An in vitro morphological study of the mouse visceral yolk sac and possible yolk sac immunocyte precursors

Mouse visceral yolk sac has been organ cultured from 9 days of gestation, a time prior to the thymus being lymphoid, until 12 days of gestation, a time after which the thymus is lymphoid. During the culture period the endodermal epithelial cells survived well, erythropoiesis diminished, endothelial-lined cavities formed in the mesodermal mass, and cells developed which have been classified as large, medium and small immunocyte precursors. The cytoplasm of the immunocyte precursors contains polysomes, spherical mitochondria, a few profiles of rough endoplasmic reticulum, occasional granules and a large Golgi complex. This study offers morphological support for the yolk sac origin of immunocyte precursors in the mouse which may seed the thymus and liver.     

Cytological distinction between clear cell carcinoma and yolk sac tumor of the ovary

Cytological characteristics of clear cell carcinoma (CCC) and yolk sac tumor (YST) of the ovary were examined and compared. Indices for differential diagnosis of these two histologically similar but biologically different tumors were analyzed based on cytological features of the tumor cells. The main results obtained were from three cases of CCC and one case of YST who underwent imprint smear or similar examinations. Seventeen cases of CCC and two of YST with positive cytology results, mainly obtained from ascites samples, were also examined. Cytological characteristics of CCC compared to those of YST were: more marked cohesiveness of tumor cells with less-marked pleomorphism, well-preserved cell borders, fine vesicular cytoplasm, evenly thickened nuclear membrane, and fewer nucleoli and multinucleated giant cells in number. Cells with large cytoplasmic vacuolation were infrequent, and hobnail-shaped tumor cells were rather few in number in CCC cases examined. On the other hand, in the case of YST, cells were more dyscohesive, with marked cellular pleomorphism, usually faint cell borders, no thickening of nuclear membrane and occasional presence of nuclear grooves. The naked cells were commonly observed. Further, nucleoli were frequently numerous in number, and multinucleated giant cells were frequently found which showed marked cellular and nuclear atypism. These characteristics found mainly in imprint smears were also relatively well preserved in specimens obtained from ascites or other metastatic sites. These findings indicate that detailed cytological examination can provide a means for differential diagnosis of CCC and YST, supplementing other clinical information.     

Hematopoiesis in the yolk sac of several representatives of the Sauropsida]

Hemopoiesis in the yolk sac of chicken and crocodile embryos was studied at different developmental stages. Primary intravascular erythropoiesis is closely related to the formation of sanguineous islands appearing in the zone of the yolk growth in the visceral mesoderm. With the development of folds enlarging the surface of the yolk resorbtion, around the vessels running within the folds there appear foci of primary granulopoiesis. The process begins in the paravasal mesenchyma which is gradually disguised by hemopoietic cells (in chicken embryos--at the stage of 8 days, in crocodiles--23 days of incubation). The granulopoiesis continues in chicken embryos during 1/3 of the incubation period, in crocodiles--during 2/3 of the incubation time. The leukopoiesis foci are developed more intensively in crocodiles as well at the size of the yolk sac folds. Leukocytic accumulations disappear in the crocodile sac after hatching (38-40 cm). The change of primary erythropoiesis for the secondary one in the chicken is preceded by the appearance of megaloblastic forms of erythrocytes with the compact homogeneous cytoplasm. They are absent from crocodiles.     

Erythropoietin-receptor expression and function during the initiation of murine yolk sac erythropoiesis

Although erythropoietin is necessary for definitive (fetal liver and bone marrow) erythropoiesis, the role of erythropoietin signaling in primitive (yolk sac) hematopoiesis has not been well defined. In situ hybridization studies have revealed that erythropoietin-receptor (EPOR) mRNA accumulation begins in mesoderm cell masses of the developing yolk sac of the neural plate stage embryo (E7.5) before the development of morphologically recognizable erythroblasts. EPOR mRNA is also present in yolk sac blood islands at early somite stages (E8.5). These findings suggest that EPOR functions during early stages of yolk sac erythropoiesis. We have used a serum-free murine yolk sac explant system (Palis et al., Blood 86:156, 1995) to investigate the initial differentiation of primitive erythroblasts from extraembryonic mesoderm cells. Exogenous erythropoietin increased both erythroblast numbers and betaH1-globin accumulation in yolk sac explants, suggesting that primary yolk sac erythroblasts are directly responsive to erythropoietin. An antisense oligodeoxynucleotide (ODN) experimental approach was used to examine the functional role of erythropoietin signaling during the initiation of yolk sac hematopoiesis in yolk sac explants. Antisense EPOR ODN produced a >50% reduction (p < 0.005) in the number of differentiating primitive erythroblasts, >95% reduction in betaH1-globin accumulation (p < 0.001), and a >50% reduction (p < 0.01) in the number of CFU-E and BFU-E compared with missense EPOR ODN-treated and untreated control explants. Antisense EPOR ODN also blocked the increase in primitive erythroblast number induced by exogenous erythropoietin. We conclude that erythropoietin/EPOR signaling is functionally active during the initial proliferation and differentiation of primary yolk sac erythroblasts. These results also suggest that other growth factor signaling cascades are active during the onset of mammalian erythropoiesis.     

