Scanning electron microscopy of stromal cells of human placental villi throughout pregnancy

Summary Morphological changes in fixed stromal cells and Hofbauer cells were studied throughout pregnancy in different types of placental chorionic villi by scanning electron microscopy. In the mesenchymal villus the fixed stromal cells were characterized by thin cytoplasmic processes. Hofbauer cells exhibited blebs on their surface. Large sail-like processes with a crescent profile which surrounded well developed stromal channels and a small cell body typified the small reticulum cells of the immature intermediate villus. The Hofbauer cells here displayed blebs, microplicae and large lamellipodia. Short cytoplasmic expansions and a large cell body characterized the fibroblasts present inside the stem villus. Hofbauer cells were rare, having blebs or a few short lamellipodia. The mature intermediate villus contained small and large reticulum cells. The latter had a much larger cell body than the small ones and displayed a few short cytoplasmic processes partly delimiting narrow incomplete stromal channels. Occasional Hofbauer cells with small microplicae and/or blebs were present. The small reticulum cells and fibroblasts present in the terminal villus showed similar morphological features as above. However, the former exhibited less developed cytoplasmic extensions and therefore no stromal channels were observed. In the terminal villus, the morphology of the rare Hofbauer cells was similar to that found in the mature intermediate villus.     

A three-dimensional study of the normal human placental villous core

Summary In order to study the three-dimensional ultrastructure of the Hofbauer cells, human placentae from the 6th to the 21 st week of gestation and also from the end of pregnancy were cryofractured and observed by scanning electron microscopy. Hofbauer cells were found in the villous core at all the gestation stages examined. Their surface morphology was characterized by lamellipodia, funnel-like structures, blebs and microplicae. This pleomorphic aspect was probably related to functional or environmental conditions. In addition, thin cytoplasmic processes connected the Hofbauer cells with each other and with the components of the villous stroma. Fractured Hofbauer cells revealed large vacuoles in the cytoplasm; the vacuoles were smaller in size both at the beginning and at the end of pregnancy. This study further attests to the macrophagic nature of these cells.     

Ultrastructural studies on white adipocyte differentiation in the mouse mammary gland following estrogen and relaxin

White adipocyte differentiation was studied ultrastructurally in the mouse mammary gland following stimulation with 17beta-estradiol and relaxin. These hormones have been previously demonstrated by us to induce hyperplasia and hypertrophy in the adipose cells of the mouse mammary gland. Following hormone treatment new fat cells are formed around the growing ducts. In these sites there is a close relationship between blood vessel growth and adipocyte development, and stromal areas featuring embryonic fat organs can frequently be found. In the sites of adipocyte differentiation the most numerous cell types are undifferentiated mesenchymal cells and preadipocytes, while fibroblasts and macrophages are much less common. No endothelial cells or pericytes were found detaching from the blood capillary walls. There were no fibroblasts or macrophages containing intracellular lipid deposits. Actively degranulating mast cells were frequent. The above findings strongly suggest the direct origin of adipocytes from perivascular mesenchymal cells, without the intermediate stages of well-differentiated fibroblasts, macrophages, endothelial cells or pericytes. The earliest morphologically recognizable stage in adipocyte differentiation is a 'pale preadipocyte', characterized by its irregular shape due to cytoplasmic processes, clear cytoplasmic matrix, well-developed organelles (especially ribosomes and Golgi apparatus), few and small lipid droplets, numerous pinocytotic vesicles and a very thin basal lamina. The next stage in adipocyte differentiation is a 'dark preadipocyte' showing a denser cytoplasmic matrix, reduced organelles and more abundant lipid accumulations. The pinocytotic vesicles are very numerous and the enveloping basal lamina is still thin. The subsequent maturation stages are the well-known globular multivacuolated adipocyte and finally the mature univacuolated, signet-ring, white adipocyte.     

Macrophages in Pacinian corpuscles

The presence of macrophages in the outer bulb region of mouse, monkey and human Pacinian corpuscles was demonstrated by light and electron microscopy. In the normal, nontreated, Pacinian corpuscles, a few particular cells were located in the spaces between lamellae of the outer bulb. These cells contained numerous vesicles and vacuoles, and various cytoplasmic processes. When horseradish peroxidase (HRP) was injected locally or systemically, many HRP-positive cells, which were considered to be similar to the particular cells described above, were found in the outer bulb region of the corpuscles. Electron microscopy revealed that these cells contained HRP in vesicles and vacuoles, suggesting that they were macrophages vigorously taking up exogenous HRP. Macrophages in the Pacinian corpuscles are considered to work as scavengers to keep the inner environment of the corpuscles clear and constant with regard to its macromolecular content.     

