Ultrastructural localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

Fine structural changes and localization of phosphatases in the epithelium of the duodenal crypt of X-irradiated mice

Summary The ultrastructural localization of acid phosphatase has been studied in the stem cells of duodenal crypt of X-irradiated mice. After a transitional increase of the cytochemically detectable activity of this enzyme, in the first hours after irradiation, the reaction appears less and less important in the cells. These observations are in agreement with the morphological alterations of the ultrastructures provoked by the irradiation. The meaning of these modifications is discussed.This work was done thanks to the contract C.E.N./A.I.E.A. No. 347/RB and thanks to grants from the Fonds de la Recherche Scientifique Fondamentale Collective.     

Ultrastructural localization of several phosphatases with cerium

Cerium ions have been used as the capture agent for inorganic phosphate released during the enzymatic hydrolysis of phosphate-containing substrates by a variety of phosphatases. Cerium phosphate reaction product accumulation is proportional to the amount of enzyme present in a cell-free model system. Ultrastructurally, cerium phosphate reaction product appears as a very fine electron-dense precipitate. Cerium appears to be a better capture agent for inorganic phosphate than lead in that reaction product is usually more uniform and more consistently reproducible when cerium is used. Furthermore, nonspecific deposits of reaction product that are commonly encountered in lead-based phosphatase reactions are virtually nonexistent when cerium is the capture agent.     

Lysozyme localization in human gastric and duodenal epithelium

Summary The distribution of lysozyme in normal gastric and duodenal mucosa was studied by light- and electronmicroscopic immunocytochemical techniques (direct enzyme-labeled antibody method).In the duodenal mucosa, lysozyme was found in the Paneth cells and the epithelial cells of Brunner's glands. Electron-microscopically, lysozyme was found in rough endoplasmic reticulum and perinuclear spaces, which were assumed to be protein-synthesizing organelles, and also in the secretory granules of Paneth cells. Additionally, lysozyme was detected in the stomach in mucinous granules and in some parts of the rough endoplasmic reticulum within the epithelial cells of the pyloric glands, the mucous neck cells of the fundic glands, and in several surface epithelial cells of the plyoric and fundic regions.This suggests that some quantity of lysozyme in gastrointestinal secretion originates from the gastric and duodenal glands, and that it acts as a defense mechanism in the gastrointestinal tract.     

Ultrastructural localization of a chondroitinase-sensitive, cuprolinic blue-positive filament in developing mouse lung

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, the lungs of mice ranging from the late fetal stage (17 days of gestation) to the puberal stage (27 days) were surveyed for their proteoglycans. A large Cuprolinic Blue-positive filament is present within the connective tissue of lungs of late fetal and young postnatal mice. It is mostly located at the boundary between large extracellular matrix structures and electron microscopically empty areas, but sometimes also at the surface of fibroblast-like cells. The stainability of the filament disappears after treatment with chondroitinase ABC or chondroitinase AC, but not after treatment with nitrous acid. The Cuprolinic Blue-positive structure appears to be most abundant around 2 days postnatally. From day 10 on, its number decreases dramatically, and it can be no longer observed in the lungs of 27-day-old mice.     

Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts.

The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies.     

Ultrastructural aspects of mouse cecal epithelium

Summary The ultrastructure of the cecal epithelium of the mouse resembles that of more distal parts of the colon. A brief discussion is presented of the possible significance of apical cytoplasmic granules to the glycocalyx and of the close association of microorganisms with the lining epithelium.This research was supported by N.I.H. grant GM-15289. The author thanks Dr. F. J. Agate for the scanning electron micrograph.     

Ultrastructural cytochemistry of membrane-bound phosphatases in preimplantation mouse embryos.

The time of appearance and the ultrastructural localization of the enzyme activity of alkaline phosphatase (AlkPase), 5′-nucleotidase (5′Nuc), Mg²⁺-ATPase, transport ATPase, cyclic AMP phosphodiesterase (cAMP-PDase), and adenylate cyclase (AC) were investigated in unfertilized eggs and in mouse preimplantation embryos. Enzyme activity was associated only with the plasma membrane. AlkPase activity appeared only in limited areas of the plasma membrane of one-cell embryos and increased in the eight-cell and morula stages. In blastocysts, the enzyme activity was concentrated mainly in the trophoblast cells. 5′Nuc activity appeared first in four- or eight-cell embryos and the highest activity was observed in trophoblast cells in the blastocyst and in plasma membrane between cells forming inner cell mass. Mg²⁺-activated ATPase activity was present in all embryos and in unfertilized egg plasma membrane. Transport (Na⁺K⁺)-ATPase appeared only in the closely apposed membranes of adjacent cells in morulae and blastocysts. A very low cAMP-PDase activity appeared between adjacent cells in two-cell embryos, and the highest activity was observed on the outer surface of the plasma membrane of trophoblasts. AC was the only enzyme whose activity was located on the inner (cytoplasmic) side of the plasma membrane and appeared as early as the one-cell stage embryo. The relation between the time of the appearance of enzyme activity and the preparation of embryos for implantation and upon embryonic proliferative activity is discussed.     

