The precise subcellular localization of Dlg in the Drosophila larva body wall using improved pre-embedding immuno-EM.

Discs-large (Dlg) plays important roles in nerve tissue and epithelial tissue in Drosophila. However, the precise positioning of Dlg in the neuromuscular junction remains to be confirmed using an optimized labeling method. In this study, we improved the method of pre-embedding immunogold electron microscopy without the osmic tetroxide procedure, and we found that Lowicryl K4 M resin and low temperature helped to preserve the authenticity of the labeling signal with relatively good contrast. Dlg was strongly expressed in the entire subsynaptic reticulum (SSR) membrane of type Ib boutons, expressed in parts of the SSR membrane of type Is boutons, weakly expressed in axon terminals and axons, and not expressed in pre- or postsynaptic membranes of type Is boutons. In muscle cells and stratum corneum cells, Dlg was expressed both in the cytoplasm and in organelles with biomembranes. The precise location of Dlg in SSR membranes, rather than in postsynaptic membranes, shows that Dlg, with its multiple domains, acts as a remote or indirect regulator in postsynaptic signal transduction.

Histomorphometric and immunohistochemical study of the goat reticulum during prenatal development.

This study sought to describe the morphological changes taking place in the goat reticulum during prenatal development, using histomorphometric and immunohistochemical techniques. A total of 140 goat embryos and foetuses were used, from the first stages of prenatal life until birth. Differentiation of the reticulum as a separate compartment of the primitive gastric tube was observed at 35 days of prenatal life (23% gestation). By 38 days (25% gestation) the reticular wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Primary reticular crests were visible at 59 days (38% gestation) as evaginations of the epithelial stratum basale, marking the earliest histological differentiation of future reticular cells. Secondary reticular crests were observed at 87 days (61% gestation). Corneum papillae first became apparent on the lateral surface of primary reticular crests at 101 days (64% gestation). The muscularis mucosae was visible by 101 days (64% gestation) in primary reticular crests. Neuroendocrine cells were detected by synaptophysin at 64 days (43% gestation), while glial cell markers (glial fibrillary acidic protein and vimentin) were observed at 64 days (43% gestation) and 38 days (25% gestation), respectively. The peptidergic innervation markers such as neuropeptide Y and vasoactive intestinal polypeptide were detected at 75 days (50% gestation). In conclusion, prenatal development of the reticulum - like that of the rumen - appears to take place somewhat earlier in goats than in sheep or cattle, but at a similar rate to that reported in deer.

Structural, histochemical and immunocytochemical study of the forestomach mucosa in domestic ruminants.

The forestomach plays an important role in the digestion physiology of ruminants. The aim of this study is to clarify the morpho-functional role of the mucosa in each of the three compartments of the forestomach in three domestic ruminants species, viz cattle, buffalo and sheep, by means of structural, histochemical and immunocytochemical methods, including transmission electron microscopy, light microscopy and scanning electron microscopy. These methods were chosen to demonstrate the indirect evidence for the presence of nitric oxide (NO) employing NADPHd and nitric oxide synthase I (NOS I). The various cell layers of the forestomach epithelium are described and illustrated in detail. An intense NADPHd staining was observed in the granulosa, spinosa and basal layers of the epithelium, in particular in the cytoplasm over the nucleus. NOS I immunoreactivity was found in all specimens of the forestomach mucosa. The results of this study might reflect a possible role of NO in delaying the onset of cellular apoptosis in the forestomach mucosa of the domestic ruminants, by playing a role in the production of cell energy.

Morphological changes associated with the development of the rumino-reticulum in growing lambs fed different rations.

Morphological changes associated with rumino-reticular development was compared in two groups of SA Mutton Merino lambs (n = 12) fed different diets at 3-5 weeks, 17-19 weeks and 31-33 weeks of age. Diet groups were identified as MMH or MHH according to the sequence at which the full-milk replacement (M) or hay (H) diet were fed to the lambs over the three study phases (phases I, II and III) preceding each age period. Prominent differences in the morphology (size and volume) and function (pH, proteolytic activity and microbial population) of the rumino-reticulum were observed in indicator lambs (n = 2) killed at every age period and also between milk-fed and hay-fed lambs. The size of the rumino-reticulum was rudimentary at 3-5 weeks of age and remained underdeveloped in lambs at 17-19 weeks of age which had received a full-milk replacement diet during phase II. One lamb, slaughtered at 3 weeks of age, showed a large distended rumen with severe sloughing of the surface cells of the stratum corneum. The size of the rumino-reticulum increased in size (2 x) in lambs which were fed hay relative to the milk-fed lambs during phase II and reached adult proportions in all lambs at 31-33 weeks of age. Ultrastructural examination showed that rumen papillae were more developed in lambs fed hay during phase II when compared to those of milk-fed lambs. Rumen papillae were best developed in phase III lambs.

