Quantitative studies of pinocytosis. I. Kinetics of uptake of (125I)polyvinylpyrrolidone by rat yolk sac cultured in vitro

A method is described for the in vitro culture of 17.5-day rat visceral yolk sac. Tissue survival was good as judged by light and electron microscopy. The rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone by the tissue was constant both within and between experiments. Within the concentration range 0.15-24 mug/ml, the 125I- labeled polyvinylpyrrolidone neither stimulated nor inhibited pinocytosis. The system offers many advantages in the quantitative study of the physical basis of pinocytosis.     

Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.     

Ethanol-induced inhibition of pinocytosis and proteolysis in rat yolk sac in vitro

Pinocytic capture of 125I-labelled polyvinylpyrrolidone and of formaldehyde-denatured 125I-labelled bovine serum albumin by 17.5-day rat visceral yolk sacs incubated in vitro was rapidly and strongly inhibited by low concentrations (0.01 and 0.05%, v/v) of ethanol. The induced inhibition of pinocytosis was readily reversible, but a marked lag was observed before ethanol-exposed tissue regained its full proteolytic capacity towards the exogenous protein. These observations suggest that the acute administration of ethanol to a pregnant rat may give rise to concentrations of ethanol in the maternal blood and/or uterine fluid that induce dysfunction of the yolk sac. In late gestation such inhibition of yolk-sac function may interfere with the transfer of passive immunity across the yolk sac. If similar dysfunction is induced earlier in gestation, in the period before the chorioallantoic placenta is functional, this could cause a transient period of inhibition of histiotrophic nutrition that may be important to the pathogenic mechanism of action of ethanol as a teratogen.     

Differential effect of zinc on teratogen-induced inhibition of pinocytosis by cultured rat yolk sac

Pinocytosis as measured by the uptake of 125I labelled PVP by the isolated cultured day 12 rat yolk sac was observed to be linear over a 4 h incubation period and to proceed at a rate of approximately 2.5 microliters/mg protein/h. Cadmium, anti-visceral yolk sac antibody (AVYS) and trypan blue all inhibited pinocytosis in a concentration-dependent fashion when added to the culture medium, although at low concentrations trypan blue was slightly stimulatory. The effect of zinc on the inhibition of pinocytosis by these three teratogens was studied. It was observed that zinc ameliorated the inhibitory effects of cadmium and AVYS, but had no effect on inhibition by trypan blue. These results indicate that the previously demonstrated protective action of zinc against cadmium-induced yolk sac dysfunction is not specific to that agent but extends to inhibition of pinocytosis by AVYS, and further suggest that, because of its refractoriness to zinc, trypan blue-induced inhibition of pinocytosis by yolk sac occurs by a mechanism different from that effected by cadmium and AVYS.     

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and ¹²⁵I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[¹²⁵I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of ¹²⁵I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of M_r 120 000 was internalized by macrophages somewhat more rapidly than copolymer of M_r 46 000, but was excluded from the yolk sac.     

The pinocytosis of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by rat yolk sac cultured in vitro.

The rates of uptake of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by 17.5-day rat yolk sac cultured in vitro were studied. Over a 6.5h period each substrate was accumulated at a constant and reproducible rate of approx. 2microliter/h per mg of protein. After accumulation in vitro, the three substances were released from the tissue into substrate-free medium at low rates. Sucrose present in the medium at concentrations up to 10 mg/ml was without effect on the accumulation of either [14C]sucrose or 125I-labelled poly(vinylpyrrolidone), but at higher concentrations inhibited the uptake of both substrates. Some batches of colloidal [198Au]gold had a significantly higher Endocytic Index (up to 5 microliter/h per mg of protein). The Endocytic Index of such a batch decreased with increasing substrate concentration, but colloidal gold did not decrease the Endocytic Index of 125I-labelled poly(vinylpyrrolidone). It is concluded that the three substrates enter the yolk sac by pinocytosis in the liquid phase. Those batches of colloidal [198Au]gold with higher Endocytic Indices are considered to enter also by adsorption on membrane binding-sites.     

