemistry.

The enzyme 3beta-hydroxysteroid dehydrogenase plays a crucial role in the steroidogenic process in the adrenal gland. In the present study we tried to characterize its localization and developmental changes in the rat adrenal cortex during the postnatal period, using immunohistochemical methods. The development of the different zones evidenced specific particularities: the zona glomerulosa almost lacked 3beta-HSD in the first days after birth; then, 3beta-HSD increased, attaining a maximum around day 20 and afterwards it decreased again and remained less intense than the neighbouring zona fasciculata up until adulthood (65 days of age). The zona fasciculata was already intensely stained at birth and the expression of 3beta-HSD increased rapidly reaching a maximum after 2 weeks of life and that level was maintained from then on. The inner part of the zona fasciculata and the zona reticularis both of which develop postnatally were faintly immunostained before day 20. The expression of 3beta-HSD increased after that age to become approximately as intense as in the outer zona fasciculata and so remaining until day 90. The development of the zona glomerulosa was parallel to the secretion of aldosterone. The same did not occur with the zona fasciculata as the intensity of staining during the first 14 postnatal days was accompanied by very low levels of corticosterone.

The development of the adrenal cortex in the rat. An immunohistochemical study.

The Inner Zone Antibody (IZAb) is a monoclonal antibody which interacts with an antigen found predominantly in rat adrenal inner cortical zones. Since its expression increases after ACTH treatment the antigen may have a role in steroidogenesis although, so far, this has not yet been fully characterised. Due to its molecular weight, it cannot be any of the known cytochrome P450 proteins. In this study we examined the expression of IZAb in male and female rats throughout their postnatal development and in aged animals. In a different set of animals, blood was collected for hormonal assays and the adrenals stained with classical methods. The staining with IZAb was clear from the first post-natal day. The zona glomerulosa which was always present at birth, was easily distinguished and unstained. The staining in the inner zone cells was fainter at birth and increased progressively until postnatal day 20. Afterwards these cells were remarkably stained at all ages. Medullary cells were also present from birth although they were generally found in clusters instead of constituting a well defined zone. Cortical cells appeared in the medullary zone at all ages after its complete development. The zona glomerulosa increased in size until approximately postnatal day 40 while the inner zones increased until day 70. The area of the cortex was significantly different between the two sexes from day 50 onwards and this was predominantly due to differences in the zona fasciculata. Corticosterone levels increased until approximately day 25 in the male rat and until day 45 in the female.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural studies on the prenatal development of the rat adrenal cortex

The ultrastructural changes observed during the intrauterine life of the rat adrenal cortex are described. Adrenals of rat fetuses from 16 1/2 to 22 1/2 days of gestation were used. Between Days 16 1/2 and 17 1/2 most mitochondria had vesicular and tubular or lamellar cristae, and a few vesicular cristae only; endoplasmic reticulum spaces were large, and there were numerous free ribosomes and scarce lipid droplets. From 18 1/2 to 22 1/2 days of development, adrenal cells showed an increasing number of mitochondria and lipid droplets and a gradual decrease in the area occupied by the reticulum; mitochondria with vesicular cristae became more numerous. Variations in volumetric and surface densities of the cell organelles and the numeric density of mitochondria were assessed by stereological methods. These showed an increase in mitochondrial volume and number at all successive periods studied and an increase in surface density of mitochondria, endoplasmic reticulum spaces, and lipid droplets between 16 1/2 and 17 1/2 and 18 1/2 and 20 1/2 days. Endoplasmic reticulum volumetric density was significantly diminished after 18 1/2 to 20 1/2 days. Since the adrenal organelle changes described herein coincided with the beginning of adrenal steroid secretion, it is suggested that they represent the morphologic counterpart of such metabolic phenomena.