Changes in the ultrastructure and fibrogenic activity of rat dermal fibroblasts as affected by various doses of hydrocortisone

By means of electron microscopical morphometry and populational analysis it has been stated that hydrocortisone acetate, injected subcutaneously twice a week in dose 5 mg/kg of the body mass, produces decrease in number of intensively collagen-producing fibroblasts and increase in the part of fibroblasts and destroying cells in population of fibroblasts only in young (2-week-old) animals. The dose 10 mg/kg produces similar changes both in young and in mature (2-month-old) animals. In the young animals given hydrocortisone in dose 5 mg/kg of the body mass, the mean summational area and extension of the granular endoplasmic reticulum membranes in the fibroblast section diminishes, the summational area of the mitochondrial section decreases in the section of one cell, and the derm with less thickness, in comparison with the derm of intact animals, is formed, while in mature animals, given hydrocortisone in a small dose, all the parameters mentioned do not significantly differ from the control. Hydrocortisone in dose 20 mg/kg decreases all quantitative parameters of dermal fibroblasts both in young and mature animals. The data of the correlative analysis give evidence on the presence of a strong positive connection between inhibition of the synthetic apparatus of fibroblast development under hydrocortisone effect and decrease of the derm thickness, forming during the postnatal period of ontogenesis.     

Dynamics of the ultrastructure of fibroblasts during pre- and postnatal development of the dermis in the rat according to electronic morphometry

Fibroblasts have been investigated in 18-day-old embryos, in newborns, in 2-week-old and in 2-month-old rats of postnatal development. Comparison of fibroblasts population is specific for each stage. Average section area of the fibroblasts nucleus is progressively decreasing during ontogenesis. Average section area of cytoplasm reaches its maximum by the end of the second week of the life and then decreases sharply, and therefore the nucleus/cytoplasm ratio in the fibroblasts is minimal at the age of two weeks. The Golgi complex section area decreases significantly in mature animals as compared to the young ones. Summational extent of the granular endoplasmic reticulum membranes in the fibroblast section in the 2-week-old animals is 2.5 times as great as the corresponding parameters in the fetuses, newborns and mature animals. This parameter is greatly informative for determining the fibroblast type in accordance with its synthetic activity.     

Changes in the ultrastructure of the chloride cells of sturgeon during adaptation to a hypertonic medium]

Alterations in the ultrastructure of chloride cells of young sturgeons Acipenser guldenstadti Brandt in the process of adaptation to hypertonic medium (S=105%) were under study. In the process of this adaptation thread-shaped long mitochondria became shorter, their random position was substituted by orientation in parallel to the axis of the cell. The amount of mitochrondria increased. A great number of vacuoles appeared in the cytoplasm. The character of the endoplasmic reticulum changed. The obtained data on the ultrastructure of chloride cells of the sturgeon confirm and supplement earlier light microscopy investigations of chloride cells of the sturgeon. The changes of the ultrastructure of chloride cells are interpreted as functional and adaptational ones.     

The secretion of collagen by insects: uptake of [3H]proline by collagen-synthesizing cells in Locusta migratoria and Galleria mellonella.

