Pinocytosis in the rat visceral yolk sac effects of temperature, metabolic inhibitors and some other modifiers

Low temperature, 2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone, ¹²⁵I-labelled bovine serum albumin or colloidal ¹⁹⁸Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the oresence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.     

In vitro studies on the effect of yolk sac antisera on functions of the visceral yolk sac: I. Pinocytosis and transport of small molecules

The production of congenital malformations by the administration of teratogenic antisera to pregnant animals has been reported from many laboratories. This work has focused our attention on the importance of the yolk sac placenta in supporting the rat embryo during early organogenesis and the significance of yolk sac dysfunction in rodent teratogenesis. The studies reported in this article deal with the effect of teratogenic antisera on the process of yolk sac transport; specifically pinocytosis (as measured by 14C-sucrose uptake) and small-molecule transport utilizing 14C-alpha-aminoisobutyric acid (AIB) and 3H-2-deoxyglucose (DOG). We sought to determine whether several different yolk sac localizing antibodies interfere with these transport processes, and, if so, which transport processes were most affected. The results of the experiments indicated that teratogenic antisera interfered with the process of pinocytosis in the yolk sac and that pinocytosis can be reduced as much as 40%. Nonteratogenic antisera, even when they localized in the yolk sac, did not interfere with the process of pinocytosis. Furthermore, the teratogenic antisera did not interfere with the transport of small molecules (either AIB or DOG) in the yolk sac. These results indicated that while fluorescent localization of an antiserum in the yolk sac did not invariably indicate the potential for teratogenicity, it is likely that the reduction in pinocytosis may directly correlate with the teratologic and embryopathic events. This work reaffirms the view that the yolk sac in important during rodent organogenesis and that yolk sac dysfunction can play an important role in the development of congenital malformations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature, metabolic inhibitors and some other factors on fluid-phase and adsorptive pinocytosis by rat peritoneal macrophages.

Low temperature, NaF and 2,4-dinitrophenol could each abolish the pinocytic uptake of 125I-labelled poly(vinylpyrrolidone) or colloidal [198Au]gold by rat peritoneal macrophages cultured in vitro. Cytochalasin B caused only partial inhibition, even at 10 microgram/ml, and colchicine (10 or 25 microgram/ml) inhibited uptake of colloidal [198Au]gold much more than that of 125I-labelled poly(vinylpyrrolidone). Dibutyryl cyclic AMP and ouabain were without effect on uptake of 125I-labelled poly(vinylpyrrolidone), and slight stimulation was seen with ATP and theophylline. Uptake of 125I-labelled poly(vinylpyrrolidone) was abolished by EGTA (5mM), but restored by adding CaCl2 (5mM). The results appear not to support the conventional criteria for the division of pinocytic phenomena into macropinocytosis, requiring a metabolic energy supply and cytoskeletal components, and micropinocytosis, requiring neither.     

Multiple antigens in the rat visceral yolk sac induce teratogenic antisera

Preparative isoelectric focusing was used to fractionate the supernatant from a homogenate of day 19 rat visceral yolk sac. Three fractions, of pI ranges 3.5-5.0, 5.0-7.0, and 7.0-9.0, were isolated and used to immunize rabbits, by four or six weekly injections, each containing 5 mg protein. The resulting antisera were all teratogenic when injected into rats on day 9 of gestation, but widely differing potencies were observed. The most potent antiserum was that against yolk sac components focusing in the pI 7.0-9.0 range: An optimum teratogenic dose of 50 mg protein per kg body weight was observed, and a dose of 100 mg/kg was shown to cause 100% embryonic resorption. Antiserum against the fraction focusing in the pI 3.5-5.0 range was the least teratogenic: A significant incidence of embryonic malformation and death was seen only at doses of 600 mg/kg and above. The two fractions that yielded the more teratogenic antisera were refocused over narrower pH ranges, yielding four subfractions in the pI 5.0-7.0 range and eight subfractions in the pI 7.0-9.0 range. Antisera against each of these 12 fractions were raised in rabbits; most of these antisera were shown to be teratogenic, although of differing potencies. It is concluded that the yolk sac contains many antigens that can elicit antibodies with teratogenic and yolk sac-localizing properties.     