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.     

Maternal transferrin uptake by and transfer across the visceral yolk sac of the early postimplantation rat conceptus in vitro

Uptake and transfer of maternal transferrin by rat embryos during organogenesis in vitro was investigated using radiolabelled rat transferrin and rocket immunoelectrophoresis. Colloidal gold to which rat transferrin was adsorbed was used as an electron microscopical marker in order to follow the route taken by internalised transferrin across the visceral yolk sac. Culture of rat conceptuses from 9.5 to 11.5 days of gestation in rat or human sera resulted in the passage of rat or human transferrin from the culture medium into the extraembryonic coelom as determined by quantitative immunoelectrophoretic analysis of exo-coelomic fluid. The concentration of human transferrin which was transferred to the exo-coelomic fluid of conceptuses cultured in whole human serum at 10.5 days and 11.5 days of gestation was similar to the concentration of rat transferrin in the fluid of conceptuses cultured in rat serum which had been diluted with Hanks' saline to 50% in order to match the levels of transferrin found in human serum. Growth of rat embryos in 50% rat serum was identical to embryonic growth in 100% rat serum. Uptake of radiolabelled rat transferrin by the visceral yolk sac at 11.5 days of gestation, following culture for 60 min in radiolabelled medium, was much greater than nonspecific uptake of radiolabelled bovine serum albumin. Accumulation of radiolabelled transferrin by the embryo was reduced by the inclusion of unlabelled transferrin into the culture medium. Uptake of transferrin adsorbed 18 nm gold particles was mediated by attachment to coated pits on the apical cell surface of the extraembryonic endoderm. Transferrin-adsorbed gold colloid was internalised via coated vesicles and found in cisternal structures of the peripheral and juxtanuclear areas, as well as in smooth and coated vesicles deep within the cell. The intercellular presence of gold particles in the endodermal layer of the visceral yolk sac and their presence in the mesoderm after 60 min of incubation suggested that passage of transferrin was rapid and mediated by vesicular evagination from the extraembryonic endoderm. These findings suggest that maternal transferrin is the primary source of transferrin for the early rat embryo and its passage to the exo-coelom and embryo is mediated by specific receptors on the apical surface of the extraembryonic endoderm.     

Plasma protein and apolipoprotein synthesis by human yolk sac carcinoma cells in vitro

Summary Three human yolk sac carcinoma cell lines were characterized for the expression of several markers. Each of the cell lines expressed alpha-fetoprotein, without detectable levels of chorionic gonadotropin, and the level of alpha-fetoprotein expression increased dramatically when the cultures were held without passage for extended periods. The secretion of a number of plasma proteins was documented by metabolic labeling, immunoprecipitation, and gel analysis. The major plasma proteins detected were alpha-1-antitrypsin, alpha-fetoprotein, transthyretin,β-2 microglobulin, and plasminogen, with lower levels of transferrin and complement C4 released. Apolipoproteins B, E, and A1 were secreted in high levels as well and were found in the form of lipoprotein particles. Time course experiments on the synthesis of apolipoproteins E and A1 indicated that, as with alpha-fetoprotein, the level of synthesis increased substantially when the cultures were held without passage. The results indicate that these yolk sac carcinoma cells display a protein expression profile similar to that observed for the human yolk sac, and the possibility that the cells may have the potential to differentiate is discussed. This investigation was supported in part by Program Project HL 28972 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

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.     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

Adsorptive pinocytosis of 125I-labelled lactate dehydrogenase isoenzymes H4 and M4 by rat yolk sacs incubated in vitro.

