Effects of exogenous ACTH on the rat fetal adrenal cortex: a histochemical and electron-microscopical observation.

ACTH was administered subcutaneously to rat fetus directly at the late stage of fetal development and acute reaction on the fetal adrenal cortex was observed histochemically and electron microscopically. By administration of ACTH the adrenal cortex became remarkably hyperemic and there were swollen cells in all layers, particularly in the middle and inner layers (corresponding to the zona fasciculata and reticularis in adult rat). Marked reduction of lipid, enlarged mitochondria with increased vesicular cristae and increased smooth-surfaced endoplasmic reticulum (SER) were characteristic. The alterations of mitochondria preceded the change of SER, and thereafter mitochondria showed rapid degeneration. The outer layer (corresponding to the zona glomerulosa in adult rat) also showed similar changes by ACTH to those of the other two layers. These results indicated that the fetal adrenal cortex of rats was exogenous ACTH-reactive and its reaction which was different from that of adult cortical cells, seemed to be specifically related to the development and differentiation of the cells.     

Electron microscopic studies on the effect of ACTH and flavin adenine dinucleotide on adrenocortical atrophy of hypophysectomized rat.

Electron microscopic observation was made on the outer fasciculata cells in the adrenal cortex of hypophysectomized rats receiving 10 mg of FAD and/or 0.3 mg of ACTH intraperitoneally once a day for 5 consecutive days from 24 hours after hypophysectomy. The simultaneous administration of FAD and ACTH to the hypophysectomized rat was more effective for preventing adrenocortical atrophy induced than the administration of ACTH alone. This effect appeared as clear cells with low electron density. While the characteristics induced by hypophysectomy were the decrease in number of smooth-surfaced endoplasmic reticulum (SER) and mitochondria and also crista of mitochondria being tubular. The clear cells showed a less degree of their characteristic. From this fact, it is considered that the external FAD acts against fasciculata cells in the adrenal cortex of hypophysectomized rats as a coenzyme for flavin enzyme under ACTH and decreases oxidation-reaction in mitochondria and oxidative phosphorilation reaction in SER, being induced by hypophysectomy.     

ELECTRON MICROSCOPIC STUDIES ON THE EFFECT OF ACTH AND FLAVIN ADENINE DINUCLEOTIDE ON ADRENOCORTICAL ATROPHY OF HYPOPHYSECTOMIZED RAT

Electron microscopic observation was made on the outer fasciculata cells in the adrenal cortex of hypophysectomized rats receiving 10 nag of FAD and/ or 0.3 mg of ACTH intraperitoneally once a day for 5 consecutive days from 24 hours after hypophysectomy.
The simultaneous administration of FAD and ACTH to the hypophysectomized rat was more effective for preventing adrenocortical atrophy induced than the administration of ACTH alone. This effect appeared as clear cells with low electron density. While the characteristics induced by hypophysectomy were the decrease in number of smooth-surfaced endoplasmic reticulum (SER) and mitochondria and also crista of mitochondria being tubular. The clear cells showed a less degree of their characteristic. From this fact, it is considered that the external FAD acts against fasciculata cells in the adrenal cortex of hypophysectomized rats as a coenzyme for flavin enzyme under ACTH and decreases oxidation-reaction in mitochondria and oxidative phosphorilation reaction in SER, being induced by hypophysectomy. ACTA PATH. JAP. 27: 637˜645, 1977.     

A morphometric study of the reversal of ACTH-induced hypertrophy of rat adrenocortical cells after cessation of treatment