Effect of glucagon on pinocytosis by the yolk sac of the rat

The uptake of macromolecular markers by fluid pinocytosis in the rat yolk sac was inhibited by glucagon, with half-maximal effect at a hormone concentration of approximately 3 X 10(-8) M. Glucagon had no effect on the cellular distribution of the marker subsequent to its uptake. Rates of uptake promptly returned to normal when the yolk sacs were transferred from a glucagon-containing to a glucagon-free medium. Epinephrine also inhibited, but only at much higher concentrations. The effect of the latter was augmented by theophylline. Insulin (10(-6) M) had no effect when added alone or with an inhibitory level of glucagon (10(-7) M). The presumption that the hormone effect was mediated by cyclic AMP was supported by the findings that the cellular levels of cyclic AMP were elevated in the presence of glucagon and that dibutyryl cyclic AMP could replace glucagon as an effective inhibitor. The conclusion that the hormone effect was on uptake rather than on subsequent regurgitation was based on the linearity of accumulation in both the presence and absence of glucagon and the inability of glucagon to stimulate loss of invertase from preloaded cells. Colchicine and vinblastine also inhibited uptake. This finding and those of others which are discussed suggest the possibility that effects of cyclic nucleotides on certain cell functions may involve their regulation of microtubular status.     

Two types of stretch-activated channels coexist in the rabbit corneal epithelial cell

Ion channels contribute to the regulation of cellular function through control of the membrane potential and intracellular concentration of various ions. We examined stretch-activated channels in the corneal epithelial cell. Patch clamping was applied to enzymatically dissociated corneal epithelial cells to characterize their stretch-activated ion channels. The plasma membrane was stretched by applying suction to the patch pipette in cell-attached or inside-out patch configuration. The ion selectivity, voltage-dependence, and stretch-dependence were examined. Two kinds of stretch-activated channel events were observed; the previously-reported large conductance (L) channel and a novel small conductance (S) channel. The probability of recording L vs. S channels in the cell-attached configuration was about 2:1. The L channel was potassium selective with single channel conductance (gamma) of about 160 pS under the symmetrical (150 mm K+) solution. The S channel was permeable to Na+ and K+ with gamma of about 20 pS under the same conditions. Both L and S channels showed little activity in the absence of suction applied to the recording pipette. Channel activity was evoked by suction (negative pressure) stronger than -20 mmHg in both channels. The open probability (Po) and the mean current increased in proportion to further applied stretch and did not saturate for applied suction as strong as -80 mmHg, the pressure at which the gigaseal started to break. Thus, two types of stretch-activated channels coexist in corneal epithelial cells; a potassium-selective L channel and non-selective S channel. The contribution of these channels to the membrane potential is discussed.     

Immunohistochemical identification of ito-cells with smooth muscle cell anti-actin in normal and pathological liver

Eighteen patients with chronic hepatitis of viral and alcoholic etiology investigated and core needle biopsy of the liver was performed. Microscopical investigation consisted of pathological diagnosis, grading and staging of chronic hepatitis and identification of ito cells with smooth muscle cell anti-actin. Ito cells were noted mainly around portal spaces as small cells with vacuolated cytoplasm. Their number is higher in normal liver and chronic persistent hepatitis. Few ito cells were observed in active forms and they are absent in almost all cases with cirrhosis. In chronic hepatitis of alcoholic origin, ito cells are large and their cytoplasma contains many vacuoles. A strong correlation between the number of ito cells and the stage of the chronic hepatitis was noted but it was not possible to correlate the same parameter with Knodell score.     