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

Summary 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.Supported by NIH Grant AI 13486-01     

Experimental manipulation of the rodent visceral yolk sac

The visceral yolk sac (VYS) is an especially important placental organ in the rodent because it is the primary source of exchange between the embryo and mother during early organogenesis before the chorioallantoic placenta circulation is established. The VYS is involved with nutritional, endocrine, metabolic, immunologic, secretory, excretory, and hematopoietic functions. The VYS also plays a role in steroid metabolism and interacts with a variety of blood-borne factors: parathyroid hormone, glucocorticoids, insulin, and vitamin D metabolites. The importance of the VYS during development is emphasized by the embryotoxicity resulting from exposure to agents which cause VYS dysfunction when administered to the pregnant animal during organogenesis. Several experimental procedures have provided useful information concerning a variety of VYS functions from early organogenesis to term: Culture of the Embryo, Fetal Incubation, Culture of the Fetus, Giant Yolk Sac, Short- and Long-Term Culture of the Yolk Sac, Modified Ussing's Chamber, Single or Double Diffusion Chamber, and the use of Heterologous Rodent Visceral Yolk Sac Antibodies. Since human yolk sac pathology has been associated with developmental toxicity and spontaneous abortion, it is important to discover whether there are some common functional roles among different mammalian species and to determine if other experimental animal models can be used to study the possible contribution of human yolk sac dysfunction to some human reproductive problems.     

Synthesis, secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac.

Electron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d less than 1.006 g/ml, d = 1.006-1.020 g/ml and d = 1.020-1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with performed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.     

Retinol-binding protein synthesis and secretion by the rat visceral yolk sac. Effect of retinol status

Studies were conducted to explore in rats the role of retinol in the regulation of the synthesis and secretion of retinol-binding protein (RBP) by the visceral yolk sac compared to the liver. Previous studies have shown that in retinol deficiency, hepatic RBP secretion is specifically inhibited, whereas hepatic RBP synthesis rate is unchanged. Retinol-depleted, retinoic acid-supplemented female rats were mated, and maternal liver, fetal liver, and visceral yolk sac were obtained at 14 days of gestation (retinol-depleted group). A group of identically treated, retinol-depleted rats were repleted with retinol on the 14th day of gestation, and the same tissues were collected 6 h later (retinol-repleted group). Normal female rats were used as controls. RBP was assayed by radioimmunoassay and RBP mRNA levels by RNase protection assay using a rat RBP cDNA clone. RBP levels in the visceral yolk sac were elevated 10-fold in the retinol-depleted as compared to the control rats and had declined to near normal values in the retinol-repleted animals. The relative levels of RBP mRNA in the visceral yolk sac were very similar in all three groups of rats. Thus, as in the liver, in the visceral yolk sac retinol deficiency inhibits RBP secretion without altering RBP mRNA levels. In the visceral yolk sac, as in the liver, retinol status appears to regulate RBP secretion specifically, without affecting the rate of RBP biosynthesis.     

Preparation and developmental toxicity of monoclonal antibodies against rat visceral yolk sac antigens

Thirty clones producing monoclonal antibodies (MCAs) to rat visceral yolk sac (VYS) antigens have been prepared. These MCAs localized by immunofluorescence in the VYS endoderm in vitro and were tested for developmental toxicity by intraperitioneal injection of ascites fluid into pregnant rats on day 9 of gestation. Five of the hybridomas produced MCAs that induced embryonic death, malformation, and growth retardation; the other MCAs had no developmental toxicity. Five MCAs, three teratogenic and two nonteratogenic, were tested for their ability to inhibit pinocytosis in the isolated day 17-VYS. Only the teratogenic MCAs were inhibitory, providing further evidence for the hypothesis that teratogenic antibodies interfere with the nutritional supply to the embryo.     

Location and identification of the collagen found in the 14.5-d rat embryo visceral yolk sac

The collagens associated with 14.5-d rat visceral yolk sacs were localized and identified by a variety of procedures. Morphological examination showed that both the visceral epithelium and mesothelium rested upon thin basement membranes, whereas the majority of the extracellular matrix consisted of a stroma containing occasional cells and abundant banded fibrils. Immunohistochemistry at the electron microscope level showed that the basement membranes specifically cross- reacted with antibodies directed against mouse basement membrane components, whereas the stroma specifically cross-reacted with antibodies directed against rat type I collagen. Extractions of acellular visceral yolk sacs and subsequent analyses showed that type I collagen components were prevalent. Furthermore, in vitro biosynthetic studies showed only the presence of type I procollagen components (or their conversion products) and alpha-fetoprotein. These findings, taken together with our previous studies on the 14.5-d rat parietal yolk sac, provide us with protein markers for studying the origin of cells in rat parietovisceral yolk sac carcinomas.     