Seasonal changes in the ultrastructure of the vascular cambium of Robinia pseudoacacia

 The ultrastructure of the vascular cambium of Robinia pseudoacacia L. was examined in trunk tissues collected over a 2 ¹/₂ year period. During dormancy, fusiform cells are densely cytoplasmic with many small vacuoles and centrally located nuclei. Mitochondria are round to oval in sectional view. The plastids are variable in shape, have few internal membranes, and generally lack starch grains. The plasmalemma is smooth in outline. Proteinaceous material occurs in the vacuoles and many lipid droplets are scattered throughout the ground substance. Smooth tubular ER, often highly dilated, predominates, but short segments of rough ER are also present. Abundant free ribosomes are evenly distributed throughout the ground substance and the dictyosomes are inactive. Microtubules are parietal and have various orientations. During reactivation, the plasmalemma becomes irregular in outline and begins to form invaginations. Concurrently, the proteinaceous material disappears, the vacuoles begin to fuse, polysomes appear, and the dictyosomes begin to produce vesicles. During the period of cambial activity, fusiform cells are highly vacuolate, and the nuclei are centrally located. The mitochondria are round, oval, or elongate. Now the plastids contain phytoferritin, starch grains, or both. Many large invaginations of the plasmalemma intrude into the vacuole, pushing the tonoplast inward and pinching off into the vacuole, which lacks proteinaceous material. Lipid droplets are scarce. Most ER is rough, and ribosomes are generally aggregated as polysomes. Dictyosomes are actively producing vesicles. During the transition to dormancy, the fusiform cells gradually assume the appearance typical of the dormant cambium.     

Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix

We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy- requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p- [dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.     

The leafy stems of Sphagnum (Bryophyta) contain highly differentiated polarized cells with axial arrays of endoplasmic microtubules

Contrary to the long-held belief that, internal to the cortical sterome, the central region of Sphagnum stems comprises unspecialized parenchyma, the present light- and electron-microscope study has revealed that these cells in fact have a highly specialized cytoplasmic organization. Their key features are: ( a ) the absence of large central vacuoles; ( b ) a spindle-shaped nucleus positioned internally; ( c ) a prominent axial system of endoplasmic microtubules associated with the nucleus, mitochondria, pleomorphic vacuoles, and membrane-bounded tubules and vesicles; ( d ) a distinct cytoplasmic polarization, with the cellular region near the capitulum being richer in organelles than the basal region; and ( e ) a high frequency of plasmodesmata in the cross walls with an enlarged median region containing no discernible desmotubule. Such a distinctive combination of cytological features has been hitherto only described for putative food-conducting cells in bryoid mosses. The results introduce a major new character common to Sphagnum and bryoid mosses and strongly suggest that this cytological organization underlines cellular specialization in symplasmic transport.     

AUTOPHAGIC VACUOLES PRODUCED IN VITRO : I. Studies on Cultured Macrophages Exposed to Chloroquine

Mouse macrophages exposed to 30 µg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1–4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.     

A freeze-fracture study of two types of collagen-phagocytosing cell in the post-partum rat endometrium

In the post-partum rat endometrium, ultrastructural distinction could be made between stromal cells (fibroblast-like cells) and macrophages, especially by the freeze-fracture technic. The stromal cells were characterized by a well-developed rough-surfaced endoplasmic reticulum (RER) and intercellular junctions, while the macrophages had many vacuoles and vesicles, but no intercellular contact with each other. The freeze-fracture image showed that the stromal cells had many low linear elevations and gap junctions on the cleaved plane of the cell membranes, while the macrophages had no linear elevations or intercellular junctions. The cell membranes of the stromal cells had more intramembranous particles (IMP) (P-face 697 +/- 63/micrometers 2, E-face 303 +/- 52/micrometers 2) than those of the macrophages (P-face 467 +/- 50/micrometers 2, E-face 217 +/- 35/micrometers 2). It was confirmed that these two types of cell phagocytosed collagen fibrils.     