Parallel Doppler assessment of yolk sac and intervillous circulation in normal pregnancy and missed abortion

This study assessed yolk sac morphology and vascularity and intervillous blood flow in normal early pregnancy and missed abortion. Transvaginal colour and pulsed Doppler were used in a prospective analysis of 87 normal pregnancies and 48 missed abortions between 6 and 12 weeks gestation. The Kruskal-Wallis rank test was used to calculate the difference in yolk sac diameter and vascularity visualization rate between gestational weeks. Repeated measures analysis of variance was used for comparison of the intervillous circulation between groups. The growth of the yolk sac was considered statistically significant between gestational weeks 6 and 9, being most prominent between 9 and 10 weeks of gestation. Vascularity of the yolk sac, characterized by low velocity and absence of diastolic flow, was demonstrated in 67 per cent of normal pregnancies. Yolk sac blood flow was detected in 19 per cent of the patients with missed abortion. Doppler analysis of the intervillous circulation demonstrated decreased peak velocity of the continuous flow in patients with missed abortion for gestational weeks 11 and 12. It is concluded that progressive decrease of yolk sac vascularity coincides with visualization of more prominent colour-coded areas within the intervillous space. In patients with missed abortion, such changes do not occur.     

Primary yolk sac tumor of the endometrium: a case report and review of the literature

We report a case of ergot-induced peripheral vascular insufficiency of the lower limbs and review the vascular complications, angiographic findings and the different modalities of treatment. The following case report highlights the clinical features and course of ergot toxicity, and the difficulty in early diagnosis.     

Hematopoietic precursor cells within the yolk sac tumor component are the source of secondary hematopoietic malignancies in patients with mediastinal germ cell tumors

BACKGROUND: Patients with mediastinal germ cell tumors (MGCT) have a high incidence of hematologic malignancies unrelated to cytotoxic chemotherapy. It has been suggested that these leukemic conditions originate from a MGCT progenitor cell capable of undergoing non-germ cell (hematopoietic) differentiation. METHODS: To assess this hypothesis, histologic material from six patients with MGCTs associated with leukemia was examined using monoclonal and polyclonal antibodies capable of labeling cells of the different marrow cell lineages. RESULTS: Morphologically identifiable hematologic cells were found within the yolk sac tumor component of the MGCT in four of these patients. In three of the four cases, the cells consisted of poorly differentiated blast cells, whereas in the fourth, clusters of erythroblasts were identified. The leukemic cells within the MGCT and in the bone marrow had similar morphology, constant expression of the early progenitor cell marker CD34, and variable expression of more mature myeloid, monocytic, erythroid, and megakaryocytic markers. Three cases expressed p53, a nuclear protein associated with neoplastic transformation in a wide range of malignancies, including testicular cancers, but which rarely is reported in leukemias. Karyotype of the leukemia was assessed in five cases: two showed an i(12p), a cytogenetic marker of GCT not identified in the usual cases of leukemia. CONCLUSIONS: The results support the hypothesis that these leukemic conditions originate in the MGCT through a mechanism of differentiation from a yolk sac tumor-derived progenitor cell, with subsequent homing to the marrow.     

Localisation of proteins in coated micropinocytotic vesicles during transport across rabbit yolk sac endoderm

Rabbit yolk sac splanchnopleur exposed in utero to IgG-HRP and IgG-ferritin conjugates, rabbit and bovine anti-HRP antibodies, free HRP, ferritin and human IgG, was examined ultrastructurally in an attempt to determine whether or not coated micropinocytotic vesicles are involved in selectively transporting immunoglobulins across yolk sac endodermal cells. Human, rabbit and bovine IgG-HRP conjugates, rabbit anti-HRP antibodies, free HRP and human IgG, all become localised in coated micropinocytotic vesicles. Differences were observed in that only human IgG and rabbit anti-HRP antibodies could be located in the intercellular space and bovine IgG-HRP conjugate could not be detected in coated micropinocytotic vesicles in confluence with the lateral and basal plasmalemma. Bovine anti-HRP anti-bodies, IgG-ferritin conjugates, and free ferritin, could not be observed in coated micropinocytotic vesicles. All proteins were detected in macropinocytotic vesicles, and dense bodies resembling phagolysosomes. Results are discussed in the light of a proposal that selection occurs at the cell surface during formation of coated micropinocytotic vesicles and is not linked to intracellular proteolysis.     

