The ultrastructure of functional mouse adrenal cortical tumor cells in vitro.

Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitchondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5-20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.     

The Ultrastructure of Functional Mouse Adrenal Cortical Tumor Cells in vitro

Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitochondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5–20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.     

Population dynamics of mitochondria. I. A model for the role to ACTH in the degradation of adrenocortical mitochondria

An allosteric substance has been supposed to be present in the adrenocortical cell and to be involved in the degradation of the adrenocortical mitochondria only when it is present in the cytoplasm as a free form. An allosteric effector has also been assumed to be synthesized in the adrenal cortex strongly depending on the ACTH supply. The allosteric effector combines hypothetically with the allosteric substance to form an association product. In its bound form, the allosteric substance is assumed to be inactive in the degradation reaction of mitochondria. With these assumptions a differential equation has been obtained to describe the decay process of those mitochondria. An algorithm has been developed to compute the dynamical fate of the mitochondria in a simple, iterative way. Experimental results on the mitochondrial decay in the rat adrenal cortex after hypophysectomy have been fitted to the differential equation in a satisfactory manner. It has been stressed that the present hypothesis constitutes in its essence a new working hypothesis on the maintenance of adrenocortical mitochondria under normal physiological conditions.     

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.     

The role of the cytoskeleton in the regulation of steroidogenesis

The slow step in steroid synthesis involves the transport of cholesterol from lipid droplets in the cytoplasm to the first enzyme in the pathway—the cytochrome P450 that converts cholesterol to pregnenolone (P450scc) which is located in the inner mitochondrial membrane. ACTH stimulates this intracellular transport of cholesterol in adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells) and this effect of the trophic hormone is inhibited by cytochalasins, by anti-actin antibodies and DNase I suggesting that the response to ACTH requires a pool of monomeric (G-) actin that can be polymerized to F-actin. Recent studies have shown that lipid droplets and mitochondria of adrenal cells are both attached to intermediate filaments. Moreover ACTH reorganizes the cytoskeleton and changes the shape of the cell. These observations suggest a mechanism for transport of cholesterol that involves reorganization and contraction of actin microfilaments which may, in turn, cause movement of droplets and mitochondria together through their common attachment to intermediate filaments.     

Structural characteristics and innervation of chromaffin tissue in the adrenal gland of the axolotl

Each adrenal gland of the Axolotl consists of a strip lying all along the medio-lateral edge on the ventral surface of the kidney. The gland is composed of interrenal cells (IC) and chromaffin cells (CC). The IC contained a great number of pleomorphic lipid droplets, smooth endoplasmic reticulum and elongated mitochondria with tubulo-vesicular cristae. Two types of CC, always disposed in clusters and exhibiting long cytoplasmic processes were described according to the electron density, size and shape of granules distributed in their cytoplasm; noradrenaline cells (NA) and adrenaline cells (A). The innervation and ultrastructural differences from the adrenal gland of other Anurans were discussed.     

Organogenesis of the pituitary, adrenal, and lung at birth in the wallaby, Macropus rufogriseus

The ultrastructure of the pituitary, the adrenal, and the lung was examined in the newborn wallaby, Macropus rufogriseus. Tissue from six wallaby neonates (less than 8 hr of age), two near-term fetuses (26 days after removal of suckling pouch young [RPY]), and a two-day-old pouch young was examined; and tissue levels of cortisol in the adrenal glands of five neonates and a near-term fetus (26 days) were measured by radioimmunoassay. At birth the adenohypophysis comprised the bulk of the pituitary gland. The pars distalis was well vascularized and many cells contained electron-dense, membrane-bound granules. The adrenal glands lacked specific zones but comprised two distinct populations of cells. The cytoplasm of one cell type contained electron-dense, membrane-bound granules, similar to those observed inside catecholamine-secreting cells of the adrenal medulla; the other cell type possessed large amounts of smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae. These features are characteristic of cells which are actively synthesizing steroid hormones. The concentration of cortisol was 0.58 ng/adrenal in the wallaby at birth. The fetal lungs near term were at the glandular stage of development, and epithelial differentiation of type I and type II pneumocytes was imminent although attenuation was not evident. The canalicular neonatal lung did not contain true alveoli, but type II pneumocytes contained osmiophilic lamellar inclusions of surfactant. The fetal pituitary and adrenal are functional at birth and are thus capable of initiating parturition and of influencing lung maturation in the fetus.     