Electron microscopic observations on the involution of the human corpus luteum of menstruation

Summary The involution of the granulosa lutein cell in the human corpus luteum is characterized by a dilatation of agranular endoplasmic reticulum vesicles and tubules. This process continues until the whole cell is filled with large vacuoles and the cytoplasm is reduced to thin strands between the vacuoles. The contents of the latter are of low electron density in contrast to the very electron dense lipid droplets in vascularization, bloom and early involution phase. Light microscopical evidence shows that the contents of the vacuoles must be lipid and the lower electron density might be explained by the relative decrease in phospholipids and increase in cholesterol and cholesterol esters during involution. Simultaneously processes of focal cytoplasmic degradation resulting in autophagic vacuoles occur in the cells. These lead in some cases to the formation of residual bodies which can be identified with lipofuscin granules. Finally, the degenerating cells disintegrate and part of the debris, including the lipofuscin granules are phagocytosed by macrophages, the so-called fluorocytes of Hamperl. During involution an amorphous substance with some protofilaments and collagen fibrils is deposited in the spaces between the shrinking lutein cells. This is the fibrohyalin material which will form the bulk of the corpus albicans.We are grateful for the assistance given by Dr. J. M. Moyes of the Women's Hospital, Crown Street, Sydney and the staff of King George V. Hospital at Sydney with the supply of the material.     

阔口尖毛虫形成包囊期间细胞超微结构的观察

阔口尖毛虫形成囊期间,细胞质内出现条带状或管产产的内质网和由不同大小的囊泡组成的包囊壁前体。并且,前体的产生与内质网有关;细胞质内发生自噬泡消化现象,这是细胞将原有结构和能量进行贮存,利用的一种重要形式;大核向细胞质突出形成阿米巴形结构,这与大核向细胞质排出部分核物质有关。     

Ultrastructure of the tubular nephron of Testudo graeca (Chelonia). A comparison between hibernating and non-hibernating animals

The tubular nephron of hibernating and non-hibernating specimens of Testudo graeca (Chelonia) was studied by means of conventional light and electron microscopy and histochemistry. The tubular nephron was composed of proximal, intermediate, distal and collecting tubules in both hibernating and non-hibernating animals. The cells of the proximal tubule showed long microvilli, cytoplasmic vacuoles, a developed endoplasmic reticulum and abundant mitochondria. Fat droplets were also observed. The intermediate segment was lined by ciliated and non-ciliated cells. The lining cells of the distal tubule presented few microvilli, abundant dense mitochondria and clear vesicles of mucous appearance in the terminal portion. Collecting ducts are composed of mucous and non-mucous cells. Mucous cells presented strong reaction to the histochemical techniques detecting sialo- and sulpho-mucins. During hibernation, a progressive vacuolar degeneration of the endoplasmic reticulum was observed in all the segments of tubular nephron, which may be caused by a massive intake of extracellular water into the cell.     

Phagocytic activity of the stellate cells in the anuran pars intermedia

Summary In an attempt to study further the stellate cell and its functions, the ultrastructure of this cell type in the neurointermediate lobe of the bullfrog, Rana catesbeiana, was examined in both organ and dissociated-cell culture. The cytoplasmic activity of stellate cells from neurointermediate lobes incubated 3 1/2 or 5 1/2 h was greater than that of those in vivo. Mitochondria and bundles of cytoplasmic filaments were numerous, in addition to prominent, well-developed Golgi complexes with associated vesicles. The most striking ultrastructural feature was the presence of phagocytic vacuoles that contain cellular debris. The stellate cells were seen to form cytoplasmic processes that phagocytosed this extracellular debris identifiable as belonging to the secretory cells of the pars intermedia. The stellate cells from the dissociated-cell preparations were also seen to contain debris within phagocytic vacuoles. In those neurointermediate lobes transplanted for 3 1/2 to 4 days into the anterior chamber of the eye, the stellate cells demonstrated similar phagocytic ability, but the phagocytic vacuoles contained material that seemed to be at a later stage of degradation. In all three of these conditions, the stellate cells were not seen to release this cellular debris nor were they seen to undergo cell division. These glial-like stellate cells of the pars intermedia acted as macrophages in all three of these experiments. There is now, therefore, a need to determine under what conditions, if any, these stellate cells function in vivo as macrophages.Supported by NSF Program for Small College Faculty Engaged in Research at Larger Institutions and Department of Energy — Associated Western Universities Faculty Participation Program. The authors thank Dr. W. Ferris and Dr. J. Berliner for the use of the electron microscopy facilities at the University of Arizona and Nuclear Medicine Laboratory, UCLA, respectively. Warm thanks are due to Ms. Ruth Cole for technical assistance     