The effect of a prolonged (7-day) ACTH administration on rat zona fasciculata cells and its reversal after cessation of treatment was investigated by morphometry. ACTH treatment caused a notable cell hypertrophy, which was mainly due to the increase in the volume of the mitochondrial compartment and to smooth endoplasmic reticulum (SER) proliferation, and a conspicuous rise in the basal level of corticosterone. After cessation of ACTH administration, rat zona fasciculata cells underwent a time-dependent atrophy, so that after 5 days they resembled those of control animals, and the blood concentration of corticosterone reverted to the base-line value. The cell atrophy was provoked by the decrease in the volumes of the mitochondrial compartment and SER, and was associated with a striking time-dependent accumulation of dense bodies. Stereology demonstrated that during the first two days after ACTH withdrawal the decrease of SER prevailed over that of the mitochondrial compartment, while the reverse occurred during the remaining three days. The increase in the volume of dense-body compartment, though largely due to the accumulation of residual bodies, was mainly coupled with a rise in the volume of the microautophagic-vacuole compartment during the first two days after ACTH cessation and with an increase in that of the macroautophagic-vacuole compartment during the following three days. The hypothesis is advanced that both micro- and macroautophagy play a role in the reversal of ACTH-induced hypertrophy of rat zona fasciculata cells after cessation of treatment, the first process being mainly involved in the elimination of SER, and the second one in the degradation of mitochondria.     

Investigations on the turnover of adrenocortical mitochondria. VI. An autoradiographic study of the effect of ACTH on the radioactivity decay in the mitochondrial compartment from the zona fasciculata of 3H-thymidine injected rats.

The radioactivity decay of the mitochondrial compartment from the zona fasciculata of the adrenal cortex of 3H-thymidine-injected rats was followed by high resolution autoradiography. The number of days in which the radioactivity of the mitochondrial compartment was reduced to a half was calculated from the semilogarithmic plots of radioactivity versus time. Since DNA is a very stable molecule, it was assumed that this parameter can be an estimate of the half-life of adrenocortical mitochondria. The half-life of mitochondria from the zona fasciculata of the normal rat averaged 11.17 days, and ACTH was found to increase significantly this figure to about 15 days. It is hypothesized that the ACTH-elicited stimulation of the growth of rat adrenal zona fasciculata mitochondria involves not only hypertrophy and proliferation of the organelles (Nussdorfer et al., '74b), but also the slowing down of the degeneration rate of mitochondria.     

Effect of the adrenal inhibitor trilostane on the morphology of the adrenocortical cells of Dahl salt-sensitive and Dahl salt-resistant rats.

The effects of trilostane on the adrenal cortex of Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were investigated morphometrically, histochemically, ultrastructurally and biochemically. The statistical analysis indicated that trilostane induced a significant increase in the adrenal weight and the surface area of cells and nuclei in the zona fasciculata (ZF) of the adrenal cortex of DS and DR rats (P less than O.OI). DS rats treated with trilostane revealed marked accumulation of large amounts of lipid droplets and a decrease in the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the ZF. Ultrastructurally, the mitochondria of the ZF in DS rats treated with trilostane revealed swelling of matrix with a loss of cristae and occasional interruption of the membranes of mitochondria. Some of them had a continuity with lipid vacuoles or SER, presenting a characteristic 'feather'-like appearance. Other characteristic findings in DS rats treated with trilostane were a marked villous proliferation of plasma membranes with numerous dense bodies, occasional coated pits, and pinocytic vesicles in the outer portion of the ZF. In DS and DR rats the plasma level of ACTH increased, and corticosterone decreased significantly (P less than O.OI) after treatment with trilostane. These morphological alterations were considered to be an expression of the inhibitory effects of trilostane on the adrenal steroidogenesis in DS and DR rats, more especially in DS rats. Simultaneously there were confirmed morphological alterations in the cells of the ZF, reflecting the feedback stimulation of endogenous ACTH. The cells of the ZF of DS rats were more responsive to suppression by trilostane than those of DR rats.     

Effects of the hypolipidemic drug nafenopin on the zona glomerulosa of the rat adrenal cortex: morphological counterparts of functional alterations

The effects of nafenopin, a hypolipidemic drug, on the zona glomerulosa of the rat adrenal cortex were investigated. Chronic nafenopin treatment significantly lowered serum cholesterol level, but did not alter blood aldosterone concentration, though the biosynthesis of adrenal cortico-steroid hormones seems to be largely dependent upon a continuous uptake of cholesterol from plasma lipoproteins. Stereology showed that the treatment provoked a notable lipid droplet depletion, coupled with a significant proliferation of smooth endoplasmic reticulum (SER) profiles. Since SER is known to be involved in the endogenous synthesis of cholesterol, the hypothesis is advanced that SER hypertrophy is a compensatory response enabling zona glomerulosa cells to maintain an adequate level of aldosterone output even in the absence of a normal supply of exogenous cholesterol.     