Selective cytotoxicity of two rodent T cell lymphomas to rat yolk sac tumours involves a retroviral envelope protein expressed by the lymphoma

OBJECTIVE: To test a model for the study of inequalities in hospitalizations in the city of Ribeirao Preto (SP), understanding them to be due both to the social position of inpatients and also to health care policies in Brazil. MATERIAL AND METHOD: Using a hospital information system in existence for more than 25 years in the city of Ribeirao Preto-SP, 56.293 hospitalizations of municipal inhabitants occurring in some of the 12 general hospitals in 1993, were studied. Using the Brazilian occupancy classification for mortality, these inpatients were grouped on 6 occupational levels, as in the British classification: professional, intermediate, qualified non manual, qualified manual, partially qualified and unqualified. RESULTS AND CONCLUSIONS: Two-thirds of the inpatients had no place in the i.e. did not belong to the economically active population--and consisted of housewives, pensioners, children and students--and one third had some economic activity and thus belonged to the economically the active population. A close association was found between social strata and the classification of the hospital financing system into private, private group clinic and public health system patients. There were differences in hospital parameters as well as in morbidity patterns between these groups. The inequalities relating to average age, average age of hospital deaths, mean lengths of stay, hospital mortality, re-internment and frequency of diseases are discussed. This model allows the social position of the inpatient to be estimated using the hospital financing system, including also those patients with no economic activity, which covers the majority of the population. Social mechanisms created to compensate for inequalities in the welfare state do not cancel out the social differences.     

Morphological and immunohistochemical identification of neurons and their targets in the guinea-pig duodenum

Nerve circuits within the proximal duodenum were investigated using a combination of immunohistochemistry for individual neuron markers and lesion of intrinsic nerve pathways to determine axon projections. Cell shapes and axonal projections were also studied in cells that had been injected with a marker substance. Several major neuron populations were identified. Calbindin immunoreactivity occurred in a population of myenteric nerve cells with Dogiel type II morphology. These had axons that projected to other myenteric ganglia, to the circular muscle and to the mucosa. All were immunoreactive for the synthesizing enzyme for acetylcholine, choline acetyltransferase, and some were also immunoreactive for calretinin. Myenteric neurons with nitric oxide synthase immunoreactivity projected anally to the circular muscle. These were also immunoreactive for vasoactive intestinal peptide, and proportions of them had enkephalin and/or neuropeptide Y immunoreactivity. It is suggested that they are inhibitory motor neurons to the circular muscle. A very few (about 2%) of nitric oxide synthase-immunoreactive neurons had choline acetyltransferase immunoreactivity. Tachykinin (substance P)-immunoreactive nerve cells were numerous in the myenteric plexus. Some of these projected orally to the circular muscle and are concluded to be excitatory motor neurons. Others projected to the tertiary plexus which innervates the longitudinal muscle and others provided terminals in the myenteric plexus. Two groups of descending interneurons were identified, one with somatostatin immunoreactivity and one with vasoactive intestinal peptide immunoreactivity. The two most common nerve cells in submucous ganglia were neuropeptide Y- and vasoactive intestinal peptide-immunoreactive nerve cells. Both provided innervation of the mucosa. There was also a population of calretinin-immunoreactive submucous neurons that innervated the mucosal glands, but not the villi. Comparison with the ileum reveals similarities in the chemistries and projections of neurons. Differences include the almost complete absence of nitric oxide synthase immunoreactivity from vasoactive intestinal peptide-immunoreactive interneurons in the duodenum, the projection of calbindin-immunoreactive Dogiel type II neurons to the circular muscle and the absence of tachykinin-immunoreactivity from these neurons.     

Developmental expression of extracellular glutathione peroxidase suggests antioxidant roles in deciduum, visceral yolk sac, and skin