The uptake of [H3]proline by collagen-secreting cells of the locust, Locusta migratoria, and wax-moth, Galleria mellonella, has been investigated by electron autoradiography. The locust cells are around the ejaculatory duct and they secrete collagen in the young adult male, while the wax-moth cells are those which produce the dorsal mass of connective tissue on the abdominal nerve cord during the late pupal stage. The cells were exposed to [H3]proline either by injection of the [3H]proline into the insect, or as a pulse while the tissue was maintained in a culture medium. The tissues were fixed at differeing experimental times after exposure to the [3H]proline. The resulting electron autoradiographs were subjected to quantitative analysis, and the silver grain distribution was determined as the relative number of grains per unit area over a series of tissue compartments. When the results of this analysis for the matrix, rough endoplasmic reticulum and Golgi complexes of the two tissues were plotted against experimental time, it was seen that the relative number of grains per unit area over the rough endoplasmic reticulum decreases while that over the matrix increases; statistical analysis has shown that these changes are significant. For the Golgi complexes, however, the theoretical variances are much greater, due to the small relative area occupied by this organelle. There is little evidence for anything other than random sampling fluctuations in the relative numbers of grains per unit area, and hence it is unlikely that the time course of the label over the Golgi complexes follows that over the rough endoplasmic reticulum. The conclusions drawn from these experiments are firstly that a large portion of the labelled protein passes straight from the rough endoplasmic reticulum to the matrix, but that a smaller portion of the labelled material might pass from the rough endoplasmic reticulum to the Golgi complexes and thence to the matrix. It is assumed that collagen comprises most of the protein which passes straight from the rough endoplasmic reticulum to the matrix, and while there is no evidence to exclude collagen from the material passing through the Golgi complexes, it is probable that other proteins and glycosaminoglycans are also present in this labelled material. These conclusions about the intracellular pathway for collagen secretion are similar to those derived from recent studies of some vertebrate fibroblasts. There is, however, conflicting opinion about the intracellular pathway of collagen and it is pointed out that there is diversity in collagen-synthesizing cells, which may account for the differences in the intracellular pathways for collagen secretion which have been proposed.     

Adrenocorticotropic (ACTH) cells of rats after head irradiation.

The heads of intact rats and of rats 15 days after adrenalectomy were irradiated with 1200 R. Some of the adrenalectomized animals with irradiated heads were treated with 5 mg of deoxycorticosterone acetate. In adrenocorticotropic cells of adrenalectomized rats 15 and 30 days after irradiation, the decreased amount of the granular endoplasmic reticulum and the Golgi regression imply a considerably decreased protein synthesis. The finding of an increased number of granules and of granules larger than normal suggests that they are the result of slowed down transport and secretion of the products of these cells' activities. After head irradiation an increased number of degenerative cells was evident in the pituitary of both intact and adrenalectomized rats. The degree of the degenerative changes and the number of degenerative cells was higher 30 than 15 days after irradiation and both were decreased if the adrenalectomized rats were treated with deoxycorticosterone acetate.     

Methylcholanthrene-induced Sarcoma and Normal Muscle Fibroblasts of the Mouse — a Comparison of their Ultrastructure

Electron microscope observations of cells from methylcholanthrene induced tumours of the mouse and mouse muscle fibroblasts, grown in vitro , show five main differences in ultrastructure. The surface of the normal fibroblast is smooth when compared with that of the tumour cell; the leading lamella of the normal fibroblast has fewer ruffles (lamellipodia) and fewer cell-substrate plaques than the tumour cell; the tumour cell does not have a layer of cortical filaments like the normal cell but has well marked filamentous tracts in the main body of the cytoplasm and the endoplasmic reticulum of the normal cell is far less abundant and wellorganized than that of the malignant cell. The fact that all these differences are possibly related to the cell surface may be of significance when considering the changes brought about during carcinogenesis.     

The morphology and ultrastructure of antennal lobe cells from pupal honeybees (Apis mellifera) growing in culture

A culture technique for the in vitro growth and differentiation of antennal lobe cells from the honeybee, Apis mellifera, is described and the ultrastructure of the growing cells is analysed. Two types of cell are present in the cultures and from their morphology and ultrastructure they can be identified as glial cells and neurones. The neurones have a granular cytoplasm, abundant endoplasmic reticulum and a small, densely stained nucleus. They produce long processes with varicosities that contain dense-core and clear vesicles. In contrast the glial cells have clear cytoplasm, little endoplasmic reticulum and a distinct cytoskeletal organisation. These cells produce short, flat processes that spread over the surface of the culture dish. Although a number of cell contacts have been identified in the cultures no synapses have yet been seen. These cultures provide a good in vitro model for an analysis of the interactions between cells derived from the antennal lobe of the honey bee.     