Multiple pigmented adrenal cortical nodules associated with Cushing's syndrome. Histochemical, ultrastructural and quantitative studies

Multiple pigmented adrenocortical nodules were found in a 25-year-old woman associated with Cushing's syndrome, whose laboratory data indicated that the adrenal cortex had been functioning autonomously and adrenocorticotrophic hormone (ACTH) from the pituitary gland as suppressed. The surgically removed left adrenal gland disclosed multiple black nodules measuring up to 3 mm in diameter and histologically consisting of large "compact cells" which contained numerous yellow-brown pigments, but adjacent cortical cells were not atrophied. This kind of adrenal lesion is generally regarded as nodular hyperplasia of the cortex. The present case revealed scanty lipid and markedly increased activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and glucose-6-phosphate-dehydrogenase (G6PD). Ultrastructural study showed abundant cytoplasm with a large number of mitochondria, well-developed smooth-surfaced endoplasmic reticulum (SER), less rough-surfaced endoplasmic reticulum (RER), lysosomes, and numerous granules in cells of the nodules. Mitochondria varied in size and shape up to occasional giant mitochondria. SER was vesicular or tubular forming a network of anastomosing tubules. Granules varied greatly in size from 400 millimicrons to 6 microns in diameter, with diverse electron densities, mostly exhibiting the structural features of lipofuscin. The ultrastructural features resembled those in black adenoma associated with Cushing's syndrome ever reported. Concentration of cortisol was increased in the tissue where numerous black nodules were contained.     

Effects of glucocorticoids on differentiation of zona glomerulosa of fetal adrenal cortex of rats.

The tissue differentiation of the zona glomerulosa of the fetal adrenal cortex of rats was studied by giving experimental treatments to the fetus in vivo. A low-glucocorticoid-condition was given to the fetus by bilateral adrenalectomy of pregnant rats for removing exogenous glucocorticoids from the fetus, and by brain aspiration of the fetuses for removing the fetal pituitary gland (ACTH) and endogenous glucocorticoids. When the fetus was placed under a low-glucocorticoid-condition for the last couple of days of gestation, poor differentiation of the zona glomerulosa occurred specifically in the fetal adrenal cortex. The degree of the poor differentiation seemed to be proportional to the duration of the low-glucocorticoid-condition. Supplemental administration of glucocorticoids could prevent this poor differentiation of the zona glomerulosa. These results indicate that the tissue differentiation of the zona glomerulosa of the fetal adrenal cortex depends much on glucocorticoids.     

Degeneration of the rat and canine adrenal cortex caused by alpha- (1,4-dioxido-3-methylquinoxalin-2-yl) -N-methylnitrone (DMNM)

The antibacterial drug alpha- (1,4-dioxido-3-methylquinoxalin-2-yl)-N-methylnitrone (DMNM) given at a dose of 22.5 mg/kg/bid to 4 dogs for 14 days caused diminished adrenal cortical reserves as determined by decreased plasma corticol (3 dogs) and lower aldosterone levels (4 dogs) following the intravenous infusion of ACTH. A dose of 100 mg/kg/day of DMNM administered to rats for 31 or 35 days resulted in significant decreases in blood glucose. Histologically, the adrenal glands of both species treated with DMNM for a maximum period of 21 days (dogs) and 35 days (rats) had widespread granular and vacuolar degeneration of the cortex. The degeneration, as demonstrated in treated rats, began in the zona reticularis and inner regions of the zona fasciculata and eventually involved the entire cortex including the zona glomerulosa. As a result of treatment, significant ultrastructural alterations within cells of the rat and canine adrenal cortex consisted of degeneration of the mitochondria and an increase in the numbers and lipolysis of lipid droplets. The ultrastructure of the zona reticularis and fasciculata was most severely affected.     