Ultrastructural differentiation of the intestinal epithelium in the bandicoot Perameles nasuta

Summary The principal cells of the epithelium in the small intestine of the marsupial Perameles nasuta were studied with the electron microscope. The cells in the lower parts of the crypts are undifferentiated and have a high nucleo-cytoplasmic ratio and an abundance of free ribosomes. As the cells move upwards to take their place in the surface epithelium covering the mucosal folds their nucleo-cytoplasmic ratio and the number of free ribosomes decrease, the cells elongate and develop a brush border, a system of microtubules in the apical cytoplasm, a terminal web, terminal bars and desmosomes.The brush border develops from a series of cell processes interdigitating with those from the opposite cell. Spaces arising between the cell processes gradually separate the contiguous cells and the cell processes become microvilli which increase in number and become uniform in size and shape. The Golgi complex gives rise to small vesicles with a different membrane structure than that of the Golgi membranes themselves. It is suggested that the microtubules do not arise as tubular invaginations of the surface membrane but that they develop from the Golgi vesicles.     

Ultrastructural localization of some phosphatases in the prothoracic gland of the insect Leucophaea Maderae

Summary The cytochemical localization of acid phosphatase and thiamine pyrophosphatase activity was studied by light and electron microscopy in prothoracic gland cells of the cockroach Leucophaea moderae. Nymphal and young adult animals were used.Prominent sites of acid phosphatase activity included large membrane-bounded dense bodies or lysosomes, and certain cisternae of the Golgi apparatus. The results suggest a possible difference in the enzymatic activity toward glycerophosphate and aromatic phosphates as substrates.Thiamine pyrophosphatase activity was localized in elements of the Golgi apparatus and endoplasmic reticulum, and in lysosome-like dense bodies. This latter activity was abolished by sodium fluoride treatment, whereas the phosphatase activity in the Golgi apparatus and endoplasmic reticulum is unaffected by such inhibition.The cytochemical results confirm through direct evidence the suggestions of Scharrer (1964), that the large dense bodies present in the prothoracic gland cells are lysosomes, and that their activity may be related to stages in the life history of the glands. Furthermore, the lysosomes or their derivative structures may play an essential role in the autolysis of the prothoracic glands toward the end of their active period.The enzymatic activity of the endoplasmic reticulum may indicate the involvement of this organelle in the metabolism of steroid-like precursor materials necessary for the synthesis of ecdysone.This study was supported by U.S.P.H.S. grants 5 T1-MH-6418 and NB-05219, and grant RO 1-AM-3984 to Dr. Berta Scharrer. I would like to express my appreciation to Dr. Scharrer for her encouragement and assistance during this study. I also wish to thank Mrs. Sarah Wurzelmann for her competent technical aid.     

Ultrastructural immunocytochemical localization of fibronectin in the developing rat lung

In a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra- and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.     

Ultrastructural localization of acid phosphatase in nonhuman primate vaginal epithelium

Summary The vagina of the rhesus monkey is lined by a stratified squamous epithelium. However, little is known regarding the cytochemical composition of its cell organelles and the substances found in the intercellular spaces. In this study we have examined the ultrastructural distribution of acid phosphatase in the vaginal epithelium. In basal and parabasal cells reaction product was found in some Golgi cisternae and vesicles and in a variety of cytoplasmic granules. Reaction product was also found in some, but not all, membrane-coating granules. In the upper layers of the epithelium, the membrane-coating granules extruded their contents and acid phosphatase was localized in the intercellular spaces. The possible roles of acid phosphatase in keratinization, desquamation, or modification of substances in the intercellular compartment are discussed.     

Ultrastructural localization of carbonyl reductase in mouse lung

Summary The immunocytochemical localization of tetrameric carbonyl reductase in the mouse lung was determined by an electron-microscopical immunogold procedure using monospecific antibodies against the enzyme. The labelling of carbonyl reductase was observed within the mitochondria of the ciliated and non-ciliated cells of the bronchioles and the type II alveolar pneumocytes, and the density of labelling in the non-ciliated cells was higher than those in the other cells. No significant labelling was detected over other compartments of the epithelial cells. The labelling was undetectable in the type I alveolar cells, alveolar macrophages and connective tissue cells of the lung. These results clearly indicate the localization of carbonyl reductase to the mitochondrial matrix of these epithelial cells, of which the non-ciliated bronchiolar cells contained particularly high amounts of the enzyme.     

Ultrastructural localization of carbohydrates in Reichert's membrane of the mouse

In the present investigation, we examined the role of trophoblast and parietal endoderm cells in the synthesis of carbohydrate-containing components of Reichert's membrane. To eliminate the function of Reichert's membrane as a filter between maternal and embryonal tissues we carried out our examination under in vitro conditions. Parietal yolk sac from mouse embryos on day 9 post coitum (p.c.) were cultivated for 0 to 5 days. Because tannic acid enables a complex formation between carbohydrates and osmium we chose the fixation with this acid for the ultrastructural study. Electron microscopy showed that for assembly of Reichert's membrane, trophoblast cells produce and then release components that were detected as tannic acid-positive granules both in the Reichert's membrane and in the vacuoles of the trophoblast cells. To localize specific carbohydrates we used postembedding-gold-lectin histochemistry on LR-Gold^R-embedded tissues. Strong binding sites for the lectins WGA (Triticum vulgare), RCA I (Ricinus communis) and Con A (Canavalia ensiformis) were observed in Reichert's membrane and trophoblast cells but not in the parietal endoderm cells. The LTA (Lotus tetragonolobus)-binding pattern was positive in the membrane and its adjacent cells but that of the LFA (Limax flavus) was negative in the parietal endoderm cells and very weak in Reichert's membrane and trophoblast cells. Our results demonstrate that trophoblast cells are involved in the construction of Reichert's membrane through the production and release of specific glycoconjugates.