Synthesis,secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac

Summary Electron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d<1.006 g/ml, d=1.006–1.020 g/ml and d=1.020–1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with preformed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.Abbreviations apo apolipoprotein - ER endoplasmic reticulum - IDL intermediate density-lipoprotein - LDL low density-lipoprotein - VLDL very low density-lipoprotein - PBS phosphate-buffered salt solution - RER rough endoplasmic reticulum - TEM transmission electron microscopy     

The rat visceral yolk sac internalizes maternal transferrin and secretes hydrolyzed products towards the fetus

The uptake of transferrin by the rat visceral yolk sac membranes, and the fate of this protein, were measured in a two-chambered system which allowed access to both surfaces of these membranes, i.e. that facing the maternal compartment and that facing the fetal compartment. 125I-labeled transferrin was internalized by the maternal surface of the visceral yolk sac but not by the fetal surface. Following internalization, this transferrin was degraded and the amino acids were secreted exclusively towards the fetal compartment. Transcytosis of intact transferrin was not detected in either direction. These results suggest that transport across the rat visceral yolk sac bound to maternally derived transferrin is not a major mechanism of iron transport in vivo. These results support a role for the visceral yolk sac in fetal metabolism, or supplying the fetus with amino acids derived from degradation of specific maternal plasma proteins, in this case, transferrin.     

Cell physiology of the rat visceral yolk sac: a study of pinocytosis and lysosome function

The rat visceral yolk sac is active in pinocytosis. Macromolecules accumulated by the tissue are, in general, routed to the lysosomes, where they either accumulate (if non-digestible by the lysosomal enzymes) or are degraded to their monomeric components. The yolk sac cells engage in adsorptive pinocytosis, which leads to the preferential uptake of macromolecules bearing certain surface features, such as a hydrophobic or a cationic domain. Substrates that enter the yolk sac by adsorptive pinocytosis can in some cases act as bivalent ligands, carrying in a second substance by "piggy-back" pinocytosis. Pinocytosis and intralysosomal digestion of plasma proteins by the organogenesis-stage rat embryo play an important nutritional role, supplying a high proportion of the embryo's amino acid requirement. Teratogenic effects can be induced by substances that inhibit either pinocytosis or intralysosomal proteolysis at this sensitive stage of gestation.     

Effects of N-methyl-N'-nitro-N-nitrosoguanidine and deoxycholic acid on processes of tumorous transformation of rat visceral yolk sac. In vitro and in vivo studies

The possibility to use the rat visceral yolk sac as a model for the study of processes of cell transformation was studied. Yolk sac teratocarcinomas could be induced using the method of in vitro culture of yolk sacs in a medium containing a direct carcinogen and a tumor promoter with subsequent transplantation under the renal or testicular capsule of syngeneic rats. Biochemical, electron-microscopic and immunohistochemical methods were used to study the characteristic changes that accompanied cellular transformation. It was shown that even a short-term (3 h) exposure of the yolk sac cells to N-methyl-N'-nitro-N-nitrosoguanidine with or without deoxycholic acid in vitro decreased significantly the rate of yolk sac transport and changed their developmental potential with manifestation of carcinogenic antigens (polyclonal keratins, monoclonal vimentin and smooth muscle actin). This cancerous transformation was promoted following their in vivo transplantation into special anatomic sites.     

Comparative enzyme histochemical study on the visceral yolk sac endoderm in the rat in vivo and in vitro