EFFECTS OF GLUCOCORTICOIDS ON DIFFERENTIATION OF ZONA GLOMERULOSA OF FETAL ADRENAL CORTEX OF RATS

The tissue differentiation of the zona glomerulosa of the fetal adrenal cortex of rats was studied by giving experimental treatments to the fetus in vivo. A low-glucocorticoid-condition was given to the fetus by bilateral adrenalectomy of pregnant rats for removing exogenous glucocorticoids from the fetus, and by brain aspiration of the fetuses for removing the fetal pituitary gland (ACTH) and endogenous glucocorticoids. When the fetus was placed under a low-glucocorticoid-condition for the last couple of days of gestation, poor differentiation of the zona glomerulosa occurred speciflcally in the fetal adrenal cortex. The degree of the poor differentiation seemed to be proportional to the duration of the low-glucocorticoid-condition. Supplemental administration of glucocorticoids could prevent this poor differentiation of the zona glomerulosa. These results indicate that the tissue differentiation of the zona glomerulosa of the fetal adrenal cortex depends much on glucocorticoids.     

Differentiation of the fetal adrenal cortex of rats--its experimental observation in vivo.

The development and tissue differentiation of the adrenal cortex of rats were studied by giving experimental treatments to the fetus in vivo. A low-ACTH-condition was given to the fetus by administrating Dexamethasone through its mother, or directly into the subcutaneous tissue of the fetus, or by decapitation, brain aspiration or hypophysectomy. When the fetus was given the low-ACTH-condition for the last 5 days or more, the hypoplastic adrenal cortex developed specifically. It had nearly normal zona glomerulosa (ZG), and poorly differentiated zona fasciculo-reticularis (ZF-R) without differentiated ZF-R. The observing day was settled at 21 1/2 days old. The shorter the period of low-ACTH-condition was, the more thickened the differentiated zone (ZF-R) grew. On the contrary, the poorly differentiated layer became thinner in proportion to the period of low-ACTH-condition. The poorly differentiated cortical cell had ACTH-reactivity. It seems that the development and tissue differentiation of the ZF-R depend on fetal own ACTH, and poorly differentiated cortical cells appear below the ZG, and differentiate by ACTH and were gradually pushed inside. The zona glomerulosa develops and differentiates without dependence of ACTH.     

Electron microscopic studies of the effect of ACTH and flavin-adenine dinucleotide on adrenocortical atrophy of rats treated with dexamethasone phosphate.

In a previous paper the authors described the morphologic observations that the concomitant administration of ACTH and flavin-adenine dinucleotide (FAD) to hypophysectomized rats exerted a more potent preventive effect on atrophy of the adrenal cortex of the animals than the single administration of ACTH. The present study was made to electron-microscopically observe the effect of concomitant administration of ACTH and FAD on atrophy of the adrenal cortex induced with the administration of dexamethasone (Dx). The zona fasciculata of the adrenal gland of rats treated with Dx+ACTH+FAD was morphologically closer in cell organelles such as smooth-surfaced endoplasmic reticulum, mitochondria and chylomicrons to that of control animals than the counterpart of animals treated with Dx+ACTH only. The zona fasciculata of the adrenal cortex of animals treated with Dx+FAD was morphologically similar to that of animals treated with Dx only. These findings suggested that FAD would potentiate the adrenocorticotropic action of ACTH through its physiologic action.     