Alpha-fodrin in the adrenal gland: localization by immunoelectron microscopy

Localization of fodrin, the brain equivalent of spectrin (a protein constituent of the erythrocyte membrane cytoskeleton), was investigated at the ultrastructural level in rat adrenal gland. By use of an affinity purified antibody directed against the alpha-fodrin subunit, all chromaffin cells, cortical cells, nerve fibers, and their surrounding Schwann cells were found to be labeled close to the cytoplasmic side of their plasma membranes. The labeling appeared more intense for chromaffin cells, and secretory granules and mitochondria were frequently found to be associated with the zone containing alpha-fodrin in these cells. The immunostained zone was estimated to extend 230 +/- 70 nm into the cytoplasm. This localization is discussed in terms of what is known of the properties of spectrin, and possible roles of the molecule in the chromaffin cell are suggested.     

ULTRASTRUCTURE, STEROIDOGENIC POTENTIAL, AND ENERGY METABOLISM OF THE SNELL ADRENOCORTICAL CARCINOMA 494 : A Comparison with Normal Adrenocortical Tissue

Electron microscope studies were carried out with the adrenocortical carcinoma 494 and normal adrenal cortex tissue. The mitochondria of the tumor cells showed marked differences when compared with mitochondria from fasciculata cells of the normal adrenal cortex. These differences were primarily related to mitochondrial number and crista structure. Corticosterone production in isolated tumor cells was extremely low and neither ACTH nor dibutyryl cyclic AMP had any stimulatory effect. Normal adrenal cells showed at least a tenfold increase under identical conditions. In the presence of corticosteroid precursors the amount of corticosterone produced by the tumor cells was much less than that produced by normal cells. The results indicate a reduced capacity for 11β-hydroxylation in the tumor mitochondria and a possible reduced capacity for biosynthetic steps before the 11β-hydroxylation reaction. Glycolysis in isolated tumor cells was also lower than in normal cells. Isolated tumor mitochondria oxidized succinate normally with a good degree of coupling with phosphorylation. However, unlike normal adrenal mitochondria, the tumor mitochondria showed little or no oxygen uptake with other Krebs cycle substrates. These data suggest that the tumor mitochondria may be lacking in the flavoprotein dehydrogenases responsible for the oxidation of NADH and NADPH, although other components of the respiratory chain may be intact.     

Glucose repression and autolysis ofSaccharomyces cerevisiae cells: alterations in the cytochemical localization of acid phosphatase

Summary Allerations in the localization of acid phosphatase inSaccharomyces cerevisiae during glucose repression and during autolysis have been studied. Cell morphology becomes distinctly changed after only 2 h in the presence of high glucose concentration while after 3 h of glucose repression the majority of the mitochondirial structures resemble promitochondria. Yeast cells repressed for 6 h contain almost completely degraded mitochondrial structures and numerous lipid droplets in the central vacuole and cytoplasm. Destruction of mitochondria is accompanied by the accumulation of acid phosphatase in these organelles and in the cytoplasm whereas its activity in the central vacuole is lowered, most probably because of the leakage of the enzyme into the cytoplasm.No preferential breakdown of mitochondria is observed during autolysis. On the contrary, mitochondria are apparently the last to be degraded. Digestion of cytoplasmic regions and membranous elements occurs intravacuolarly after sequestration by protrusions of the central vacuole which are formed at the initial stages of autolysis. Acid phosphatase is not released from the central vacuole, suggesting indirectly that vacuole enzymes do not migrate into the cytoplasm during autolysis.     

