Effects of mevinolin,an inhibitor of cholesterol synthesis,on the morphology and function of differentiating and differentiated rat adrenocortical cells in primary culture

Summary Mevinolin, an inhibitor of cholesterol synthesis, was used to study the effect of endogenous cholesterol synthesis on the morphology and function of differentiating and differentiated fetal rat adrenocortical cells grown in primary culture. Upon adrenocorticotrophic hormone (ACTH) stimulation under conditions in which endogenous cholesterol synthesis was inhibited but exogenous (lipoprotein) cholesterol was available, the cells differentiated normally from glomerulosa-like to fasciculata-like cells; the steroid hormone secretion was maximally induced. Under conditions in which cholesterol synthesis was maximally inhibited by mevinolin and the cells had no access to exogenous cholesterol, the cells did not differentiate into fasciculata-like cells; the ACTH-induced steroid response was highly suppressed under these conditions. The addition of either human low-density lipoprotein (LDL) or high-density lipoprotein (HDL₃) to the culture medium restored the ACTH-induced differentiation and steroid secretion. Thus, in the absence of exogenous cholesterol, endogenous cholesterol synthesis was a prerequisite for differentiation. In cultures grown in the presence of exogenous cholesterol and ACTH with mevinolin-inhibited cholesterol synthesis and high steroid output, an increase in cytoplasmic lipids was evident, suggesting upregulation of LDL and HDL receptors. The results also demonstrated that induction of phenotypic differentiation from glomerulosalike into fasciculata-like cells can proceed in the presence of a cholesterol synthesis inhibitor like mevinolin; this differentiation in the absence of endogenous cholesterol synthesis is accompanied by the appearance of cytoplasmic cholesterol ester droplets, typical of fasciculata cells.     

Biphasic effect of ACTH on growth of rat adrenocortical cells in primary culture

Summary The proliferation rate of differentiating fetal rat adrenocortical cells was studied in primary culture. In this system, stimulation with ACTH induces differentiation of zona glomerulosa-like cortical cells into zona fasciculata-like cells. Incorporation of bromodeoxyuridine (BrdU) was studied immunocytochemically by use of anti-BrdU antibody, and the proliferation rate was counted from the monolayer colonies of adrenocortical cells. After 21 days of cultivation in the absence of ACTH, the proliferation rate of zona glomerulosa-like cells was 10%. The rate slowly declined to 1% at the age of 100 days during continuous cultivation in the absence of ACTH. Stimulation with ACTH induced a strong inhibition in the proliferation rate (down to 2% during the first 24 h). Treatment with ACTH during the following 48 h led to an extremely intense proliferation of adrenocortical cells at a proliferation rate of 25%. Continuous treatment with ACTH up to 100 days led to a persistent growth of adrenocortical cells, and a proliferation rate over 2-fold higher than in control cells cultivated in the absence of ACTH. Thus, ACTH is the principal growth-promoting factor also in vitro, as has been found in in vivo studies. This growth effect is mediated by a biphasic course; at the beginning of differentiation the effect is inhibitory and is followed by a persistent stimulation of the growth of adrenocortical cells.     

Effects of chronic exercise on the ultrastructure of the adrenocortical cells in the rat

Summary Four groups of male rats were exercised for periods of 2, 4, 6, and 8 weeks with controls in each group. As a result of chronic exercise there was an increase in the width of the zona fasciculata of the adrenal cortex. Also, there was an increase in the number and size of the mitochondria, and an increase in the quantity of smooth endoplasmic reticulum, and during the first 4 weeks of exercise an increase in the number of lipid droplets in the zona fasciculata. The close relationship between the smooth endoplasmic reticulum and the mitochondria, and the relationship between the smooth endoplasmic reticulum and the lipid droplets suggests a possible means for a transport mechanism for movement of precursors between these organelles.This research was supported in part by a Public Health Research Career Development Award KO4 GM42,355 from the National Institute of General Medical Sciences     

