Histophysiology of the zona fasciculata of the adrenal cortex of albino rats

Three phases have been detected in the fascicular zone of the white rat adrenal cortex. During the first phase (normoemia) blood stream is rather moderate, lipids accumulate in adrenocorticocytes. This results in increase of their volume and in decrease of nuclear-cytoplasmic relations. The second phase (functional hyperemia) is characterized with an elevated blood stream and plethora, maximal parameters of nuclear volume and nuclear-cytoplasmic relations, decreasing content of lipids and volume of adrenocorticocytes. During the third phase (functional hypoemia) parameters characterising intensity of blood stream, nuclear volume and nuclear-cytoplasmic relations decrease.     

Morphometric study of the postnatal development of the human adrenal cortex]

Under study was the developmental dynamics of a number of morphometrical parameters of the adrenal cortex of man. The number of glandular cells in the organ was found to considerably decrease at late developmental periods, the size of nuclei in them diminished. These factors are considered to be responsible for the drop of functional activity of the adrenal cortex in elderly and senile age. The increase of the cell size during the growing of the organism is thought to be an important factor of the growth of the adrenal cortex of man.     

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.     

Morphology and ultrastructure of the adrenal gland in Bactrian camels (Camelus bactrianus)

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.     

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.     

Changes in the thymus of peripubertal rats induced by centrally applied somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

Changes in the Thymus of Peripubertal Rats Induced by Centrally Applied Somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.     

Investigations on the turnover of adrenocortical mitochondria

The effects of chronic administration of ACTH and dexamethasone on the morphology of mitochondria in zona glomerulosa cells of the rat adrenal cortex were investigated by stereological techniques. It was found that the volume of the mitochondrial compartment as well as the surface of the outer and inner mitochondrial membranes were significantly increased or decreased in relation to the number of days of ACTH- or dexamethasone-treatment. In ACTH-administered rats, the average volume of individual mitochondria decreased significantly up to the 6th day of treatment and then showed a conspicuous increase from the 6th to the 15th day, whereas in dexamethasone administered animals this parameter, after a small increase during the first 6 days of treatment, displayed a significant decrease. The number of mitochondria per cell showed a dramatic increase during the first 6 days of treatment with ACTH and continued to increase, but only slightly, with the subsequent treatment. In contrast, this parameter showed a parabolic decrease as a function of the duration of treatment in animals receiving dexamethasone. In the light of evidence showing that dexamethasone blocks ACTH-release, these findings are discussed and interpreted to indicate that ACTH is involved in the maintenance and stimulation of the growth and proliferative activity of mitochondria in rat adrenal zona glomerulosa.     

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     

Proliferation-associated oxygen consumption and morphology of tumor cells in monolayer and spheroid culture.

The oxygen consumption rate, proliferative activity, and morphology of EMT6/Ro mouse mammary sarcoma cells in monolayer and multicellular spheroid culture have been investigated in a comparative study. During the transition of monolayer cells from the exponential into the plateau growth phase, there is a distinct decrease in the cellular volume that is associated with a corresponding decrease in the proliferative and respiratory activity of the cells. The decline in cell volume is mainly due to a decrease in the content of cytoplasm, whereas the size of the nucleus is only slightly reduced. A concomitant decrease in the number of mitochondria per cell obviously accounts for the reduction in cellular oxygen uptake. Despite a continuous decrease of cell proliferation from the surface to interior regions of EMT6 spheroids reflected by a gradient in tritiated thymidine labeling, volume-related oxygen consumption is rather uniform in viable regions of these aggregates. The finding can be explained by the results of the morphometric evaluation showing a uniform volume density of mitochondria, i.e., of oxygen-consuming sites within these spheroids.     

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.     

The effects of chronic SRIH-14 and octreotide administration on the pituitary-adrenal axis in adult male rats

The effects of chronic treatments with SRIH-14 and octreotide on pituitary corticotropes (ACTH cells) and on the adrenal cortex of male Wistar rats were examined. Adult males received two daily s.c. injections of 20 microg/100 g of body weight of either SRIH-14 or octreotide for 28 consecutive days. ACTH cells were studied using a peroxidase-antiperoxidase immunocytochemical procedure. Morpho-metry was used to evaluate the changes in cell and nuclear volumes (microm3) and volume densities (%) of ACTH-immunoreactive cells. The adrenal cortex was analyzed by histological and morphometric methods. A significant (p<0.05) decrease in body weight and in the absolute weights of the pituitary and adrenal glands was observed in both treated groups. Morphometric parameters of ACTH cells in both treated groups were not significantly (p>0.05) different than in control rats. The absolute volumes of the adrenal gland and adrenal cortex were significantly (p<0.05) decreased in both treated groups. The absolute and relative volumes of the zona glomerulosa (ZG), as well as the cellular and nuclear volumes of the ZG were significantly (p<0.05) decreased in the both treated groups. In rats treated with SRIH-14 and octreotide, the absolute and relative volumes of the zona fasciculata (ZF) and zona reticularis (ZR), as well as their stereological parameters, did not change significantly (p>0.05). The aldosterone levels in the SRIH-14 and ocreotide-treated groups were significantly (p<0.05) decreased - by 13% and 19%, respectively. The concentration of ACTH and corticosterone did not change significantly. Together, these findings show that SRIH-14 and octreotide administration affected the morphological characteristics of the adrenal ZG in a similar manner, and brought about a decrease in plasma aldosterone concentration. These treatments did not affect pituitary ACTH cells or adrenal ZF and ZR functioning.     

