Superficial cell differentiation during embryonic and postnatal development of mouse urothelium.

After drastic urothelial destruction around birth and around postnatal day 6, mouse urothelial renewal starts each time de novo. The differentiation of superficial cells during urothelial restoration was followed for the first time from embryonic day 15 to postnatal day 6 by the detection of differentiation markers: cytokeratins, uroplakins and apical membrane specialization. The differentiation markers of short-lived superficial cells were studied before and after urothelial destruction. Three distinctive types of superficial cells, typical for certain developmental period, were characterised: cells at low differentiation stage with microvilli and cilia, expressing CK7 and CK18, detected on embryonic day 15; cells at advanced differentiation stage with star-like arrangement of prominent membrane ridges, expressing CK7 and CK20, present between the two urothelial destruction events; highly differentiated cells with typically jagged apical surface, expressing CK7 and CK20, found twice during development. This cell type appears for the first time on embryonic day 18 as the terminal stage of embryonic differentiation. It was found again on postnatal day 6 as an initial stage of differentiation, leading toward terminally differentiated cells of the adult urothelium. Our work proves that apical membrane specialization is the most valuable differentiation marker of superficial cells.

Autophagy-related vacuoles in mouse gallbladder epithelium.

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.

The role of the Golgi apparatus during terminal differentiation of mouse urothelial surface cells.

The development of the Golgi apparatus in the surface cells of mouse urinary bladder during embryonic development was investigated by electronmicroscopic cytochemistry. The distributions of NADPase and TPPase activities were studied in the urinary bladder during day 15 to day 18 of gestation. At the early embryonic stage, the products of the NADPase and TPPase reactions were visible exclusively in 1 to 2 medial and/or trans Golgi saccules. The strongest increment of NADPase and TPPase positive Golgi cisternae was detected at day 17 when the activity of the urothelial cells was very prominent. At this age, NADPase activity was detected also in lysosomes and on the apical surface of the urothelial cells. The highest distribution pattern of NADPase and TPPase activities observed at this stage rapidly decreases at day 18 of fetal life. The results suggest that the organization of the Golgi apparatus reflected the intensity of the processes occuring in the urothelial cells during gestation.

Loss of endothelium mediated vascular relaxation as a response to various clamping pressures. Part II. Direct measurements of clamping pressures and scanning electron microscope study.

BACKGROUND: We used novel equipment for measuring the direct tip pressure (P) of a clamp arm on the vessel wall and studied the relationship between endothelial injuries and various clamping pressures. METHODS: A strain gauge was applied to the surgical clamp arm and connected via amplifier to a 12-bit analogue-digital converter on a PC--MSDOS computer. In the on-line in vivo measurements on rat thoracic aorta a momentary peak clamping pressure (MPCP) as well as the lower stationary clamping pressure (SCP) was defined. Clamping forces of two clamps commonly used in cardiovascular microsurgery were tested in the experiment on rat thoracic aortas: clamp A had the tip pressure p = 0.60 N/mm2 and clamp B p = 5.16 N/mm2. After 15 minutes of occlusion, the thoracic aorta was excised and scanning electron microscopy studies for aortas clamped with clamp A and clamp B were performed. RESULTS: Great endothelial lacerations with complete disruption of the endothelial layer in the rings clamped with the clamp B were seen, but no disruption in rings clamped with clamp A. CONCLUSIONS: The clamping vessel wall injuries, particularly in endothelial layers, depend on the momentary peak clamping pressure as well on the lower stationary clamping pressure.

Loss of endothelium-mediated vascular relaxation as a response to various clamping pressures.

