The fine structure of the newborn rat adrenal cortex

Summary The structural changes of the zona juxtamedullaris of the rat adrenal cortex at birth, have been examined by the light and the electron microscope. In this zone clusters of medullary cells lying among the strands of cortical tissue were observed. In the inner portion of the zona juxtamedullaris two types of adrenocortical cells were found: light and very-dark cells. The latter are smaller than the light cells and are always in close connection with the medullary tissue. The ultrastructural features of the very-dark cells suggest that these elements are in degeneration. This finding supports the hypothesis that at birth there is a partial degeneration of the rat zona juxtamedullaris, i.e. the zone corresponding to the fetal zone of some mammalian species.It is proposed that in all mammalian species at birth there is a partial regression of the zona juxtamedullaris and that the regression of the fetal zone is only the quantitative increase of this phenomenon. This hypothesis is discussed in relation to numerous data demonstrating that there are enzymatic conditions in the rat during fetal life, which permit a discrete hypertrophy of the adrenal cortex.The author wishes to express his appreciation to Dr. A. Gambino and to Mr. G. Gottardo for technical assistance.     

Gestational changes in hamster adrenal cortex: Morphometric and ultrastructural stereologic studies

Summary In the hamster, the weight of the adrenal glands increases during the course of gestation, with the highest value at day 5. In comparison to non-pregnant control animals, there were no changes in the volume of the zona glomerulosa (ZG) and zona fasciculata (ZF), while the volume of the zona reticularis (ZR) increased notably. The average volume of ZG-cells rose at day 5 of pregnancy and thereafter gradually decreased to that of control hamsters. A marked drop in the volume of ZF-cells was seen at days 5 and 10 of pregnancy, whereas at day 15 the cells were larger than in controls. At day 5 of pregnancy, a conspicuous increase in the cell volume was found in ZR, followed by lower values at day 10 and again higher than in control hamsters at day 15. The total number of parenchymal cells in hamster adrenal cortex increased at day 10 of gestation, then underwent a marked decrease, reaching the control value at the final day of pregnancy; this drop was mainly due to a reduction in the number of ZF-cells. The changes in the cell volume were paralleled by rather proportional changes in the volume of the mitochondrial compartment and in the quantity of smooth endoplasmic reticulum. The volume of the lipid-droplet compartment significantly rose in the course of gestation in both ZF- and ZR-cells. The cortisol output by adrenal homogenates gradually decreased during pregnancy.Study performed in partial fulfillment of the Ph.D. Thesis of Magdalena Nowak     

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

Summary 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, which 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 -glucuronidase).     

Unusual mitochondrial ultrastructure in the pig adrenal cortex

Unusually large mitochondria with few cristae were observed in the cells of the boundary layer between the zonae fasciculata and reticularis of the pig adrenal. These mitochondria occasionally contained parallel arrays of beaded filaments which appeared to be composed of repetitive electron opaque particles, measuring 10 to 11 nm in diameter. The possibility that these filaments are arranged in closely packed arrays of tubular structures with a central filament is discussed.     

On the innervation of the rat and pig adrenal cortex

Summary The innervation of the adrenal cortex of the rat and the pig is investigated with the electron microscope. Nerve fibers containing synaptic and two types of dense-cored vesicles come into contact with endocrine cells. There are no specialized pre- and postsynaptic membranes. The synaptic cleft is about 200 Å wide. Generally the basement membrane between nerve and cell is absent. These observations are discussed on the base of more recent experimental findings. Small fibers having an average diameter of about 0.2 to 0.5 and containing only tubules and filaments are considered to represent parts of an afferent nervous system.Dedicated to Prof. v. Kügelgen on the occasion of his 60th birthday.Supported by the Deutsche Forschungsgemeinschaft.     