ELECTRON MICROSCOPIC STUDIES OF THE EFFECT OF ACTH AND FLAVIN-ADENINE DINUCLEOTIDE ON ADRENOCORTICAL ATROPHY OF RATS TREATED WITH DEXAMETHASONE PHOSPHATE

In a previous paper the authors described the morphologic observations that the concomitant administration of ACTH and flavin-adenine dinucleotide (FAD) to hypophysectomized rats exerted a more potent preventive effect on atrophy of the adrenal cortex of the animals than the single administration of ACTH. The present study was made to electron-microscopically observe the effect of concomitant administration of ACTH and FAD on atrophy of the adrenal cortex induced with the administration of dexamethasone (Dx). The zona fasciculata of the adrenal gland of rats treated with Dx+ACTH+FAD was morphologically closer in cell organelles such as smooth-surfaced endoplasmic reticulum, mitochondria and chylomicrons to that of control animals than the counterpart of animals treated with Dx+ACTH only. The zona fasciculata of the adrenal cortex of animals treated with Dx+FAD was morphologically similar to that of animals treated with Dx only. These findings suggested that FAD would potentiate the adrenocorticotropic action of ACTH through its physiologic action.     

Structure and dynamics of adrenal mitochondria following stimulation with corticotropin releasing hormone

The mitochondria of rat adrenals were investigated qualitatively and quantitatively in different functional states of the adrenal cortex. Following stimulation of the animals with corticotropin releasing hormone (CRH), the corticosterone serum levels reached a maximum 1 hour after stimulation with CRH. The amount of inner mitochondrial membrane within the zona fasciculata increased showing a biphasic time course, with a first maximum 2 hours and a second maximum 8 hours after stimulation. In contrast, a significant rise of mitochondrial volume occurred only 24 hours after CRH stimulation. Therefore, the dense vesicularization of mitochondrial cristae may constitute an early process to enhance the steroidogenic capacity of these cells. Within cells of the transition zone between zona glomerulosa and zona fasciculata, we could depict a special type of mitochondria with characteristic crescent-like cristae only seen after stimulation with CRH. This type of mitochondria may represent an intermediate form between mitochondria of zona glomerulosa and zona fasciculata underlining the impressive transformational capacity of adrenocortical mitochondria. After hypophysectomy, zona fasciculata cells contained mitochondria with tubular inner membranes, representing a hypofunctional state. In contrast, the hypofunctional state after hypophysectomy and the hyperfunctional state after stimulation of the adrenal cortex via CRH injection did not appear to correlate with the morphology of mitochondria from the zona reticularis and adrenal medulla.© Willey-Liss, Inc.     

DIFFERENTIATION OF THE FETAL ADRENAL CORTEX OF RATS - ITS EXPERIMENTAL OBSERVATION In Vivo

The development and tissue differentiation of the adrenal cortex of rats were studied by giving experimental treatments to the fetus in vivo. A low-AGTH-condition was given to the fetus by administrating Dexamethasone through its mother, or directly into the subcutaneous tissue of the fetus, or by decapitation, brain aspiration or hypophysectomy. When the fetus was given the low-ACTH-condition for the last 5 days or more, the hypoplastic adrenal cortex developed specifically. It had nearly normal zona glomerulosa (ZG), and poorly differentiated zona fasciculo-reticularis (ZF-R) without differentiated ZF-R. The observing day was settled at 21 1/2 days old. The shorter the period of low-ACTH-condition was, the more thickened the differentiated zone (ZF-R) grew. On the contrary, the poorly differentiated layer became thinner in proportion to the period of low-ACTH-condition. The poorly differentiated cortical cell had ACTH-reactivity. It seems that the development and tissue differentiation of the ZF-R depend on fetal own ACTH, and poorly differentiated cortical cells appear below the ZG, and differentiate by ACTH and were gradually pushed inside. The zona glomerulosa develops and differentiates without dependence of ACTH. ACTA PATH. JAP. 27: 759-773, 1977.     

Structure and dynamics of adrenal mitochondria following stimulation with corticotropin releasing hormone.