Extracellular glutathione peroxidase (EGPx) is a secreted selenium-dependent enzyme that reduces hydroperoxides and organic hydroperoxides. Selenium deficiency in females is associated with infertility and spontaneous abortion, suggesting a role for selenium-requiring proteins during embryonic development. To gain insight into functions of EGPx in vivo, we determined sites of murine EGPx synthesis by in situ hybridization during embryogenesis and in adult tissues. At E7.5 of development, high EGPx expression was found in the maternally derived deciduum, with lower levels of accumulation in the embryonic visceral endoderm. At E9.5, the major sites of expression were the yolk sac endoderm and heart musculature. By E16.5, EGPx mRNA expression persisted in yolk sac endoderm but also accumulated significantly in atrially derived myocytes, ossification centers, adipose tissue, intestinal epithelium, and in a ventral-to-dorsal gradient in developing skin. Glutathione peroxidase activity due to EGPx protein was identified in the fluids surrounding the developing mouse embryo at midgestation. The expression of EGPx in tissues at the maternal-fetal interface--deciduum, visceral yolk sac, and skin--suggests that EGPx may serve to protect the embryo from oxidant damage. In adult mice, we identified the S1 segment of the kidney proximal tubules as the primary site of EGPx mRNA accumulation, with lower EGPx levels in atrial cardiac muscle, intestine, skin, and adipose tissue. These findings suggest that EGPx may serve a wider antioxidant role than previously recognized in the interstitium of multiple localized tissues, particularly those associated with the active transport of lipids.     

Regulation of metallothionein expression in human amnion epithelial and mesenchymal cells

OBJECTIVE: In this study the potential for metallothionein expression in amnion epithelial and mesenchymal cells in response to cadmium was evaluated. STUDY DESIGN: Levels of metallothionein messenger ribonucleic acid were evaluated in freshly separated amnion epithelial and mesenchymal cells and in amnion cells in culture. RESULTS: The levels of metallothionein messenger ribonucleic acid in human amnion mesenchymal cells freshly isolated after delivery of term pregnancies were greater than those in epithelial cells of the same tissue. The levels in mesenchymal cells in monolayer culture at confluence also were greater than those in confluent epithelial cells propagated from the same tissue. In response to treatment with cadmium (100 nmol/L to 50 micromol/L), which is inhaled in cigarette smoke, the levels of metallothionein messenger ribonucleic acid in both cell types increased markedly in a dose-dependent manner, but the level was greater in epithelial cells at all concentrations of cadmium chloride tested. With cadmium chloride (10 micromol/L), the level of metallothionein messenger ribonucleic acid increased by as much as 1000-fold in epithelial cells and 10-fold in mesenchymal cells compared with untreated (control) cells. Dexamethasone and tetradecanoyl phorbol acetate also acted to increase the levels in amnion epithelial and mesenchymal cells but not nearly to the levels effected by cadmium. CONCLUSION: These findings are indicative that metallothionein expression in amnion epithelial cells is exquisitely sensitive to cadmium in concentrations similar to those in amniotic fluid of pregnancies of women who smoke cigarettes. We hypothesize that increased levels of metallothionein in amniotic fluid and amnion epithelial cells will bind and thereby may limit the availability of copper to the Cu++-dependent enzyme lysyl oxidase in mesenchymal cells and thereby impair the cross-linking of interstitial collagens, which is effected by this enzyme.     

Immunohistochemical study of proliferating mesenchymal spindle cells in heterogenous soft tissue lesions

Clinics associated with psychology training programs have potential to generate important research data. The expectation that research be experimental in nature has limited the pursuit of research in training clinics. This type of efficacy study is not feasible in most clinics, where too much variation occurs as a result of training and administrative demands. Numerous process-outcome studies have been effectively conducted in training clinics. This article reviews some of these studies, identifies problems related to research in training clinics, and makes recommendations for the development of an enhanced research agenda for training clinics.     

Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.     

Effects of NH4Cl and nocodazole on polarized fibronectin secretion vary amongst different epithelial cell types

The extracellular matrix protein fibronectin was found to be secreted by three polarized epithelial cell lines Madin-Darby canine kidney (MDCK), Caco-2 and LLC-PK1. About 54 and 46% of fibronectin was secreted from the apical and basolateral cell surfaces, respectively, in MDCK cells. In Caco-2 and LLC-PK1 cells, the majority (about 92-93%) of fibronectin secretion occurs from the basolateral cell surface, with the remaining 7-8% from the apical surface. In all three cell types, NH4Cl was found to inhibit basolateral secretion (resulting in enhanced apical secretion), while total fibronectin secretion was not significantly affected (although a delay in secretion was observed). Nocodazole reduced total fibronectin secretion to about 70% of control levels in MDCK and Caco-2 cells, with significant inhibition on secretion from both surfaces. In contrast, total fibronectin secretion was enhanced by nocodazole in LLC-PK1 cells. Furthermore, the majority of fibronectin secretion was redirected to the apical cell surface in LLC-PK1 cells. These observations demonstrate that the nature as well as the extent of the effects of NH4-Cl and nocodazole on polarized fibronectin secretion varies amongst different epithelial cell types.