The circahoral rhythm of the functional activity of rat adrenocorticocytes]

Functional manifestations of biological ultradian rhythms in the adrenal cortex were first confirmed on the cytologic level. The cycle of the ultradian rhythm in the cells of the adrenal cortex fascicular zone consisted was determined to involve three phases: accumulation of lipids, delipoidization, and the rest period. A definite volume of cells and their nuclei, certain nuclear-cytoplasmic relations, the number of liposomes and mitochondria on the square unit are characteristic of each phase. The activity of cells is especially high during the second phase which is characterized by the increase in nuclear sizes and nuclear-cytoplasmatic relations, by the decrease in cell volume and in the number of liposomes in their cytoplasm; this is indicative of the rised synthesis and secretion of hormones. It is underlined that the cell volume increases when liposomes are accumulated in the cytoplasm to decrease upon their disappearance.     

Light and electron microscopic immunocytochemistry on the localization of 3 beta-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.     

Ultrastructural pathology of the adrenal gland in Cushing's syndrome

The ultrastructural pathology of the adrenal glands was studied in fifteen cases of Cushing's syndrome. Some specific features correlated with the pathological aspects of adrenals were found. In the hyperplastic adrenal cortex the cytoplasms contained a rich smooth endoplasmic reticulum and many mitochondria. Increased lipid-pigment complexes were found especially in the compact cells. In adenomas, the clear cells showed large lipid vacuoles; the compact cells presented anisomorphous mitochondria, a well developed smooth endoplasmic reticulum and many pigment bodies. The nuclei of adenomatous cells were irregular, with deep invaginations. In adrenal carcinomas, the pleomorphism of nuclei, nucleoli, mitochondria, and smooth endoplasmic reticulum was more obvious. Absolutely reliable characteristics proving malignancy at ultrastructural level do not, however, exist. The steroidogenic activity of both hyperplastic and tumoral adrenal glands can be assessed using the agranular endoplasmic reticulum and the mitochondria as functional parameters.     

Incorporation of regulatory peptide hormones by individual cells of the adrenal cortex: Prolactin-adrenocorticotrophin differences

Recent refinements in methodology now permit the study of endogenous peptide hormones in their individual target cells. The investigations reported here deal with the question of whether endogenous ACTH can be detected in its target cells in the highly active adrenal gland of the normally lactating rat. This question was examined with immunohistochemistry. ACTH was found in both cytoplasm and nuclei of adrenal glomerulosa cells. In cells of the fasciculata and reticularis layers of the adrenal cortex, it did not appear inside nuclei but was present in the cytoplasm and on the nuclear envelope. The distribution of ACTH was compared with and found to be different from that of PRL. PRL, confirming previous findings, was not detectable at all in glomerulosa cells and, in cells of the inner cortical zones, was present in both cytoplasm and nuclei. In neither case was hormone found in the adrenal medulla. The apparent feasibility of studying peptide regulators such as ACTH and PRL in their individual target cells may be a focal point for an acceleration of our understanding of how these peptides work.     

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.     

Effects of tropic hormones on ultrastructure and synthesis of androgens in adrenals of male pseudohermaphrodite rats

Summary Ultrastructural and biochemical study of the adrenals in the pseudohermaphrodite (tfm) rat reveals hypertrophic adrenocortical cells. The cytoplasm of the cells in the zonae fasciculata and reticularis contains an exceptionally well developed smooth endoplasmic reticulum closely applied to mitochondria and lipid droplets. Mitochondria are more numerous than in normals and have especially abundant tubular cristae. More lipid droplets (appearing as empty vacuoles) are surrounded by pleomorphic mitochondria.The incubation study indicates that the capacity of rat adrenal cortex of producing androgens is greater in tfm than in normal animals. Hypophysectomy and castration result in a significant decrease in androgen biosynthesis by tfm rat adrenals and cause a reduced concentration of plasma testosterone. Administration of tropic hormones to hypophysectomizedcastrated rats appears to stimulate their adrenal androgenesis. It is suggested that in tfm rats the higher than normal luteinizing hormone (LH) together with adrenocorticotropic hormone (ACTH) stimulates the hypertrophy of cellular organelles in the adrenal cortex and causes an accompanying increase in androgenic steroids which may be responsible, at least in part, for the increased level of plasma androgens.     

