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 ACTH on circadian periodicity of nuclear volume and mitotic division in the zona fasciculata externa of the adrenal cortex of mice.

In cells of the zona fasciculata externa of the adrenal cortex of mice, the maximal value of nuclear volume is observed in evening and night time, while the mitotic peak occurs in the early part of the day. Ten day subcutaneous injection of 1.5 units of ACTH twice in 24 hr produced nuclear hypertrophy and stimulation of mitotic activity of cells of the zona fasciculata externa. The circadian periodicity of nuclear volume in mice injected with ACTH is disturbed, while the circadian rhythm of mitotic activity is retained.     

The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex

Summary The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.     

A correlated thin-section and freeze-fracture analysis of guinea pig adrenocortical cells

Comparison of the fine structural features of guinea pig adrenocortical cells as seen in thin sections with those revealed by freeze-fracture confirms the structural appearance of steroid-secreting cells as interpreted from thin sections and reveals significant new features of the membranous organelles. Smooth-surfaced endoplasmic reticulum appears as a network of tubules, interwoven or in parallel, and as cisternae, fenestrated and non-fenestrated. These elements are tightly packed in the deeper cortical cells, excluding other organelles from their domain. Tubules and fenestrated cisternae possess randomly distributed intramembranous particles on their PF faces, while closely packed non-fenestrated cisternae possess aggregates of particles interspersed with aparticulate regions on their PF faces. These differences in particle distribution suggest functional specialization among the various forms of reticulum. Mitochondria appear as elongated structures of varying shape. Freeze-fracture reveals that all their cristae have circular origins from the inner membrane. Sinuous tubules, which appear as tubules in section, and straight tubules, which appear as lamellae in section, arise from single sites. Flattened sac-like cristae may have multiple circular origins. Definite contact points seen between inner and outer membranes may facilitate passage of molecules, including steroids, into the mitochondrial compartments. Lysosomes and peroxisomes, which are easily identified in thin sections with the aid of cytochemistry, are difficult to identify with certainty by freeze-fracture. Single membrane-bound granules of slightly smaller diameter than mitochondria may represent lysosomes. Smaller granules interconnected with the tubular reticulum, as well as dilated regions of this organelle, may represent peroxisomes. Plasma membranes show no indication of tight junctions but do have abundant gap junctions which show a zonal differentiation: small gap junctions throughout the cortex, medium-sized regularly shaped gap junctions in zona fasciculata externa, and large irregular gap junctions in zona fasciculata interna and zona reticularis. The large junctions cover planar areas as well as surfaces of projections of one cell into another. Such junctions may allow passage of ions as well as of low-molecular-weight substances between the cells, facilitating or even amplifying the response to trophic hormone stimulation.     

Effects of prolonged infusion with endothelin-1 on the function and morphology of rat adrenal cortex

A week of SC infusion with endothelin-1 (ET-1) (0.2 microgram.kg-1.hr-1) lowered PRA and raised plasma aldosterone (A) concentration in rats. Kalaemia and the plasma levels of ACTH and corticosterone (B) were not affected. Prolonged ET-1 administration caused a notable hypertrophy of zona glomerulosa (ZG) and its parenchymal cells, without inducing any apparent change in zona fasciculata. Stereology showed that ZG cell hypertrophy was mainly due to the increase in the volume of the mitochondrial compartment and to the proliferation of smooth endoplasmic reticulum (i.e., the two organelles in which the enzymes of steroid synthesis are contained). Isolated ZG cells from ET-1-infused animals evidenced a notable enhancement in their basal production of A and B. The secretory responses of ZG cells to the maximal effective concentrations of their three main stimulators (ACTH, angiotensin-II and K+) displayed comparable increases. These findings indicate that ET-1, when chronically administered, is able to specifically enhance the growth and steroidogenic capacity of rat ZG, and suggest that the mechanism underlying this ET-1 effect involves stimulation of the de novo synthesis of both the steroidogenic enzymes and the membrane framework in which they are located.     

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.     

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.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Effect of beta-endorphin on the steroid production of isolated zona glomerulosa and zona fasciculata cells

Small doses of β-endorphin (10^?11?10^?5M) decrease corticosterone production of zona fasciculata cells but fail to influence steroid production of zona glomerulosa cells. 10^?4M β-endorphin increases corticosterone production of both zones. The stimulating effect of ACTH on zona fasciculata corticosterone- and zona glomerulosa aldosterone production was decreased by β-endorphin (10^?9?10^?7M). Conclusion: β-endorphin might modulate both basal and ACTH stimulated corticosterone secretion.     

Effect of pituitary intermediate lobe extract on steroid production by the isolated zona glomerulosa and fasciculata cells

The effect of intermediate lobe extract (ILE) on aldosterone and corticosterone production of the zona glomerulosa cells and on corticosterone production of the zona fasciculata cells was investigated. The slope of the dose-response curve of ILE dilution was steeper than that of alpha h 1-39 ACTH measured on zona glomerulosa steroid production. The ED50 of both ILE and ACTH was lower when measured on zona glomerulosa than on zona fasciculata steroid production. It is supposed that a hormone (or some other substance) in ILE alters the sensitivity to ACTH of the zona glomerulosa cells.     