In vitro effects of puromycin aminonucleoside on the ultrastructure of rat glomerular podocytes

Summary Puromycin aminonucleoside (PAN)-induced nephrosis in rats provides a model for studying the pathogenesis of severe proteinuric conditions, such as minimal change disease. The present study used scanning (SEM) and transmission (TEM) electron microscopy to investigate the in vitro effects of PAN on rat glomerular podocytes. Slices of rat kidney were incubated for up to 3 days in Medium 199 with Hanks' salts (control) or in medium with PAN. Semiquantitative SEM analysis of glomeruli on the upper surface of kidney slices indicated that incubation with PAN (100 g/ml and 500 g/ml) decreased the number of microvilli on podocyte cell bodies (days 1, 2 and 3), increased the number of glomeruli showing flattening of podocyte cell bodies and major processes (days 2 and 3), and increased the number of glomeruli showing surface membrane blebbing on podocyte foot processes (day 3) (p<0.001 in all cases). TEM morphometry revealed that incubation with 500 g/ml PAN retarded significantly (p<0.001 at days 2 and 3) the loss of podocyte foot processes observed in control cultures. Whilst the SEM changes to podocyte ultrastructure largely mimic those seen in PAN nephrosis in vivo, the retardation of foot process loss runs counter to the major TEM change observed in vivo.     

Investigations into the mechanism of hormone release by rat adrenocortical cells

Summary Vinblastine treatment blocks corticosterone release from rat adrenal zona fasciculata without impairing hormone synthesis, and induces the formation of acid phosphatase-positive granular clumps at the juxta-sinusoidal pole of the cells. Autoradiography shows that ACTH administration to vinblastine-treated animals mobilizes the 3H-cholesterol stored in the lipid droplets and leads to a noticeable labelling of the granular clumps. The possible significance of these granules is discussed.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.     

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.     

Effects of corticosteroid-hormones on the smooth endoplasmic reticulum of rat adrenocortical cells

Summary The ultrastructural changes occuring in adrenocortical cells of prednisolone-treated rats were evaluated by morphometric methods. They consist mostly in a conspicuous decrease in the smooth reticulum surface and in the lipid droplets. The diminution of the smooth reticulum accounts for about 60 % of the decrement in cellular volume and in adrenal weight. Since numerous enzymes of corticosteroid-synthesis are localized in the microsomal fraction of adrenocortical cells, it is reasonable to suggest that the decrement of these organelles is the morphological expression of the cellular deficit in hormone-synthesis.The possible mechanism of reduction of the reticulum membranes is discussed in relation to the probable regulation mechanism of the adrenocortical secretion. It is proposed that the reticulum decrement is the result of a reduced synthesis of new membranes, due to an inhibition of protein-synthesis in adrenocortical cells.The author wishes to express his sincere appreciation to Mr. G. Gottardo and to Dr. A. Gambino for their excellent technical assistance.     

Lipid detection by malachite green-aldehyde in the dental basement membrane in the rat incisor

Summary Developing rat incisors were treated with malachite green-aldehyde fixative solution (MGA), which retains and stains lipids. We observed positive staining occurring as dots in the basement membrane. Most of these dots (2–3.5 nm in diameter) were grouped in the lamina densa but some were also present in the lamina lucida and the lamina fibroreticularis. These data provide evidence for the existence of lipids in the dental basement membrane and suggest that they are distributed together with the various groups of proteins so far detected.     