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.     

Mitochondrial dynamics in chondrocytes and their connection to the mechanical properties of the cytoplasm

BACKGROUND: The motion and redistribution of intracellular organelles is a fundamental process in cells. Organelle motion is a complex phenomenon that depends on a large number of variables including the shape of the organelle, the type of motors with which the organelles are associated, and the mechanical properties of the cytoplasm. This paper presents a study that characterizes the diffusive motion of mitochondria in chondrocytes seeded in agarose constructs and what this implies about the mechanical properties of the cytoplasm. METHOD OF APPROACH: Images showing mitochondrial motion in individual cells at 30 s intervals for 15 min were captured with a confocal microscope. Digital image correlation was used to quantify the motion of the mitochondria, and the mean square displacement (MSD) was calculated. Statistical tools for testing whether the characteristic motion of mitochondria varied throughout the cell were developed. Calculations based on statistical mechanics were used to establish connections between the measured MSDs and the mechanical nature of the cytoplasm. RESULTS: The average MSD of the mitochondria varied with time according to a power law with the power term greater than 1, indicating that mitochondrial motion can be viewed as a combination of diffusion and directional motion. Statistical analysis revealed that the motion of the mitochondria was not uniform throughout the cell, and that the diffusion coefficient may vary by over 50%, indicating intracellular heterogeneity. High correlations were found between movements of mitochondria when they were less than 2 microm apart. The correlation is probably due to viscoelastic properties of the cytoplasm. Theoretical analysis based on statistical mechanics suggests that directed diffusion can only occur in a material that behaves like a fluid on large time scales. CONCLUSIONS: The study shows that mitochondria in different regions of the cell experience different characteristic motions. This suggests that the cytoplasm is a heterogeneous viscoelastic material. The study provides new insight into the motion of mitochondria in chondrocytes and its connection with the mechanical properties of the cytoplasm.     

Short-term effects of ACTH on protein synthesis in adrenal cortex cells of young rats

Two units of ACTH were administered intraperitoneally to young 20 gm-rats which received an intravenous injection of L-leucine-3H thirteen min later. ACTH-injected rats, and control rats which received the isotope alone, were killed at 2-, 10-, 30- and 60-min intervals. Electron microscope autoradiographs in control animals showed strong amino-acid uptake at pulse time (2-min) in the cytoplasm of adrenal zona fasciculata cells. Label was shared between the endoplasmic reticulum (ER) and mitochondria, and a lower but still considerable uptake was seen in nucleoli. At first chase time interval (10-min) cytoplasmic labelling declined, while nuclear and nucleolar labelling increased, both changing little thereafter, and there was a 10-30 min Golgi peak. ACTH administration provoked an overall increase in amino-acid incorporation into cytoplasm, nucleus and nucleolus at pulse time, with no changes in the distribution of the reactions among organelles. Intensification of labelling was most evident over nucleoli, the grain density of which was four-times as high as in controls. The short-term increase in ER and mitochondrial protein synthesis observed after ACTH injections was considered to be consistent with the hypothesis that most newly-formed proteins in these cells may be involved in the regulation of steroidogenesis. The marked increase in nucleolar labelling suggested the presence of proteins involved in RNA synthesis.     

Adrenal glucocorticoid function and cytoplasmic dehydrogenase activity in the presnece of mechanical and toxic liver damage]

Against the background of low steroid metabolism in the liver there was noted some decrease in the rate of corticosterone synthesis by the adrenal gland sections, and also, a decrease in the dehydrogenase activity in the cytoplasm of adrenal cells in male rats 48 hours after partial hepatectomy, as compared to the sham-operated animals. These changes resulted from suppression of the central mechanisms of stress realization due to the lowered steroid metabolism. Intraperitoneal administration of carbon tetrachloride (0.1 ml per 100 g of body weight) at the same periods led to a significant intensification of the steroidogenesis in the adrenal tissue and to the activation of NAD-dependent dehydrogenases in the cytoplasm. The role of toxic injury of the glands in the changes of the functional state of the adrenal cortex cells is discussed.