The contraction/relaxation responses of thoracic aortal rings clamped with two clamping pressures to potassium chloride (KC1), noradrenaline and carbachol were studied using a scanning electron microscope (SEM) to ascertain endothelial lacerations. Clamp A had the tip pressure PA = 0.60 N/mm2 and clamp B PB = 5.16 N/mm2. In 15 Wistar albino rats, weighing 328 +/- 19 g (mean +/- SD), the thoracic aorta was occluded for 15 min and then three vascular rings (2 mm wide) were excised. The proximal unclamped ring served as a control. The aorta diameter was calculated from the circumference of distal rings 1.61 +/- 0.01 mm (n = 15, dmin = 1.51 mm, dmax = 1.70 mm). The rings were challenged with cumulative additions of KC1 (10-80 mmol/l) to measure the contraction. Then cumulative relaxation on the administration of carbachol (0.01-100 mumol/l) as a response to noradrenaline precontraction (0.1 mumol/l) was determined. A significant loss (P < 0.05) of vascular relaxation in all clamped rings (clamped with PA and PB clamping pressures) was seen. No significant differences (P > 0.05) were observed for contraction between clamped and control rings clamped with clamp A, however the rings clamped with clamp B showed significantly reduction of contraction (P < 0.05). No significant differences were seen from control rings between groups A and B (P > 0.05), as well as from clamped rings between groups A and B (P > 0.05) for both the contraction and relaxation parts of the experiments. With SEM, great endothelial lacerations with complete disruption of the endothelial layer in the rings clamped with the clamp B were seen, but no disruption in rings clamped with clamp A. Therefore endothelial vascular layers are much more susceptible to pressure injuries than was previously believed. The clamped vessel wall injuries, particularly in endothelial layers, depend on the momentary peak clamping pressure (MPCP) as well as on the lower stationary clamping pressure (SCP).

A mini organ culture as a model for studying the gallbladder epithelium of mouse.

A mini organ culture of mouse gallbladder was developed as an alternative to primary cultures of epithelial cells of this organ. Small pieces of tissue were prepared and maintained in minimum essential Eagle medium with 10% foetal calf serum, for as long as 7 days. Qualitative and quantitative ultrastructural studies have been performed using electron microscopy. The viability of cells was evaluated by stereological quantification of endocytotic vesicles containing horseradish peroxidase and labelling of exocytotic glycoproteins with tannic acid. The morphology of tissue pieces during the 1st h of culturing and tissue isolated directly from animals exhibited no significant differences. However, after 4 h in culture degradative changes became evident in many cells. At that time, endo- and exocytosis were both dramatically reduced. After 24 h, the morphology, as well as endo- and exocytosis recovered and were comparable to the parameters of the tissue in vivo or after 1 h in culture. The endocytotic activity remained unchanged from day 1 to 7 of culturing, while the number of exocytotic vesicles gradually decreased after 2 days in culture. Our results prove that mini organ culture of gallbladder is morphologically and functionally comparable with the tissue in vivo and for studies of epithelium in culture it is more convenient than primary cultures.

Cytochemical staining of vacuolar compartments in the developing small intestine of mice.

Osmium impregnation, nicotinamide adenine dinucleotide phosphatase (NADPase) and thiamine pyrophosphatase (TPPase) localisation have been applied as a cytochemical tool for the labelling of the endomembranes in the small intestinal cells of mice during embryonic development. Only cisternae of the endoplasmic reticulum were stained by prolonged osmification in the undifferentiated cells. At later developmental stage, Os-black was found in cis Golgi cisternae, small vesicles and also in the endoplasmic reticulum of some epithelial cells. At the early embryonic stage the product of NADPase reaction was visible in medial saccules of the Golgi complex and in lysosomes, while at the later stages positive staining appeared also on the apical cell surface and within the structures probably belonging to endosomal compartment. TPPase activity was constantly present in trans Golgi cisternae of the cells during the studied period of fetal life. These results indicate that the cytochemical markers for Golgi staining can also be found in several other endomembrane systems. They give the insight into various connections between the particular intracellular compartments occurring at different developmental stages.

The morphology of parietal peritoneum: a scanning electron micrograph study.

From 1988 to 1992, 114 patients with end-stage renal failure were treated with continuous ambulatory peritoneal dialysis (CAPD). In 30 patients (18 men, 12 women, age 31-80 years), 40 scanning electron micrographs (SEM) of parietal peritoneal tissue, obtained with biopsy, were performed: in 20 patients at the time of the first catheter implantation, in 14 patients after catheter removal (because of peritonitis in 12 patients and drainage problems in 2 patients), and in 6 patients during catheter reinsertion. In uremic patients two types of mesothelial cells were observed: hexagonal and elongated. In some patients microvilli were abundant and covered the whole surface of mesothelial cells; in other patients microvilli were lacking. Wide openings (stomata) between mesothelial cells were found in some cases, which were wider in patients with peritonitis. During peritonitis, microvilli disappeared, and mesothelial cells were covered with fibrin, leukocytes, and erythrocytes instead. In the majority of patients with peritonitis, mesothelial cells were totally peeled away, or removed, leaving a denuded surface of fibrous tissue. A recovery of the parietal peritoneum was observed in one patient at the time of peritoneal catheter reinsertion: a complete mesothelial regeneration with abundant microvilli appeared. In other patients the surface was denuded, without microvilli or mesothelial cells, covered with fibrin and fibrous tissue. Despite observed changes of the parietal peritoneum with SEM during the course of CAPD and peritonitis, changes may be reversible due to regeneration of mesothelial cells. Prolonged changes after discontinuation of peritoneal dialysis may persist in patients without mesothelial cell regeneration or with a defective process of fibrinolysis.