Dynamic features of cells expressing macrophage properties in tissue cultures of dissociated cerebral cortex from the rat

Summary Two previously identified forms of macrophage were investigated in primary cultures of cerebral cortical cells. Dynamic features were revealed through time-lapse video recording and aspects of macrophage function were assessed. The two cell forms were shown to be different pre-mitotic stages of a single cell type. The cell cycle for these cells involved an initial large, flat, quiescent cell which retracted to yield a slightly rounded form with numerous processes. This latter form lost processes and developed profuse filopodia as it became very rounded just prior to division; both resulting daughter cells then regained the initial large flat appearance. These cells possessed several properties of macrophages, including phagocytosis, nucleoside diphosphatase enzyme, and CR3 receptors. These properties were transient, expressed just before and after mitosis, but subsequently down-regulated in the flat daughter cells. Because of this feature, it was difficult to determine the exact size of this cell population; however, the observed rate of proliferation suggests it may be substantial. It is suggested that these cells correspond to non-microglial macrophages of brain tissue and, because of their significant down-regulation, they may be difficult to detect. This may be important in studies of brain accessory immune cells in tissue culture.     

Culture and characterization of dental follicle cells from rat molars

Summary Because the dental follicle is necessary for the eruption of teeth of limited eruption, it was the objective of this study to determine if the cells of the follicle could be cultured in vitro. To achieve this, dental follicles and associated enamel organs were dissected from the first and second mandibular molars of 6–7-day-old rats (secretory stage of amelogenesis), and then cultured in a medium that promotes fibroblast growth — the predominant cell type of the dental follicle. The cultured cells grew to confluency and were kept through 3 passages before experimentation. The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained an abundant rough endoplasmic reticulum, but did not form desmosomes. Immunofluorescent staining for anti-vimentin showed that all the cells stained and electron-microscopic immunogold labeling indicated that the antibody was associated with intermediate filaments. As revealed by SDS-polyacrylamide gel electrophoresis and Western blotting, the cultured cells synthesized and secreted the extracellular matrix molecules fibronectin and procollagens. Subsequent immunofluorescence staining of permeabilized and non-permeabilized cells confirmed the presence of fibronectin and type I collagen both intra- and extracellularly. Thus, based on all the above characteristics, the cultured cells appeared to be fibroblasts derived from the dental follicle, although a few of the fibroblasts may be derived from undifferentiated mesenchymal cells interposed between the alveolar bone and follicle. Experiments now can be conducted to determine how these cultured cells respond directly to growth factors that alter the rates of tooth eruption.     

Differentiation of the melanotrophic cells of rat pituitary primordium in organotypic culture in defined medium

Summary Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a ³⁵S-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2–3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against -melanocyte-stimulating hormone (MSH), ₃ and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the MSH content of the explants and MSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in MSH secretion, which was significant after 3 days in culture, indicating that dopamine D₂ receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis.     

Effects of pimozide on the ultrastructure of the pars intermedia in the rat

Summary The effects of pimozide, a dopamine receptor-blocking agent, were studied in the pars intermedia of the rat. The animals received 100 g/100 g pimozide daily for 2, 5, 10, 15, and 20 days. Pimozide induces ultrastructural changes after 5 days of treatment. About 50% of the MSH-cells display characteristics of stimulation. Their cytoplasm is partially or totally depleted of secretory granules. The rough endoplasmic reticulum displays a network of interconnecting cisternae and ribbon-like structures. The well-developed Golgi complexes exhibit numerous dilatations of their cisternae, which contain electron-dense material. The nerve endings are not altered. Twenty days after treatment, the above-described changes have not decreased in magnitude. The present findings suggest that pimozide stimulates the mechanism of synthesis and release in some MSH-cells, most probably the elements underlying an inhibitory dopaminergic control.Supported by CONICET and CIUNC of ArgentinaMember of the Research Career of CONICET, ArgentinaFellow of CONICET, Argentina     