Ultrastructure of tissue basophils and capillaries during different periods of pre- and postnatal development of the dermis in the rat

Dynamics of ultrastructure of mast cells (MC) and that of the capillaries of the derma have been studied by means of electron morphometry methods in rats during pre- and postnatal ontogenesis. The dynamics of the Golgi complex volume, that of the mitochondria and specific granules have been calculated in the total volume of the MC membrane organelles. Certain new data have been obtained on the process of the MC specific granules formation in the derma from progranules up to the stage of a mature granule. The process mentioned is most intensive during the first weeks of the postnatal ontogenesis. At the age of two weeks, signs of an active exocytosis of the granules are noted. Judging by certain morphological signs, development of transendothelial transport of substances in capillaries takes place in parallel with formation of the specific granules and corresponds to the beginning of exocytosis of substances in the MC granules. The correlative analysis proves that formation of the MC specific granules is connected with the number of microvesicles in cytoplasm of endotheliocytes.     

Ultrastructural studies on the effects of hypergravity environment on the parathyroid glands in golden hamsters of different ages

The ultrastructure of the parathyroid glands of infantile, young, adult and senile golden hamsters subjected to a hypergravity environment was studied. In the parathyroid glands of 5-, 10- and 20-day-old, and 1- and 3-month-old golden hamsters exposed to a hypergravity environment, the Golgi complexes were significantly increased, and in 5-, 10- and 20-day-old, and 1- and 8-month-old animals exposed to a hypergravity environment, the cisternae of the granular endoplasmic reticulum appeared to be increased as compared to those of each control group. In addition, in centrifuged animals numerous prosecretory granules were observed in the Golgi areas, and many secretory granules were located in the peripheral cytoplasm. The ultrastructure of the parathyroid glands of 14-month-old centrifuged animals resembled that of 14-month-old control animals. These results suggest that the secretory activity of the parathyroid gland may be stimulated in infantile, young and adult golden hamsters subjected to a hypergravity environment and may not be stimulated in senile animals subjected to a hypergravity environment.     

THE DIFFERENTIATION OF WHITE ADIPOSE CELLS : An Electron Microscope Study

Differentiating white adipose tissue from presumptive and developing fat pads of newborn and young rats was fixed in buffered osmium tetroxide, embedded in Vestopal W, and examined in an electron microscope. Pre-adipose cells were found to be fibroblasts characterized by their spindle shape, long tenuous cytoplasmic extensions, and profuse endoplasmic reticulum. The developmental stages traced from fibroblast to mature adipose cell show a gradual change in cell shape, an accumulation of cytoplasm and non-membrane-bounded lipid, a decrease in the endoplasmic reticulum, and a change in shape of mitochondria. Transitory glycogen appears at mid-differentiation. Numerous smooth-membraned vesicles occur in the cytoplasm throughout differentiation. Pinocytosis is constantly evident. Cells of the multilocular stage are shown to differ from brown fat cells, particularly with respect to cytoplasmic membrane systems and mitochondria. No transport of particulate lipid from the lumen of the capillary to, or within, the adipose cell was detected, nor could any cell organelle be demonstrated to be visibly related to lipid synthesis and/or deposition.     

Prolactin-induced stimulation of H(3)-proline incorporation into the basement lamella of tadpole skin: light and electron microscope study

Radioautographic studies revealed that prolactin markedly stimulates the incorporation of H(3)-proline into the basement lamella of the tail fin of frog tadpole. Radioactivity in fibroblasts reached a peak at 3 hours. Thereafter, the number of silver grains in fibroblasts was reduced with concomitant increase in the basement lamella. Fibroblasts of the prolactin animals also showed extensively developed rough endoplasmic reticulum and Golgi elements. Epidermis of control animals was labelled as in the prolactin animals, although the basement lamella was sparsely labelled. Fibroblasts, not the epidermis, seem to secrete collagen into the basement lamella.     