Three-dimensional development of bovine reticular cell (Cellula reticuli).

This paper describes the three-dimensional construction of the reticular cell, cellula reticuli, in bovine reticulum and its ontogenetic development. Reticular cells of fetal suckling calf and adult tissues were investigated both at the surface of intact mucosa and macerated samples using scanning electron microscopy. At the third month of gestation, the formation of the reticular cells on the mucosal surface started from the center of the cell and just above the reticular crest. The reticular crests were observed on the surface of the macerated sample at an earlier period of gestation (third month). In the eighth month of gestation, primary, secondary and tertiary reticular crests and papillae could be observed from the mucosal surface. Macerated samples showed that those structures were already formed completely under the epithelium by the first half of the seventh month of gestation. It is suggested that the development of the reticular papillae starts from the lower to the upper parts of the reticular cell.

Análisis morfológico comparado de la región fúndica entre un cánido (canis familiaris) y un camélido sudamericano (lama glama) / Comparative morphological analysis of the fundic region between a canidae (canis familiaris) and a southamerican camelidae (lama glama)

Los hábitos alimentarios que presentan los cánidos (monogástricos) respecto de los camélidos (poligástricos), son diferentes. Los primeros consumen dietas pobres en fibras, en tanto que los camélidos consumen una dieta rica en fibra que consiste en pastos cortos y duros (bofedal). Sin embargo son capaces de aprovechar eficientemente estos alimentos. Por ello, fueron analizados comparativamente las regiones fúndicas de ambas especies, donde encontramos diferencias morfológicas adaptativas. Para la realización del presente trabajo, se obtuvieron muestras de las regionnes fúndicas, provenientes de estómagos de perro (Canis familiaris) y de llama (Lama glama), adultos. estas muestras fueron procesadas de acuerdo a las técnicas histológicas corrientes e histoquímica de Picrosirius y analizadas comparativamente al microscopio óptico. Los resultados revelaron que existen diferencias importantes en las regiones fúndicas de ambas especies en estudio. Estas diferencias se encuentran especialmente a nivel de la mucosa, donde la profundidad de las glándulas fúndicas, número de células parietales (oxínticas) y la irrigación sanguínea son mucho mayores en la llama. La pared abdomasal presenta un refuerzo de tejido conjuntivo compacto de haces paralelos, entre la serosa y la muscular longitudinal externa. Estas adaptaciones redundarían en una mayor eficiencia digestiva por parte de los camélidos, dadas las características de su dieta habitual

Ultrastructure of the epithelium of the rumen, reticulum and omasum of grey, white and black Karakul lambs.

Mortalities due to digestive disturbances occur in homozygous grey and white lambs after they have reached weaning age. Milk-filled, distended rumens, due to malfunctioning of the oesophageal groove, are found 24 h after birth. Scanning electron microscopical studies revealed that milk caused sloughing of the luminal cells in the forestomachs of the affected lambs, while no sloughing of cells was apparent in control black lambs. The purpose of this study was to compare the ultrastructure of the forestomach mucosa of grey, white and black Karakul lambs; to determine whether the sloughing of luminal cells was evident in sections; and, if possible, to find a reason for the desquamation of the cells. Samples of the forestomach of grey, white and black Karakul lambs were prepared routinely for electron microscopy and studied with a Phillips electron microscope. In all the lambs the mucosa of the forestomach was a stratified squamous epithelium consisting of a stratum basale, stratum spinosum and stratum corneum. In the grey and white lambs the luminal cells of the stratum corneum were electron dense, non-nucleated and vacuolated. Sloughing of luminal cells was observed. In the black lambs no sloughing of cells was evident and the luminal cells were moderately electron-dense, nucleated elements. Desquamation of the luminal cells in the affected lambs revealed the underlying layer with its exposed desmosomal attachment sites. This explained the differences in the appearance of the luminal cells in the three groups of lambs as revealed by the scanning electron microscope.

Intracysternal crystalline cytoplasmic inclusions in a cardiac myxoma.