Catecholaminergic innervation of the rat adrenal cortex

Summary The zona glomerulosa of the rat adrenal gland is innervated by catecholaminergic nerves. Using histofluorescence techniques, we observed catecholaminergic plexuses surrounding adrenal capsular and subcapsular blood vessels. Individual varicose nerve fibers that branched off these plexuses were distributed among adrenal glomerulosa cells. This innervation was permanently eliminated after neonatal sympathectomy with guanethidine or 6-hydroxydopamine, but was not affected by ligation of the splanchnic nerve or extirpation of the suprarenal ganglion. At the ultrastructural level, axonal varicosities were commonly observed in close proximity to glomerulosa cells and blood vessels. Nerve fibers and varicosities were found to contain small (30–60 nm) clear vesicles as well as large (60–110 nm) and small (30–60 nm) dense-cored vesicles. In tissue fixed for the dichromate reaction with or without pretreatment with the false transmitter 5-hydroxydopamine, many nerve terminals contained numerous small dense-cored vesicles which are thought to contain catecholamines. These results establish the anatomical substrate for the catecholaminergic innervation of the rat adrenal cortex.     

Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure of the sarcoplasmic reticulum (SR) in the red, white and intermediate striated muscle fibers of the extensor digitorum longus muscle of the rat was examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by the osmium-DMSO-osmium procedure.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of the A-I junction. Numerous slender sarcotubules, originating from the A-band side terminal cisternae, extend obliquely or longitudinally and form oval or irregular shaped networks of various sizes in front of the A-band, then become continuous with the tiny mesh (fenestrated collar) in front of the H-band. The A-and H-band SR appears as a single sheet of anastomotic tubules. Numerous sarcotubules, originating from the I-band side terminal cisternae, extend in threedimensional directions and form a multilayered network over the I-band and Z-line regions. At the I-band level, paired transversely oriented mitochondria partly embrace the myofibril. The I-band SR network is poorly developed in the narrow space between the paired mitochondria, but is well developed in places devoid of these mitochondria.The three-dimensional structure of the SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a much smaller total volume of SR than the mitochondria-poor white fiber. Moreover, the volume of SR of the intermediate fiber is intermediate between the two.     

Scanning electron microscopy of human cerebellar cortex

Summary Scanning electron microscopy and cryofracture technique were applied to study neuronal architecture and synaptic connections of the human cerebellum. Samples were processed according to the technique of Humphreys et al. (1975) with minor modifications. The granule cells exhibit unbranched filiform axons and coniform dendritic processes. The latter show typical claw-like endings making gearing type synaptic contacts with mossy fiber rosettes. The unattached mossy rosettes appear as solid club-like structures. Some fractographs show individual granule cells, Golgi neurons and glomerular islands. The climbing fibers and their Scheibel's collaterals were also characterized. In the Purkinje layer the surface fracture was produced at the level of the Bergmann glial cells, which are selectively removed, allowing us to visualize the rough surface of Purkinje cells and the supra- and infraganglionic plexuses of basket cell axons which appeared as entangled threads. In the molecular layer the three-dimensional configuration of the Purkinje secondary and tertiary dendritic branches was obtained. The filiform parallel fibers make cruciform synaptic contacts with the Purkinje dendritic spines. The appearance of stellate neuronal somata closely resembled that of the granule cells. The subpial terminals of Bergmann fibers appeared attached to the exterior of the folia forming the rough surfaced external glial limiting membrane.     

Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells

The membrane-bound adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Millipore filters with pores 0.22 μm in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5′-guanylyl-imidodiphosphate at a concentration of 1 · 10^?6 M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5′-guanylyl-imidodiphosphate.     

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.     

Light and scanning electron microscopic study of cerebellar cortex of teleost fishes

Summary The teleostean cerebellar cortex has been studied with respect to its cytoarchitectonic arrangement and intracortical neuronal circuits. Samples of fish cerebellum were fixed either by immersion or vascular perfusion in 5% glutaraldehyde solution and processed for light and scanning electron microscopy. The cerebellar cortex shows four distinct layers: granular; fibrous stratum; Purkinje cell; and molecular layers. In the granular layer, mossy and climbing fiber glomeruli were characterized. The mossy glomerular region appeared as polygonal, round or ovoid clews formed by the convergence of up to 17 dendritic profiles upon a thick mossy fiber branch. The en passant nature of mossy fiber-granule cell dendrite synaptic relationship was clearly appreciated. The climbing fibers showed tendril and glomerular collaterals. The latter form thin, elongated glomeruli. Remnants of a neuroglial envelope were observed in the mossy fiber glomeruli but are apparently absent from the climbing fiber glomeruli. The beaded-shape Golgi cell axonal ramifications were observed participating in the formation of both glomerular types. Velate protoplasmic astrocytes and oligodendrocytes were also identified. The fibrous stratum appeared to be formed by compact bundles of thick and thin myelinated axons, running horizontally beneath the Purkinje cell layer and apparently belonging to ascending climbing fibers and descending Purkinje cell axons. At the Purkinje cell layer a selective removal of Bergmann glial cells was observed allowing the visualization of the pericellular basket and the pinceaux. Climbing fiber stems and their tendril collaterals were seen on their way to the molecular layer ascending parallel to the Purkinje dendritic ramifications. Stellate neuron processes were found passing through the fan-like arborescence of Purkinje cell dendrites.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