Morphological modifications of knee articular cartilage in bullfrogs (Lithobates catesbeianus) (Anura: Ranidae) during postmetamorphic maturation

The organization of knee articular cartilage of the bullfrog (Lithobates catesbeianus) differs in relation to morphofunctional adaptation in many aspects from similar structures in mammals. Thus, we investigated the structural organization and distribution of the extracellular matrix components in three articular cartilage regions in the distal epiphysis of the femur and proximal epiphysis of the tibia in male bullfrogs at 7, 540 and 1,080 days after metamorphosis. Cartilage thickness and cell density decreased in all regions with age. The basophilia differed among cartilage sites during aging. Calcium deposits were detected in growth cartilage of the femur and tibia in older animals. Immunohistochemical staining for chondroitin-6-sulfate was positive in the pericellular and territorial matrix in all samples. Positive immunostaining for type I collagen was observed in the superficial layer at all ages and in ossification centers of older animals. Reactivity to type II collagen was intense and was found throughout the stroma at all ages. Ultrastructural analysis of the epiphyseal region, in young animals, showed that the cytoplasm of chondrocytes was rich in rough endoplasmic reticulum, Golgi complex and mitochondria. In old animals, were observed a reduction in the size and number of mitochondria, disintegration of rough endoplasmic reticulum, and vacuolization of the Golgi complex. The bullfrog articular cartilage presented structural and organizational changes during aging which may contribute to the functional cartilage deterioration in old animals.     

Ultrastructure of epidermal glands in neotenic reproductives of the termite Prorhinotermes simplex (Isoptera: Rhinotermitidae)

The ultrastructure of epidermal glands in neotenic reproductives of Prorhinotermes simplex is described and their development is compared among young and old neotenics of both sexes. Secretory cells forming the epidermal gland are attached to the cuticle all over the body. The glands are formed by class 1 and class 3 secretory cells and corresponding canal cells with secretory function. Class 1 cells are sandglass-like and class 3 secretory units are located among them. Class 1 cells contain predominantly tubular endoplasmic reticulum, the major part represents the smooth and the minor the rough form. Numerous electron dense granules occur in the cytoplasm, they are always disintegrated prior to be released. Class 3 secretory cells contain a large amount of vacuoles, which are always lucent in males while newly produced vacuoles are dense in females. Dense vacuoles are frequently transformed into lucent ones before being released. Canal cells are locally equipped with microvilli. The conducting canal is surrounded by an electron dense secretion of regular inner structure. The cytoplasm of the canal cell contains numerous mitochondria, rough endoplasmic reticulum and a large proportion of microtubules. The young neotenic reproductives differ from the old ones by a lower amount of secretory products. Epidermal glands probably produce substances inhibiting the occurrence of superfluous reproductives.     

Intracellular Transport and Elimination of Resin from Epithelial Duct-cells of Pinus halepensis

The ultrastructure of the resin secreting cells of root ductsof young Pinus halepensis seedlings was studied. It is suggestedthat the endoplasmic reticulum (ER) in addition to taking partin resin synthesis also plays a role in transporting the resinfrom the plastids, mitochondria and nuclear envelope to theplasmalemma. By fusing with the plasmalemma the ER releasesthe resin to the outside of the protoplast. The resin producedin the ground cytoplasm and by the Golgi apparatus seems tobe eliminated by plasmalemma invaginations. Pinus halepensis, resin secretion, root ducts, endoplasmic reticulum     

Secretion of collagen by insects: Autoradiographic study of l-proline 3H-5 incorporation by the firebrat Thermobia domestica

The biosynthesis of collagenous endoskeletal endosterna by fibroblasts was studied by electron microscope autoradiography after tritium-labelled proline administration in the firebrat Thermobia domestica at various time intervals. Autoradiographs were quantitatively analyzed and the relative concentration of label was determined for various cellular compartments. The labelling in the rough endoplasmic reticulum, ground cytoplasm, Golgi apparatus and endosterna was compared during the secretion of radioactive products. The label, initially located in the rough endoplasmic reticulum was found in the Golgi apparatus before it accumulated in the endosternum. The involvement of the rough endoplasmic reticulum and Golgi apparatus in the transport of secreted collagen is discussed, comparing our results with current knowledge of collagenous secretion.     