The mitochondria of rat adrenals were investigated qualitatively and quantitatively in different functional states of the adrenal cortex. Following stimulation of the animals with corticotropin releasing hormone (CRH), the corticosterone serum levels reached a maximum 1 hour after stimulation with CRH. The amount of inner mitochondrial membrane within the zona fasciculata increased showing a biphasic time course, with a first maximum 2 hours and a second maximum 8 hours after stimulation. In contrast, a significant rise of mitochondrial volume occurred only 24 hours after CRH stimulation. Therefore, the dense vesicularization of mitochondrial cristae may constitute an early process to enhance the steroidogenic capacity of these cells. Within cells of the transition zone between zona glomerulosa and zona fasciculata, we could depict a special type of mitochondria with characteristic crescent-like cristae only seen after stimulation with CRH. This type of mitochondria may represent an intermediate form between mitochondria of zona glomerulosa and zona fasciculata underlining the impressive transformational capacity of adrenocortical mitochondria. After hypophysectomy, zona fasciculata cells contained mitochondria with tubular inner membranes, representing a hypofunctional state. In contrast, the hypofunctional state after hypophysectomy and the hyperfunctional state after stimulation of the adrenal cortex via CRH injection did not appear to correlate with the morphology of mitochondria from the zona reticularis and adrenal medulla.     

Multiple pigmented adrenal cortical nodules associated with cushing's syndrome

Multiple pigmented adrenocortical nodules were found in a 25?year-old woman associated with Cushing's syndrome, whose laboratory data indicated that the adrenal cortex had been functioning autonomously and adrenocorticotropic hormone (ACTH) from the pituitary gland as suppressed. The surgically removed left adrenal gland disclosed multiple black nodules measuring up to 3 mm in diameter and histologically consisting of large “compact cells” which contained numerous yellow-brown pigments, but adjacent cortical cells were not atrophied. This kind of adrenal lesion is generally regarded as nodular hyperplasia of the cortex. The present case revealed scanty lipid and markedly increased activity of 3β-hydroxysteroid dehydrogenase (3β-HSD) and glucose?6?phosphate-dehydrogenase (G6PD). Ultrastructural study showed abundant cytoplasm with a large number of mitochondria, well-developed smooth-surfaced endoplasmic reticulum (SER), less rough-surfaced endoplasmic reticulum (RER), lysosomes, and numerous granules in cells of the nodules. Mitochondria varied in size and shape up to occasional giant mitochondria. SER was vesicular or tubular forming a network of anastomosing tubules. Granules varied greatly in size from 400 mm? to 6 m? in diameter, with diverse electron densities, mostly exhibiting the structural features of lipofuscin. The ultrastructural features resembled those in black adenoma associated with Cushing's syndrome ever reported. Concentration of cortisol was increased in the tissue where numerous black nodules were contained. Acta pathol. jpn. 34: 827 ～ 837, 1984.     

Ultrastructural morphometry of normal adrenal cortex and of hyperplastic adrenals in gushing’s disease

Surgical specimens of 4 normal adrenal glands and of 5 hyperplastic ones in Cushing’s disease were studied ultrastructurally. The ultrastructure of the three zones of each adrenal gland was morphometrically and statistically analyzed. Comparing the statistical data of the three zones of the normal gland, the development of smooth endoplasmic reticuium and rough endoplasmic reticuium showed an increase from the outer zona glomerulosa to the inner zona fasciculata and reticularis. Also, the mitochondria were more numerous in the inner zona reticularis than in the outer zones. In Cushing’s disease, the smooth endoplasmic reticuium, the rough endoplasmic reticuium, and the mitochondria were developed to a significantly higher degree than in normal human adrenal glands. The distribution of the cell organelles in the zona fasciculata and zona reticularis is almost alike in normal adrenal glands and in the adrenal cortex in Cushing’s disease, except that the smooth endoplasmic reticuium in the zona reticularis was less extensive than in the zona fasciculata. The volume percentages of lipid vacuoies in the hyperplastic zona fasciculata in Cushing’s disease was strongly and significantly decreased in comparison to normal adrenal glands. Our ultrastructural findings and the statistical data were in accordance with the results from animal experiments with adrenocorticotropic hormone (ACTH) stimulation and confirmed former qualitative ultrastructural findings concerning human adrenal gland changes in ACTH-dependent Cushing’s disease.