Prolonged osmification as an indicator of the differentiation process in the endomembrane system.

The technique of prolonged osmification was used in the analysis of reducing capacity of perinuclear space, endoplasmic reticulum and cis-Golgi cisternae in different epithelial cells during embryonic differentiation and immediately after the birth. Cells of the mouse gastric and intestinal epithelium and of the exocrine pancreas and mammary gland were analyzed. It was shown that endomembrane compartments exhibit high variability in their capacity to reduce OsO4 into lower valency oxides. Typical staining of cis-Golgi cisternae by osmium black does not occur before the cells achieve the developmental state in which production of specific products starts. The changes in stainability occurring from the perinuclear space and endoplasmic reticulum towards the cis-Golgi cisternae indicate a maturation pathway with no direct correlation to the chemical characteristic of the substances produced in different cell types. In the mammary gland the reduction capacity of endoplasmic reticulum disappeared with the intensive synthesis of lipids. Considering our previous results and those of other authors, the possible reasons for the observed dynamics in reducibility in particular segments of endomembraneous space are discussed.

Prolonged osmification of stomach epithelium.

Osmium impregnation was used upon the mice stomach epithelium to show possible differences in staining during differentiation. In the cells of the stratified gastric epithelium of 14-day-old mice embryos Os black was completely lacking in the Golgi complex. In some but not all cells the staining appears in the perinuclear space and in the endoplasmic reticulum. In the mucoid cells 1 and 8 days after the birth the osmiophility is not uniformly distributed throughout the endomembrane segments, except the cis face of the Golgi complex which is heavily stained. Our results indicate on the variability of the reduction capacity of particular endomembrane segments during differentiation and among the cells at the definite developmental stage.

The carbohydrates of secretory granules and the glycocalyx in developing mucoid cells.

Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin. The glycocalyx was stained with ruthenium red (RR). During differentiation of the embryonic mucoid cells the number of secretory granules increased in parallel to the increase in their carbohydrate component. NMS-stainable parts in secretory granules also had binding sites for the conjugates RCA II- and WGA-ferritin, but the binding of ConA could not be identified. The increasing quantity of NMS in secretory granules was correlated with the increased amount of PA-TCH-SP and TA-UA positive substances in the apical glycocalyx only in 14- and 18-day-old embryos. The observed uniform affinity for RR and lectin conjugates in all analysed developmental stages remains to be explained.

Nafenopin-induced proliferation of peroxisomes in the small intestine of mice.

The effect of nafenopin on the epithelial cells of the small intestine of mice was studied. After 17 days the control and nafenopin-treated groups were sacrificed. The tissues were incubated in alkaline DAB medium. Ultra-thin sections of small intestinal tissue from both groups were examined by electron microscopy. Electron micrographs were prepared and examined stereologically so that any morphologic differences in the epithelial cell peroxisomes and mitochondria between the experimental and control groups could be evaluated quantitatively. In the nafenopin-treated group proliferation of peroxisomes occurred, as indicated by significant increases in volume, and surface and numerical density of these structures compared with controls. No such alterations were found in the mitochondria. Our results show that the response of small intestinal epithelial cells to nafenopin is analogous to that produced in hepatocytes by the same drug. Hepatocyte peroxisomes are supposed to be involved in lipid metabolism and it seems that small intestinal epithelial peroxisomes play a similar role.

The development of microperoxisomes in the cells of the proximal tubules of the kidney and epithelium of the small intestine during the embryonic development and postnatal period.

Microperoxisomes were identified in the cells of proximal tubules of the kidney and small intestine of mice from 13-19 days old embryos, in suckling period and adult animals. Examination of both tissues after incubation in diaminobenzidine medium revealed anucleoid microperoxisomes closely related to the granular endoplasmic reticulum and occasionally found in close relation to it. The number of microperoxisomes within the cells considerably increases with the advanced stage of differentiation of both studies tissues. In the cells of proximal tubules the changes in shape and size of microperoxisomes are also observed with a higher stage of differentiation. These studies indicate that the formation and augmentation of microperoxisomes passes strictly parallelly with the differentiation, i.c. with the ability of cells to perform their specific functions.