Aortic smooth muscle cells in collagen lattice culture: effects on ultrastructure, proliferation and collagen synthesis

Adult pig smooth muscle cells (SMC) were isolated from the aortic media by collagenase digestion, subcultured as monolayer, and then re-integrated into a three-dimensional network of type I collagen. The contractile state characteristic for resident arterial wall SMC changed to the synthetic, fibroblast-like state. The cells reorganized the randomly orientated collagen fibrils causing the lattice to shrink. The influence of the extracellular matrix on the ultrastructure, the proliferation, and the collagen synthesis of these SMC embedded in the collagen lattice was investigated and compared to cells cultured in monolayer. The amount of total protein and collagens synthesized by SMC embedded in lattices was lowered as compared to monolayer cultures. Whereas total protein synthesis decreased continuously during the culture period, the proportion of collagen synthesis remained at a constant level. Although cells proliferated in lattices, proliferation was clearly slowed down as compared to monolayer cultures. The ultrastructure of entrapped synthetic state SMC was comparable to that of monolayer-cultured cells. Their cytoplasm was largely filled by elements of the endoplasmic reticulum, Golgi complexes and abundant mitochondria. With prolonged culture time, electron-dense granules as well as bodies containing whorled membranes could be found in the cytoplasm. These results indicate that synthetic state SMC can exhibit differential biosynthetic activity dependent on the actual matrix environment; cells seem to be able to sense the macromolecular composition of the extracellular matrix and to modify their production of matrix components accordingly.     

An ultrastructural and histochemical study of autoimmune aspermatogenesis in the rat testis

Summary Immune aspermatogenesis was induced in young rats by the method of Freundet al. (1954) and testes were studied by electron microscopic and histochemical methods. At time of sacrifice the testes of several animals were markedly atrophic as demonstrated by reduction in weight. Sections of seminiferous tubules exhibited primarily profiles of Sertoli cells but germinal elements were sparse or absent. The ultrastructure of Sertoli cells appeared to be normal except for the presence of areas of dilated smooth endoplasmic reticulum and fragments of phagocytized germ cells in the cytoplasm.Light microscopic sections showed an apparent hyperplasia of intertubular tissue. Electron micrographs revealed a moderate to extreme vesiculation of the smooth endoplasmic reticulum in many interstitial cells. Macrophages and lymphocytes were often observed in contact with these cells.There was increased localization of non-specific esterase and acid phosphatase associated with lipid bodies of the tubules and in intertubular areas.This work was supported in part by USPHS Grant FR 5391.     

Cellular differences in ring glands of flesh fly pupae as a consequence of diapause programming

The ultrastructure of the ring gland (corpus cardiacum (CC), prothoracic gland (PG) and corpus allatum (CA)) was examined in diapausing and nondiapausing flesh fly pupae. The diapause developmental state, which is environmentally regulated and coordinated by the brain-ring gland complex, is associated with differences in the ultrastructure of PG and CA cells but not in the CC. During diapause the PG and CA cells have extensive arrays of rough endoplasmic reticulum and spherical mitochondria. The PG cells also contain lipid droplets surrounded by an electron dense amorphous coat not seen in PG cells from nondiapausing pupae. In nondiapausing pupae, the PG and CA cells contain large amounts of ribosomes throughout the cytoplasm but very little rough endoplasmic reticulum and elongated mitochondria. The fact that ring glands from diapausing pupae readily incorporate (35)S-methioninc indicates that the gland is actively synthesizing proteins, thus the contrasts in ring gland ultrastructure are not due to cellular quiescence during diapause but reflect fundamental cellular and physiological differences between the diapause and nondiapause developmental program.     