The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary The choroid plexus consists of the choroidal epithelium, a derivative of the neural tube, and the choroidal stroma, which originates from the embryonic head mesenchyme. This study deals with epithelio-mesenchymal interactions of these two components leading to the formation of the organ. Grafting experiments of the prospective components have been performed using the quail-chicken marker technique. Prospective choroidal epithelium of quail embryos, forced to interact with mesenchyme of the body wall of chicken embryos, gives rise to a choroid plexus showing normal morphogenesis and differentiation. The choroidal epithelium induces the differentiation of organtypical fenestrated capillaries, which are highly permeable to intravenously injected horseradish peroxidase. The choroidal epithelium of the grafts constitutes a blood-cerebrospinal fluid barrier. On top of the choroidal epithelium, there are epiplexus cells displaying a typical ultrastructure. The experimental results show that these cells do not originate from the transplanted neural epithelium. Prospective choroidal stroma of chicken embryos does not exert a choroid plexus-inducing influence upon a quail embryo's neural epithelium isolated from parts of the brain that normally do not develop a choroid plexus. The experiments show that the choroidal epithelial cells are determined at least three days before the first organ anlage is detectable.This work was supported by the Deutsche Forschungsgemeinschaft (grant Ch 44/7-1)     

Effects of pimozide on the ultrastructure of the pars distalis in the rat

Summary The effects of pimozide, a dopamine receptor-blocking agent, were studied in the pars distalis of the rat. The animals received 100g/100 g pimozide daily for 5, 10, 15, and 20 days. Pimozide induces striking ultrastructural changes after 5 days of treatment. The number of luteotroph (LTH) cells is significantly increased; they display characteristics of stimulation. The extrusion of granules into the intercellular space via exocytosis is frequently observed. The intercellular spaces are highly dilated, forming a lacunar system filled with an amorphous material, erythrocytes and involuted LTH cells. Transitional stages in the process of involution are observed in LTH cells. Luteotroph cells also form a syncytium. Twenty days after treatment the abovedescribed changes decrease in magnitude. The present findings suggest that pimozide stimulates the mechanism of synthesis and release in the luteotroph cells, an effect that is less evident with longer treatment.Supported by CONICET and CIUNC of ArgentinaMember of the Research Career of CONICET, ArgentinaFellow of CONICET, Argentina     

Influence of a short light pulse at night on the ultrastructure of the rat pinealocyte: a quantitative study

Summary Although it is generally known that light strongly influences N-acetyltransferase activity and melatonin production in the pineal gland, little information is available concerning morphological changes following light exposure. As exposure of rats to a short light pulse at night rapidly depresses melatonin synthesis, we decided to determine whether this experimental condition produces rapid changes in the pinealocyte organelles. A 30-min light pulse at night (six hours after lights out) provoked rapid changes in the relative volumes of some pinealocyte organelles. The volume fractions of mitochondria, Golgi apparatus and lipid droplets, and the numbers of dense-core vesicles and synaptic ribbons decreased, whereas the volume fraction of lysosomes increased. There were no differences in the volumes of granular endoplasmic reticulum and vacuoles containing flocculent material in those animals exposed to light compared with control animals. These results indicate that a short light pulse at night causes ultrastructural changes that can be interpreted as morphological features of diminished activity of pinealocytes.     

Effects of prolonged sodium restriction on the morphology and function of rat adrenocortical autotransplants

Summary Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions. The hypothesis is advanced that the lack of splanchnic nerve supply and chromaffin medullary tissue in regenerated nodules may be the cause of such an impairment.     

Postnatal changes in the ultrastructure of the rat olfactory epithelium: the supranuclear region of the supporting cells

Summary The present electron-microscopical study reports ultrastructural changes occurring in the supranuclear region of the supporting cells of the rat olfactory epithelium during the first 16 days of postnatal life. These changes are concerned with the enclosure of receptor cell dendrites and an increase in the amount of smooth endoplasmic reticulum, which has a specific distribution in the supporting cell. An increase in microvillous projections at the free cell surface is also observed. Moreover, this report demonstrates that the cytoarchitecture of the apical portion of the olfactory epithelium at the 16th day of postnatal life is similar, with respect to the relationship between supporting cells and receptor cell dendrites, to that of adult animals.