Tissue culture of rat adult decapsulated adrenal glands

Summary A method of cultivation involving both repeated trypsinisations (at room temperature) and explantation of the tissue fragments on polythene discs has been shown to be apt to the growth in vitro of rat adult decapsulated adreno-cortical tissue. This is the first time that the successful cultivation of such a tissue is reported. The technique and its applications are discussed.The effects of _1–24 corticotrophin (ACTH_1–24) on the rat adult adrenal cultures have been examined by both electron microscopy and autoradiography. Zona fasciculata and reticularis cells grown in the absence of ACTH for long terms (15–16 days) survive and proliferate as dedifferentiated elements. If ACTH_1–24 is added to the cultures, adrenocortical cells will, within 2 days, simultaneously increase their proliferation rate and differentiate. After 7 days of treatment, cortical cells exhibit not only fully differentiated but even hypertrophic morphologic features. Significant stimulations of adrenal DNA, RNA and gross protein synthesis have been found to take place at different times after the starting of the ACTH_1–24 treatment. These data are discussed in relation to the findings previously reported in literature.Rat adult adrenal gland tissue cultures are proposed as a non-previously available tool for investigations into the physiopathology of the adrenal cells to be carried out in a carefully controlled environment.A Preliminary report on part of this material was given at the annual meeting of the Société Française de Microscopie Elécronique, Nantes, May 1972.Authors wish to thank Drs. P. G. Andreis and A. S. Belloni for their skilful assistance in the autoradiographic experiments. Thanks are also due to Mr. G. Gottardo for his excellent technical help in electron microscopic work. This work was partly supported by a contract with the CNR-Italy (No 69.0172/115.3439).     

Scanning electron-microscopic studies on the three-dimensional structure of mitochondria in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure and arrangement of mitochondria in the red, white and intermediate striated muscle fibers of the rat were examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by means of the Osmium-DMSO-Osmium procedure.Beneath the sarcolemma, spherical or ovoid subsarcolemmal mitochondria show accumulations. The mitochondria are numerous and large in size in the red fibers, intermediate in the intermediate fibers, and few and small in the white fibers. Paired, slender I-band-limited mitochondria were located on both sides of the Z-line and partly embraced the myofibrils at the I-band level; they occurred in all three types of fibers. In the intermyofibrillar spaces, numerous mitochondria formed mitochondrial columns. These columns were classified into two types: 1) thick mitochondrial columns, formed by multiple mitochondria each with an intermyofibrillar space corresponding to one sarcomere in length, and 2) thin mitochondrial columns, established by single mitochondria corresponding to one sarcomere in length. In the red fibers mitochondrial columns were abundant and the ratio of the thick and thin columns was almost the same, while in the intermediate fibers most of the columns belonged to the thin type. The white fibers displayed rare, very thin columns.     

Aspects of the three-dimensional intracellular organization of mesocarp cells as revealed by scanning electron microscopy

Summary The osmium-ligand binding technique and scanning electron microscopy have been applied to the study of the three-dimensional organization of mesocarp cells of a mature avocado fruit. Using this approach the mitochondria of the cells appear as elongated, branching structures and the endoplasmic reticulum consists of a complex of tubular strands, vesiculated strands and lamellar sheets. Associations of the endoplasmic reticulum with other organelles are also apparent. It is suggested that this approach provides a valuable means to assess the structural transitions in cell organization that occur during development or with functional changes.     

