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.     

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.     

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.     

The pattern of apolipoprotein B100 containing lipoprotein subclasses produced by the isolated visceral rat yolk sac depends on developmental stage and fatty acid availability

Explants of visceral rat yolk sacs from gestational days 16, 18 and 22 were used for studying developmental changes of secretion and density distribution of lipoproteins, particularly of those containing apoB. Moreover, the influence of fatty acid supply on the amount and density distribution of secreted apolipoproteins was studied on day 18 of gestation. Active lipoprotein production was observed in yolk sacs taken on days 16 and 18 of gestation. It declined considerably on day 22 of gestation in parallel with the production of total protein, triacylglycerols and cholesterol. On all gestational days, apoB floated mainly in the LDL range ( > or = 70%) with differences in the distribution pattern of LDL subclasses. The lowest density of secreted LDL was found on day 18 of gestation (peak at d = 1.025 g/ml) followed by day 16 (peak at d = 1.035 g/ml) and day 22 of gestation (peak at d = 1.045 g/ml). ApoAIV, apoE and apoAI floated exclusively in the HDL range with a peak at d = 1.089 g/ml independently of the gestational day. After incubation of yolk sacs from the 18th day of gestation with 0.4 mM or 0.8 mM oleate, the density of secreted apoB containing particles was decreased (peaks in the VLDL and IDL density range), whereas palmitate in the same concentrations caused a redistribution of secreted apoB toward higher densities (peaks at d > or = 1.032 g/ml). Taken together, the data provide evidence that the density of LDL subclasses produced by isolated yolk sacs between days 16 and 22 of gestation depended on the gestational stage. Moreover, addition of unsaturated or saturated fatty acids to the organ culture differently affected the secretory rate and the density of lipoproteins delivered by yolk sacs on day 18 of gestation.     

Free vitamin B12 and transcobalamin II-vitamin B12 complex uptake by the visceral yolk sac of the Sprague-Dawley rat: effect of inhibitors

Exogenous free vitamin B12 or B12 bound to human transcobalamin II (TCII) accumulated in the near-term rat visceral yolk sac. The rates of their uptakes in vitro and in vivo increased rapidly with time then reached a plateau, which supports a saturable transport/binding process as the rate-limiting step for the uptake of free and TCII complexed B12. Both uptakes were significantly decreased by trypan blue, colchicine, and low temperature but not by ouabain. Such inhibition suggests that the absorption of free and bound B12 is via an endocytosis process dependent upon energy but not the magnesium-dependent sodium/potassium-activated ATPase. Thus, the role of the visceral yolk sac in vitamin transfer to the conceptus and the alterations in yolk sac function associated with birth defects and diminished growth can be integrally related.     

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.     

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.     

The effect of intrauterinely injected lymphocytes prepared from sensitized or non-sensitized guinea-pigs on the ultrastructure of rat fetal membranes (visceral yolk sac)

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8-12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (less than 0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 mug 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.     

Real time microfiberoptic redox fluorometry: modulation of the pyridine nucleotide status of the organogenesis-stage rat visceral yolk sac with cyanide and alloxan

The surface of rat visceral yolk sacs (VYS) of intact, viable rat conceptuses were continuously monitored with a microfiberoptic sensor optimized for detection of the reduced pyridine nucleotides, NADH and NADPH. Model chemical toxins, cyanide and alloxan, were used and evaluated on the basis of their differential ability to modulate NAD(H)- and NADP(H)-dependent cellular pathways, respectively. Exposure with 2 mM sodium cyanide for 5 min caused a reversible fluorescence increase of 325 arbitrary fluorescence units (AFU) and 225 AFU on Gestational Days (GD) 10 and 11, respectively. Exposure with 40 mM alloxan for 5 min resulted in a fluorescence decrease of 170 and 120 AFU on GD 10 and 11, respectively. Glutathione (GSH) levels in the VYS, as determined by HPLC, showed a marked decrease from 27.3 +/- 2.1 to 2.9 +/- 0.4 pmol/mg protein, within the 5-min alloxan exposure period on GD 10. No decrease in GSH levels was noted for the same exposure duration on GD 11. A 2-hr pretreatment with 25 microM BCNU [(1,3 bis(2-chloroethyl)-1-nitrosourea], to inhibit glutathione disulfide reductase (GSSG-Rd), resulted in an elimination of the fluorescence decrease, but still led to a significant drop in GSH levels as seen on both days of gestation. These results are consistent with overall changes in intracellular pyridine nucleotide concentrations, where the relative amounts of NADPH increase significantly and disproportionately from GD 10 to 11. The net oxidation of NADPH, through GSSG-Rd activity, appears to be responsible for the alloxan-induced decrease in surface fluorescence. Conversely, the cyanide-induced fluorescence increases appear to be the result of NAD+ reduction, mediated through the inhibition of the terminal cytochrome oxidase in the electron transport chain.     