Electron microscopy of an atrial myxoma removed at surgery from a 56-year-old woman revealed unusual intracysternal inclusions in the cytoplasm of tumor cells. These structures appeared in the rough endoplasmic reticulum and consisted of multilaminar arrays of crystalline plates of regular periodicity, about 10 nm apart. Although crystalline and other intracysternal inclusions may occur in different pathologic disorders, their occurrence in an atrial myxoma is a hitherto unreported observation.

Participation of bone marrow stromal cells in hemopoietic recovery of rats irradiated and then parabiosed with a non-irradiated litter mate. II. Scanning and transmission electron microscopic observations.

A light microscopical study on the recovery process after lethal irradiation and parabiosis has been made. Electron microscopically, in the bone marrow of lethally irradiated rats, hemorrhage occurred due to detachment of sinus endothelial cells. Afterwards, reticulum cells with small intracytoplasmic lipid droplets appeared. On day 3, these cells were rapidly replaced by the reticulum cells with large lipid droplets, and resulted in fatty marrow within 7 days. Spindle-shaped fibroblastoid reticulum cells were also observed. In the bone marrow of lethally irradiated rats parabiosed with non-treated litter mates, hemopoiesis was initiated by adhesion of nucleated blood cells to intricate fine cytoplasmic pseudopods of fat-storage cells. On days 3 to 5, in parallel with progressive hemopoietic recovery, fibroblastoid and reticulum cells with large lipid droplets decreased whereas those with small droplets increased. On day 8, reticulum cells with lipid droplets were seldom seen, and hemopoietic distribution became the same as normal. These results suggested that bone marrow stromal cells, namely reticulum, fat-storage, and fibroblastoid cells share a common cellular origin, and also that they regain their structure and function when fat-storage cells were placed in contact with hemopoietic precursor cells.

Morphometry of mitochondria in ovine forestomach epithelia: high densities of mitochondria correlate with specialised metabolic activity.

The volume density of mitochondria and the surface density of mitochondrial inner membranes were investigated in the stratified squamous epithelia of the oesophagus and forestomachs of sheep, and in the oesophagus and nonglandular part of the ventricle of rats. The higher values found for both parameters in all forestomach regions are thought to be correlated with the special metabolic activity known to take place in these epithelia.

Development of agranular reticulum in Sertoli cells of the testis of the dogfish Squalus acanthias during spermatogenesis.

Biochemical analyses of Squalus testis indicates that key enzymes involved with androgen production increase progressively from immature regions containing spermatogonia to mature regions in the late spermatid stage of maturation (Canick et al., 1983). In an effort to identify cells possessing the cytological characteristics of steroid production and to determine the structural correlates of the observed functional changes, we have carried out an electron microscopic study of Squalus testis. This report demonstrates that Sertoli cells contain a well-developed agranular reticulum, mitochondria with tubulovesicular cristae, and numerous lipid droplets. Moreover, as germ cells mature, there is an increase in abundance of agranular reticulum in the adjacent Sertoli cells. By the time of spermatid elongation, this has reached dramatic proportions and fills the Sertoli cell as a mass of tubules. These results lead us to conclude that the Sertoli cell is responsible for secretion of the increasing amounts of androgen during the spermatogenetic cycle in Squalus.

Scanning electron microscope studies of lymphatic tissues with special reference to the structure of the reticulum.

Reticular cells of the lymph nodule in the rabbit appendix and the lymphatic pulp including the germinal center in the dog mesenteric lymph node were observed under the scanning electron microscope after removal of free cells. Three dimensional architecture and arrangement of the reticular cells varied by regions in the lymph nodule. The appendix lymph nodule encapsulated with the endothelium of lymphatic sinuses was subdivided into two regions: (1) a central region of sparsely arranged stellate cells with coarse processes and (2) a peripheral zone of densely arranged stellate cells with many delicate processes. In the lymph node, the germinal center contained a loosely formed network of delicate stellate cells. Peripheral reticular cells of the nodule were thread-like, extending concentrically around the germinal center or along the sinus wall. Many lateral bridges of the threads occurred segmentally. The reticular framework of the medullary cord formed spongy meshes by anastomoses of stellate cells. Cytoplasmic perforations were observed in the sinus wall of the medullary cord. Those may be for lymphocyte migration.

Morphological and histochemical characterization of the medullary cells in the bursal follicles of the chicken.