Effects of a prolonged treatment with aminoglutethimide on the zona fasciculata of rat adrenal cortex: A morphometric investigation

Summary The effects of a 7-day administration of aminoglutethimide (AG) on the adrenal zona fasciculata were examined in normal and dexamethasone/ACTH-treated rats. There was a 70–74% decrease in the concentration of corticosterone in blood, but no conspicuous qualitative changes suggesting cell degeneration occurred. Morphometry showed that AG induced a significant hypertrophy of the zona fasciculata and its parenchymal cells only in normal animals, which was due to an increase in the volume of the mitochondrial compartment and to proliferation of the smooth endoplasmic reticulum. This response to AG was considered to be non-specific and mediated by the enhanced secretion of ACTH following the decrease in the blood level of corticosterone. AG administration significantly increased the volume of the lipid-droplet compartment and the number of intramitochondrial lipid-like inclusions in both groups of animals. These changes were interpreted as the morphological counterpart of the AG-induced block of cholesterol utilization in steroid synthesis.     

An electron microscope autoradiographic study of DOC conversion to corticosterone in the rat adrenal cortex

Summary Seven thymuses from children between 1 and 12 years were examined by electron microscopy. Biopsies had been taken during surgical correction of congenital heart defects.In all cases we found interdigitating reticulum cells (IRC) in the medulla and inner cortex. These cells resembled the IRC which have been described previously in the thymus-dependent regions of the spleen and lymph node. They were characterized by an irregularly shaped nucleus, narrow cisterns of rough endoplasmic reticulum, and widespread interdigitation and invagination of the cell membrane. The surfaces of the IRC were in close contact with those of small lymphocytes, sometimes polysomal lymphatic cells, epithelial cells, and occasionally with those of lymphatic cells containing ergastoplasm.The IRC is apparently a specific cell of thymus-dependent regions. It may be that the IRC in the thymus, lymph node, and spleen contribute to the microenvironment needed for the differentiation of T-cells.Supported by the Deutsche Forschungsgemeinschaft, SFB 111/CII and III.—We wish to thank Miss M. Neubert and Mrs. R. Köpke for their technical assistance and Mrs. M. Soehring for her help with the translation.     

Metabolism and toxicity of xenobiotics in the adrenal cortex,with particular reference to 7,12-dimethylbenz(a) anthracene

The adrenal cortex contains high amounts of detoxifying enzymes, as well as generators and protectors of reactive oxygen species. The high content of cytochrome P-450 enzymes in the adrenal cortex together with its remarkable tendency to accumulate hydrophobic substances probably contributes to the extraordinary vulnerability of the gland to a number of xenobiotics. The best studied adreno-corticolytic compounds are the potent carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) and its liver metabolite 7-hydroxymethyl-12-methylbenz (a)anthracene (7-OHM-12-MBA). Adrenocorticolysis generated by these agents in vivo as well as in vitro demonstrates high regioselective requirements and is strongly influenced by the presence of ACTH, steroids, cytochrome P-450 inhibitors and antioxi-dants. Furthermore, 7-OHM-12-MBA has been demonstrated to uniquely generate selective and massive oxidation of mitochondrial glutathione in cultured rat adrenal cells. The DMBA-induced adrenocorticolysis is thoroughly discussed in this review with particular emphasis on the metabolism of DMBA and the influence of various effectors. A working hypothesis involving a possible peroxidative mechanism is also presented.     

Irido-corneal angle of mammalian eyes: Comparative morphology of encapsulated corpuscles in odontocete cetaceans

Summary A study of the structure and distribution of encapsulated corpuscles (corpuscles of Rochon-Duvigneaud) in the irido-corneal (anterior chamber) angle in 42 adult eyes from nine species of odontocete cetaceans representing each of the five families in this group indicates a diversity of form and function. The corpuscles occur as single features or multiple groupings with each species having a distinct pattern. No definite phylogenic or environmental patterns were seen although sometimes there were similarities among related genera. The neural structure of the angle suggests that these receptors probably respond to a variety of stimuli including intraocular pressure, vitreous displacements, external muscular force applied to the sclera and cornea, and changes in the volume of the massive iris/choroid vascular system.