Summary Histochemical study of the visceral yolk-sac endoderm of the rat was performed in vitro (whole-embryo culture for 24, 48 and 72 h explanted at 9.5 days of gestation) and in vivo (10.5, 11.5 and 12.5 days of gestation) in order to compare the distribution and activity of various enzymes involved in the digestion and energy metabolism in both systems. It was shown that, both in vitro and in vivo -glytamyltransferase and dipeptidylpeptidase IV are demonstrable in the apical cell membranes (membranebound hydrolases), while acid phosphatase, dipeptidylpeptidases I, II and acid -galactosidase are concentrated in the supranuclear vacuoles (lysosomal hydrolases), and cytoplasmic lactate dehydrogenase and mitochondrial enzymes (succinate dehydrogenase, NAD-dependent isocitrate dehydrogenase, cytochrom oxidase) are localized in the whole cytoplasm and mainly in the apical cytoplasm, respectively, of the visceral yolk-sac epithelium. In vivo, the activity of all enzymes increased until 12.5 days, but in vitro, this activity increased only until 48 h after the start of culture (corresponding to 11.5 days in vivo). Comparison of the yolk sacs at 10.5 and 11.5 days in vivo with those after 24 and 48 h in vitro showed that the activities of all the investigated enzymes were almost identical. Yolk sacs which were cultured for 72 h showed lower activities of lysosomal and mitochondrial enzymes than those at 12.5 days in vivo. It is concluded that the digestive function and energy metabolism of the visceral yolk-sac epithelium are almost identical in vitro and in vivo at 10.5 and 11.5 days.Supported by the Deutsche Forschungsgemeinschaft 541/1-1)     

Comparative enzyme histochemical study on the visceral yolk sac endoderm in the rat in vivo and in vitro

Histochemical study of the visceral yolk-sac endoderm of the rat was performed in vitro (whole-embryo culture for 24, 48 and 72 h explanted at 9.5 days of gestation) and in vivo (10.5, 11.5 and 12.5 days of gestation) in order to compare the distribution and activity of various enzymes involved in the digestion and energy metabolism in both systems. It was shown that, both in vitro and in vivo gamma-glytamyltransferase and dipeptidylpeptidase IV are demonstrable in the apical cell membranes (membrane-bound hydrolases), while acid phosphatase, dipeptidylpeptidases I, II and acid beta-galactosidase are concentrated in the supranuclear vacuoles (lysosomal hydrolases), and cytoplasmic lactate dehydrogenase and mitochondrial enzymes (succinate dehydrogenase, NAD-dependent isocitrate dehydrogenase, cytochrom oxidase) are localized in the whole cytoplasm and mainly in the apical cytoplasm, respectively, of the visceral yolk-sac epithelium. In vivo, the activity of all enzymes increased until 12.5 days, but in vitro, this activity increased only until 48 h after the start of culture (corresponding to 11.5 days in vivo). Comparison of the yolk sacs at 10.5 and 11.5 days in vivo with those after 24 and 48 h in vitro showed that the activities of all the investigated enzymes were almost identical. Yolk sacs which were cultured for 72 h showed lower activities of lysosomal and mitochondrial enzymes than those at 12.5 days in vivo. It is concluded that the digestive function and energy metabolism of the visceral yolk-sac epithelium are almost identical in vitro and in vivo at 10.5 and 11.5 days.     

Differential permeability of murine visceral yolk sac to thymidine and to hydroxyurea.

Mouse embryos at the 10–12-somite stage of development were excised from the uterus either with or without the encapsulating visceral yolk sac and were incubated in vitro in 3 × 10^?7M thymidine (methyl-T, 5 μCi/ml) for 30 min or in 4 × 10^?3M hydroxyurea for 45 min with [³H]thymidine present for the last 30 min. Radioautograms of nuclei of neural epithelium enabled an estimate of the effectiveness of the barrier imposed by the visceral yolk sac membrane to the passage of thymidine and hydroxyurea.Labeling of nuclei in the neural epithelium showed that the visceral yolk sac caused a 44% decrease in frequency and a 51% decrease in intensity of label. Hydroxyurea inhibited labeling by 15% in frequency and 37% in intensity irrespective of the presence or absence of visceral yolk sac. These results show that hydroxyurea and the presence of visceral yolk sac independently interfered with labeling of the neural epithelium by thymidine and that visceral yolk sac caused a proportionally greater retardation of label than did hydroxyurea.Nuclei of the endodermal epithelium of the intervening yolk sac, following exposure to hydroxyurea, showed a labeling decrease of 44% in frequency and 77% in intensity. The inhibitory effect of hydroxyurea on yolk sac labeling, however, did not alter yolk sac permeability to hydroxyurea. The results indicate that the visceral yolk sac, by offering no detectable barrier to hydroxyurea, permits prompt teratogenic action of hydroxyurea directly upon the embryo and suggest that the visceral yolk sac is a likely candidate to account for reports that the 8.5-day mouse embryo in situ fails to label with radioisotopic thymidine.