Using light and electron-microscopy and enzyme-histochemical methods the cells in the follicular medulla of the chicken bursa of Fabricius were characterized. The following cell types were identified: lymphocytes of both B and T cell series, lymphoblasts, plasma cells, dendritic reticulum cells and histiocytic reticulum cells--the latter being the only cells phagocytosing the tracer substance used (colloidal carbon). The medullary cells observed showed electron-microscope and histochemical characteristics typical of the germinal center cells in mammalian lymphoreticular tissues. Furthermore, a distinct bursal follicular epithelial cell was identified, and so was a medullary epithelial structure resembling Hassall's corpuscle of the thymus. These findings give further support to the idea that the avian bursa has functions of a peripheral lymphoid organ as well as containing T-derived lymphocytes.

Light- and electron microscopical observations on the caecilian spleen. A contribution to the evolution of lymphatic organs.

Red and white pulp were distinguished in the spleen of the caecilian species Ichthyophis paucisulcus and Afrocaecilia taitana. The red pulp was composed of endothelium-lined sinusoids and reticular connective tissue. Between the processes of the reticulum cells, accompanied by fine collagen fibrils, the following cell types were found: lymphocytes, macrophages (frequency containing fragments of erythrocytes), neutrophils, eosinophils, mast cells and/or basophils (metachromatic granules), thrombocytes, plasma cells, pigment cells as well as cells which presumably represent blast cells. Morphological evidence suggested the formation of thrombocytes in the red pulp. Besides sinusoids, ellipsoids and peculiar arteriolar vessels with a high endothelium and a loose layer of muscle cells were observed. Veins were concentrated in the splenic periphery. White pulp consisted of arterioles which were surrounded by a lymphocyte sheath. Follicles were not identified with certainty. Occasionally mitotic figures were associated with lymphocytes. On the basis of our findings, we suggest the following functions of the caecilian spleen: destruction of aged erythrocytes, formation of thrombo- and lymphocytes as well as of plasma cells and, to a marked lesser degree, of other blood cells.

Lymphoid organs of teleost fish. III. Splenic lymphoid tissue of Rutilus rutilus and Gobio gobio.

The ultrastructure of splenic lymphoid tissue of two teleost fish, Rutilus rutilus and Gobio gobio, constitutes discrete foci around the small arteries and "melano-macrophage" centres. It contains small and medium lymphocytes, lymphoblasts, macrophages and plasma cells. Plasmacytopoiesis and macrophage-lymphocyte clusters have been described and the significance of "melano-macrophage" centres and macrophage-lymphocyte clusters is discussed.

Haemopoietic microenvironments in vitro: ultrastructural aspects.

Haemopoietically active long-term bone marrow cultures from several species have been investigated ultrastructurally. Human, tree shrew and mouse cultures generally support granulopoiesis, although recently it has been possible to convert a granulopoietic mouse culture to extensive erythropoiesis. The haemopoietic products of the cultures include granulocytes (neutrophil and basophil), mast cells, monocytes, megakaryocytes and all stages of the erythrocytic series. Plasmacytes and occasional lymphocytes have been observed in small numbers in human cultures (possible indicating retention rather than formation). The stromal elements of the adherent layer of these cultures include endothelial cells, reticulum cells, fat cells and fibroblasts. The adherent layers are responsible for the inductive microenvironment within the cultures, and show features specific for the line of differentiation. In the granulocytic cultures there is close association between developing fat cells (reticulum cells) and granulocyte precursors. Endothelial cell monolayers cover large regions of these cultures, and the areas beneath this monolayer are rich in early granulocytes. Mature granulocytes and monocytes migrate through the endothelial layer, demonstrating in vitro "transmural passage". Cultures stimulated for erythropoiesis show a considerable reduction in fat cells, in endothelial cell cover and in the numbers of classical monocytes. Erythropoiesis appears to be promoted by a close association of the entire erythrocytic series with monocytic cells, forming "erythroblastic islets" in vitro. A possible pathway of intracellular communication between differentiating haemopoietic cells and the stromal cells in their microenvironment is suggested.

Adherent bacterial populations on the bovine rumen wall: distribution patterns of adherent bacteria.

Fourteen tissue sites from the bovine reticulo-rumen were examined by scanning electron microscopy to determine the distribution patterns of bacterial populations adhering to the epithelium. Although diet variations did not appear to influence the total number of tissue-adherent bacteria present in adult Herefords, diet affected their distribution. It appeared that the distribution of the bacterial populations may be directly affected by the physical state of the digesta. The digesta may be mechanically removing adherent bacteria from the tissue surface by abrasive action. The total adherent population consisted of subpopulations with separate distribution patterns, and macropopulations of morphologically similar bacteria were occasionally observed at specific sites on the epithelial surface. Ureolytic organisms on the epithelium followed a distribution pattern considerably different from the general bacterial distribution.