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.     

Studies on the extra-hepatic effects of glucagon in the eviscerated rat

The in vivo effects of glucagon on the metabolism of extra-hepatic tissues have been investigated in eviscerated, functionally hepatectomized rats with intact kidneys. In these animals, even pharmacological amounts of exogenous glucagon did not significantly alter plasma glucose, FFA, or amino acids, compared with saline treatment. The possible secondary release of adrenal catecholamines following such doses of glucagon appeared to be similarly ineffective in increasing the peripheral tissue mobilization of substrates. It was only when the eviscerated animals were pretreated with insulin that the subsequent administration of glucagon or epinephrine elicited significant elevations in plasma FFA. The concomitant evisceration and adrenalectomy did not produce results which were significantly different from evisceration alone. Both kinds of animals required insulin pretreatment before a lipolytic response to glucagon or epinephrine could be demonstrated. This suggests that severe insulin insufficiency itself elicits almost maximum catabolism in these animals and that the further addition of other catabolic hormones such as glucagon or epinephrine cannot increase these catabolic effects, as manifest in plasma concentrations of FFA. These data show an extra-hepatic lipolytic effect of glucagon in vivo, but do not illuminate the significance of this effect in the intact animal.     

Effect of glucagon on hepatic circulation in the pig

The effect of intravenous injection of 50 mug/kg of glucagon on the hepatic circulation of the pig was studied in 12 animals. Glucagon caused an arterial pressure reduction of 11 mm Hg after two minutes and 7 mm Hg after ten minutes. The portal pressure and blood flow were not altered. The superior mesenteric arterial flow decreased by 12%. The hepatic arterial blood flow increased by 80% after two minutes and by 58% after ten minutes. There was no difference in response when anesthesia was achieved with small intravenous doses of thiopental (Pentothal) sodium or 70% nitrous oxide in oxygen and tubocurarine chloride.     

Hypothalamic influence on insulin and glucagon release in the rat

Blood glucose, plasma insulin, and glucagon levels were measured in undisturbed and free-moving rats. The insulin and glucagon levels rise in the 1st min after the beginning of food ingestion, whereas the glucose level begins to increase only in the 3rd min if carbohydrate-rich food is eaten. This early rise in insulin and glucagon level is also observed under conditions in which carbohydrate-free food is eaten. A similar release of insulin and glucagon can be obtained by injection of 0.1 microgram of norepinephrine into the ventromedial hypothalamus, but the same injection made into the lateral hypothalamus causes release of insulin only, whereas injections in other hypothalamic areas are nearly without effect. Similar injections of isoproterenol did not cause changes in insulin, glucagon, and glucose levels. It is suggested that the early insulin and glucagon responses are of reflex origin and that the ventromedial and lateral hypothalamic areas are relay stations in the reflex pathways. The lack of effect of atropine to block the insulin and glucagon responses to noradrenergic stimulation of the ventromedial hypothalamus indicates that the efferent pathway is not cholinergic.     

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.     

Immunohistochemical identification of epithelial and mesenchymal cell types in the chorioallantoic and yolk sac placentae of the guinea-pig

To define the epithelial and mesenchymal cell types of the guinea-pig placenta, immunostaining patterns were determined for the intermediate filament proteins cytokeratin and vimentin. Chorionic and yolk sac placentae were studied at 15, 20, 25, 29-30, 44-45, 55 and 65 days of gestation. Immunohistochemistry was performed on 5-microm thick sections of paraffin embedded tissue using specific antibodies against cytokeratin, a marker for epithelial cells, including trophoblast, and vimentin, a marker for mesenchymal cells and stromal decidua. Immunostaining was identified by the avidin-biotin-peroxidase technique with diaminobenzidine as the chromogen. Most of the surface of the placenta is covered by the columnar epithelium of the parietal yolk sac, beneath which is found a layer of chorionic giant cells. In the guinea-pig, a sheet of mesenchymal cells interposed between these cell layers immunostained for vimentin, a protein that is expressed only intracellularly, and had nuclei orientated parallel to the surface of the placenta. This cell layer is quite different from Reichert's membrane in the rat or mouse, which is acellular. Within the main placenta, cytokeratin immunostaining demonstrated that the trophoblasts lining the large maternal blood sinuses are different in character from the surrounding syncytiotrophoblast, confirming earlier ultrastructural observations. In the subplacenta, some trophoblast did not immunostain for cytokeratin and there was non-specific staining of cellular debris, so that immunostaining for vimentin provided the clearest indication of the maternal-fetal interface. In later stages of gestation (30-55 days), trophoblasts invading the walls of maternal arteries immunostained for cytokeratin and were vimentin negative. In early gestation, however, trophoblast invasion of the maternal vessels was indicated by cells that were immunoreactive for both cytokeratin and vimentin.     

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.