The influence of castration on cell proliferation in the adrenal cortex of female rats

Proliferation kinetics in the zones of the adrenal cortex of female rats after castration are reported. Following ovariectomy a wave of increased cell proliferation was observed in the zona glomerulosa and zona fasciculata after 1 and 2 days respectively. Increased cell proliferation in the zona reticularis was only slight. Cell proliferation returned to the level in control animals at 4 weeks. It is assumed that the regulative process following ovariectomy takes place directly in the adrenal cortex but it is possible that steroid metabolism in the liver is of importance. No relationship between the 3H-TdR labelling index (LI) and the mitotic index (MI) was observed.     

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.     

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.     

Spontaneous or experimentally induced formation of a special zone in the adrenal cortex of the adult brush-tailed possum (Trichosurus vulpecula)

The cytology and ultrastructure of the hypertrophied special zone, which is formed spontaneously in the adrenal cortex of adult female brush-tailed possums (Trichosurus vulpecula), was compared to the adrenocortical tissue in adult males in which the special zone, normally absent, was induced following castration alone or by additional treatment with follicle-stimulating hormone (FSH). The special zone in females was situated between the zona fasciculata and the zona reticularis, the latter being a rudimentary zone in this species. Special zone tissue extended as a broad band parallel to and on one side of the adrenal medulla. In the luteal phase of the reproductive cycle, the special zone cells showed ultrastructural features commonly associated with steroidogenic tissues, with many mitochondria and compact masses of smooth endoplasmic reticulum. Cytoplasmic lipid inclusions were rarely observed. In lactating females, however, the special zone cells exhibited cytological and ultrastructural features suggestive of a transformation in their morphology broadly divided into two types of cells: (1) cells at the periphery of the special zone (closest to the zona fasciculata) showed variable quantities of lipid inclusions, mitochondria with dispersed cristae, and segregation of the smooth endoplasmic reticulum into compact masses; (2) cells within the more central regions showed an increasing abundance of lipid inclusions which in many cells became the dominant feature of the cytoplasm. These special zone cells contained very little smooth endoplasmic reticulum and their mitochondria contained few cristae together with amorphous granular material within the matrix. In castrated males, special zone tissue developed between the zona fasciculata and the zona reticularis, appearing initially as focal islands of cells (8 months postcastration) and later (11 months postcastration) expanding into a single zone, probably via the proliferation and differentiation of adjacent cells of the zona fasciculata and longitudinal growth of the special zone. Similar focal aggregations of special zone cells were induced after 14 days of FSH treatment given to 2-month castrated males. In all castrated and FSH-treated castrated males, the ultrastructure of special zone cells was similar to that of special zone cells in luteal-phase female possums. The findings suggest that the formation and cellular composition of the special zone is associated with changes in the pituitary-gonadal axis and that FSH plays a primary role in the differentiation of this tissue.     

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.     

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

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.     

A comparative morphometric analysis of the effects of thyroxine and ACTH on the zona fasciculata of rat adrenal cortex.

The effects of thyroxine and ACTH on the ultrastructure of cells of the adrenal zona fasciculata were studied by morphometric analysis and compared by two-way analysis of variance. The results indicate that the effects of thyroxine and ACTH are different and independent. The statistically significant differences in effect of thyroxine and ACTH are: thyroxine causes an increase in the volume fraction of mitochondria and a decrease in the volume fraction of endoplasmic reticulum independent of ACTH; thyroxine causes a decrease in surface density of mitochondria and endoplasmic reticulum in the absence of ACTH, but in the presence of ACTH this effect is moderated by an interaction of throxine and ACTH. There were no statistically significant effects of either thyroxine or ACTH on volume fraction or surface density of liposomes.     

Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex.

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.     

Proliferation and distribution of adrenocortical cells in ACTH treated female hamsters

The aim of the study was the analysis, by means of combined stereologic methods and metaphase arrest technique of the adrenocortical growth of ACTH treated hamster. Adult female hamsters were treated daily with 50 micrograms ACTH (Synacthen Depot, Ciba) for 3, 6 and 9 days. Between days 3 and 9 nearly linear increase in adrenal gland weight was observed, mainly due to the enlargement of the fasciculata and reticularis zones. Throughout the experiment, there was no marked change in the number of the zona glomerulosa cells while from the day 3 a notable increase in the number of fasciculata and reticularis cells occurred. Prolonged ACTH treatment resulted in a significant increase in 3H-thymidine incorporation with the highest value on day 6 and subsequent drop on day 9. Also metaphase index of adrenocortical cells notably increased due to ACTH administration and again the highest value was found on day 6 of experiment. From stereologic data and from metaphase index, adrenocortical cell birth-rates in individual adrenocortical zones and in the entire cortex were calculated. There were great differences between the values calculated from metaphase index and those obtained from stereologic data, with the former significantly lower. ACTH enhances centripetal migration of parenchymal cells in the hamster adrenal cortex and causes their accumulation in the zona reticularis.