Role of glucocorticoids and progesterone in the development of rough endoplasmic reticulum involved in casein biosynthesis.

Hydrocortisone acetate injected into pseudopregnant rabbits induced casein synthesis and a parallel accumulation of casein mRNA. These effects were not accompanied by any enrichment of total RNA in the mammary cell. Hydrocortisone acetate did not favour the attachment of polysomes to endoplasmic reticulum. Casein mRNA concentration was enhanced in free and membrane-bound polysomes. After long treatments, the concentration of casein mRNA reached a plateau in membrane bound polysomes whereas it continued to be accumulated in free polysomes, suggesting that a substantial part of casein synthesis is then carried out by free polysomes. Progesterone injected with high doses of prolactin was unable to prevent the stimulatory action of prolactin on the synthesis of casein, the accumulation of casein mRNA and mammary gland growth, as judged by DNA content. By contrast, the increase in the total RNA content of mammary gland was still significantly reduced by progesterone. In addition, progesterone inhibited almost completely the formation of membrane-bound polysomes and the anchorage of casein mRNA to endoplasmic reticulum. From these data, it was concluded that the formation of the endoplasmic reticulum is not a prerequisite for the initiation of casein synthesis. Glucocorticoids do not play a major role in the formation of the endoplasmic reticulum and the Golai apparatus and in the binding of casein synthesizing polysomes to membranes. Progesteronne is capable of inhibiting preferentially and gradually the stimulation of cellular functions requiring the most potent prolactin stimulation.     

Connective tissue cells, cell proliferation and synthesis of extracellular matrix-a review.

The ubiquitous connective tissues contain a wide range of cells including fibroblasts, osteoblasts and chondroblasts. Recently it has been demonstrated that another principal cell of the connective tissue is the smooth muscle cell in several organ systems. These have been shown to be responsible for the synthesis of the connective tissue matrix components of the uterine myometrium and of the arterial system, including collagen, both elastic fibre proteins and glycosaminoglycan. Microtubule inhibitors such as colchicine and vinblastine, and iron chelators such as alpha,alpha -dipyridyl have been used to study their morphologic and chemical effects on collagen synthesis and secretion. Colchicine produces an increase in large Golgi-associated vacuoles, which sometimes contain material reminiscent of aggregates of collagen macromolecules. Vinblastine produces alterations in the endoplasmic reticulum cisternae similar to alterations seen in ascorbic acid deficiency, and alpha,alpha-dipyridyl increases the frequency of regions in cells, interpretable as potential sites of communication of rough endoplasmic reticulum cisternae with the cell surface. Ferritin conjugated anti-procallagen sera were used to localize procollagen in cells and demonstrated procollagen not only in the cisternae of rough endoplasmic reticulum but in all of the elements of the Golgi complex as well. The studies reported in this review have shown that in cell culture arterial smooth muscle will produce not only the microfibrillar protein of the elastic fibre but soluble and/or insoluble elastin as well. Recent studies on serum factors responsible for the proliferation of connective tissue cells have demonstrated that at least one of the principal factors responsible for fibroblast and/or smooth muscle cell proliferation in culture is derived from thrombocytes. Medium containing serum derived from cell-free plasma lacks most of this proliferative effect which can be reinstated when platelets are present during recalcification to form serum. This effect is due to the platelet release reaction as shown by combining supernatant factors derived from platelets exposed to purified thrombin to cell-free, plasma derived serum. Studies with macrophages have also suggested that phagocytic macrophages release factor(s) into a cell culture medium that may also participate in stimulating fibroblasts to proliferate in vitro. The means by which these factors stimulate fibroblast proliferation and connective tissue synthesis remains to be elucidated.