The effects of monensin on Golgi complex of adrenal cortex and steroidogenesis.

The ultrastructural and biochemical changes produced by monensin on zona fasciculata cells of the rat adrenal cortex are described. In this study we used adrenal cells in culture, adrenal slices and the intact animal. Monensin (1 microM) was added to the culture medium containing the cells, and to the incubation medium containing the adrenal slices, and was injected intravenously to the intact animal (0.65 mg/kg body weight). The ultrastructural alterations were similar in the three experimental conditions, and consisted of Golgi complex disorganization with dilated cisternae or large smooth vesicles. Quantitative analysis showed a significant increase of the relative volume of the Golgi area. The biochemical study demonstrated a significant decrease of corticosterone concentrations in culture medium after monensin addition, and in adrenal glands from treated rats. These results showed that monensin alters the fine structure of adrenal cortex Golgi complex and inhibits corticosteroidogenesis, which supports the probable role of the Golgi complex in the regulation of steroidogenesis.     

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.     

In vitro studies of nuclear volume of rat adrenocortical cells: lace of ACTH effect

The effect of ACTH on nuclear volume of adrenocortical cells in the zona fasciculata of rat adrenal cortex was examined in vitro. Sections of adrenal gland were incubated for 60 or 90 min in Krebs-Ringer's solution with 1% glucose in the presence of ACTH, actinomycin D, cycloheximide and aminoglutethimide. ACTH, despite its clear effect in stimulating steroidogenesis, did not exert a direct effect on the nuclear volume of cells studied. This phenomenon is not dependent upon the stimulation of steroidogenesis, since aminoglutethimide does not influence the nuclear volume of adrenocortical cells studied; rather, ACTH in the presence of aminoglutethimide leads to a decrease in their volume. Actinomycin D does not influence nuclear volume while after incubation with cycloheximide nuclei were larger than the control. The presence of ACTH did not alter this effect. These results indicate no relationship between the degree of corticosterone output and nuclear volume in rat adrenocortical cells of the zona fasciculata in vitro.     

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.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

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.     

pH-Sensitive Liposomes

Abstract

pH sensitive liposomes are lipid compositions that can be destabilized when the external pH is changed; usually from a neutral or slightly alkaline pH to an acidic pH. They are designed to circumvent delivery of liposome contents to the lysosomes of cells following internalization of the vesicle via the endocytic pathway. In the majority of compositions, a lipid containing a pH titratable group is mixed with phosphatidylethanolamine containing unsaturated acyl chains in a molar ratio (pH sensitive component/PE) of 1/4 or greater. There are five major groups of phosphatidylethanolamine containing pH-senstive lipid compositions. These can be classified by their acid-titratable component: phospholipids, acylated amino acids, fatty acids, cholesterol derivatives and miscellaneous double chain amphiphiles. The biophysical mechanism of action involves a transition of the lipids from the lamellar phase to the hexagonal phase. In cell culture, pH sensitive vesicles can increase the delivery of fluorescent markers, proteins, cytotoxic compounds, RNA and DNA into the cytoplasm. The mechanism of delivery is suggested to involve the destabilization of the liposome in the endosome as the pH is reduced from 7.4 to 5.0 and subsequent destabilization of, or fusion with, the endosomal membrane; some of the liposome contents are introduced into the cytoplasm. In most cases, the extent of liposome contents delivery into the cytoplasm is less than 1% of the amount that becomes cell associated. However further studies, with more reliable assays to differentiate cytoplasmic from lysosomal delivery, are required to place an exact value on this efficiency. The efficiency of pH sensitive liposomes in vivo is limited by stability of certain of the liposome compositions in serum and targeting to the appropriate cell. Cholesterol hemisuccinate is a particularly attractive component for in vivo use since it stabilizes the liposome when in serum at pH 7.4. The use of pH sensitive liposomes in drug delivery should continue to expand due to the increasing number of macromolecular therapeutic agents with intracellular targets.