Vacuolation: Origin and development of the lysosomal apparatus in root-tip cells

Summary The morphology of vacuolation has been investigated in root tip cells of corn using the freeze-etching technique. The genesis of vacuoles involves the following processes: a) Formation of small, endoplasmic-reticulum (ER)-derived vesicles (provacuoles); b) fusion of provacuoles resulting in the formation of small vacuoles, and followed by fusion and expansion of vacuoles; c) incorporation of large, dictyosome-derived vesicles into vacuoles by invagination of the tonoplast; d) invagination of the tonoplast resulting in the incorporation of cytoplasmic material into vacuoles. The morphological findings are correlated with biochemical data obtained from isolated vacuoles (lysosomes). Provacuoles (ER-derived vesicles) are shown to be primary lysosomes; their hydrolases arise from the ER. Vacuoles represent secondary lysosomes (digestive vacuoles) of the higher-plant cell. The metabolic role of lytic processes proceeding in the lysosomal apparatus is discussed.     

A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.     

Ultrastructural differentiation of stromal and vascular components in early macaque placental villi

In this study, closely staged placental villi from rhesus monkeys between 19 and 60 days of gestation were used to examine 1) the origin of endothelial cells and the mechanism of angiogenesis in villi; 2) the origin of placental macrophages (Hofbauer cells), and 3) the origin of the reticular cells that compartmentalize the stroma. The results did not support the concept that early stromal cells in the villi were derived by in situ delamination from cytotrophoblast. The extraembryonic mesodermal (mesenchymal) cells at the earliest of ages examined contained considerable granular ER. These cells organized into groups and formed primitive intercellular junctions, thus giving rise to the early angioblastic masses. The angioblastic masses were cellular, not syncytial; and lumen formation was not the result of intracellular vacuolization, but rather was the result of the acquisition of junctionally defined spaces. The earliest capillaries lacked intravascular blood cells and a basal lamina. Later, blood cells were evident in the lumina. At about 45-50 days of gestation, fetal capillaries began to indent the basal surface of the trophoblast. The basal lamina of the fetal capillaries still had not developed by 60 days of gestation. Between 35 and 40 days of gestation, significant morphological changes took place in the villous stroma. Evidence was obtained that the mesenchymal cells differentiated into the reticular cells that subdivided the stroma into fibril-rich and fibril-free compartments. At the same time, Hofbauer cells were observed for the first time; they occupied the fibril-free regions of the stroma. We did not observe any clear indications of intermediate stages of differentiation between other stromal cell types and Hofbauer cells. It is suggested that placental macrophages may have an origin independent of other stromal types; one possibility is that they are derived from blood monocytes as in other tissues. It is further suggested that the activities of the macrophages and reticular cells may be important in remodeling the extracellular matrix and may be related to the process of branching morphogenesis in the villi.     

Fine structure of the parotid gland in the crest-tailed marsupial-rat (Dasyuroides byrnei).

The parotid gland of Dasyuroides byrnei was examined by light microscopy, and transmission and scanning electron microscopy. The acini were composed predominantly of seromucous cells with a few mucous cells. The seromucous cells were light or dark cells containing acidophilic spherical granules of moderate to high electron density and had well-developed cytoplasmic organelles-ordinary mitochondria and large mitochondria with tubular cristae, RER with vesicular or tubular elements, and Golgi apparatus with lamellae, vesicles and vacuoles. The mucous cells had basophilic amorphous granules of low electron density, like those of ordinary mucous cells. The intercalated ducts were composed of simple cuboidal light cells having a few electron-dense granules. The striated ducts consisted of tall columnar light cells containing numerous vesicles and mitochondria with tubular cristae, the same as found in acinar seromucous cells.     

Immunohistochemical, autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein.

Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using 35S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for 35S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for 35S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

FERRITIN PARTICLES IN MACROPHAGES AND IN ASSOCIATED MAST CELLS

In a variety of tissues (lymph node and glandular stroma), mast cells have been found in close and often intimate association with macrophages containing numerous ferritin-like particles in their cytoplasm and within cytoplasmic vacuoles (siderosomes). Phagocytic vacuoles in a given macrophage differed markedly. Some contained abundant Prussian blue-reactive material and others contained periodic acid-Schiff reactive substance at the light microscope level, and ultrastructurally some were filled with ferritin particles and others were not. Ferritin-like particles have also been observed occasionally in the mast cells associated with macrophages and even within the matrix of some of the granules in these mast cells.     

Immunohistochemical,autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein

Summary Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using ³⁵S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for ³⁵S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for ³⁵S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.