The pinocytic uptake of 125I-labelled porcine lactate dehydrogenase isoenzymes H4 and M4 by 17.5-day rat visceral yolk sac incubated in vitro was saturable and binding obeyed Michaelis-Menten kinetics. The uptake characteristics of the two isoenzymes were very similar. For the H4 and the M4 isoenzymes, the dissociation constants of the protein-plasma-membrane complex were 0.62 microM and 0.84 microM respectively, and the maximum rates of uptake 0.13 and 0.26 nmol/mg of yolk-sac protein per h respectively. These findings contrast with those from studies in vivo, which show the M4 form is taken up by rat liver sinusoidal cells at a much higher rate than the H4 form, and point to different recognition systems for the adsorptive pinocytosis of simple non-conjugate proteins in yolk-sac epithelial cells and liver sinusoidal cells. Competition experiments indicate that binding of the H4 isoenzyme to the yolk-sac cells is restricted to hydrophobic interactions, whereas the binding of the M4 isoenzyme involves hydrophobic as well as positively charged sites on the protein molecules.     

Effect of deglycosylation of yeast invertase on its uptake and digestion in rat yolk sacs.

The uptake by rat yolk sacs of native invertase and invertase which was deglycosylated by treatment with endo-beta-N-acetylglucosaminidase was compared. The initial rate of uptake of the deglycosylated enzyme was severalfold greater and its accumulation leveled off much earlier than that of the native enzyme. Uptake rates of the deglycosylated and native forms of the enzyme were proportional to their concentration in the medium in the range employed and were inhibited about 85% by 10(-6) M glucagon in both cases. After preloading of yolk sacs with native invertase, the tissue level of activity remained relatively constant over a subsequent 6-h time period, while with the deglycosylated form, activity declined substantially. Since this difference appears not to be attributable to differences in thermal stability, it is suggested that the deglycosylated form of the protein is more susceptible to intracellular proteolytic digestion. In vitro studies on the digestion of these two forms of invertase by trypsin are consistent with this suggestion.     

Effects of L-asparaginase on rat embryonic development and yolk sac membrane in vitro

A comparative analysis of the teratogenic effects of L-asparaginase on 10.5- and 11.5-day rat embryos after 24 and 48 hours of exposure in vitro, respectively, were performed. Several medium concentrations of L-asparaginase (0.05, 0.25, and 1.5 IU/ml) were tested in both embryo series. Resulting embryos were submitted to morphological studies in a search for a specific route of pathogenesis. Morphological alterations of the visceral yolk sac were also studied to investigate its contribution to L-asparaginase teratogenicity in rats. Main embryonic malformations (open truncal neural tube, open encephalic vesicles, anophthalmia, lack of inversion, abnormal frontolateral protrusions, great vascular dilations at the cephalic level) and developmental retardation were already generated after the first 24 hours of culture (embryos of 10.5 days) and presented a dose-response relationship. Vascular dilations and neurulation disturbances seemed to be related to an early mesenchyme deficiency. Reduced number of mesenchymal cells was more evident in embryos of 10.5 days than those of 11.5 days, suggesting the existence of a later compensatory mechanism of cellular proliferation in the older embryo. Visceral yolk-sac endodermal cells at both embryonic stages were greatly deformed and enlarged by an increase of the high electron-dense vacuolar system. Therefore, both a blockage of the processes of lysosomal digestion and derived trophic deficiencies probably existed. A double teratogenic mechanism for L-asparaginase is postulated: a direct action mainly in younger embryos (before invagination of the embryo into the yolk sac) and a yolk sac-mediated one.     

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)     

A comparative study of the effects of polyamino acids and dextran derivatives on pinocytosis in the rat yolk sac and the rat peritoneal macrophage

Poly-l-lysine, poly-l-α-ornithine, poly-l-glutamic acid, dextran, DEAE-dextran and dextran sulphate all fail to affect greatly the rate of pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone by 17.5-day rat visceral yolk sac or rat peritoneal macrophages cultured in vitro. It is concluded that these agents do not much affect the rate of pinocytic ingestion of liquid. Polycations accelerate the accumulation of colloidal ¹⁹⁸Au in both systems, and this is ascribed to the formation of substrate · modifier complexes which either adsorb to plasma membrane, and thus gain rapid entry, or initiate another mode of endocytosis. Pinocytic uptake of formaldehyde-denatured ¹²⁵I-labelled bovine serum albumin was accelerated by poly-l-lysine and poly-l-ga-ornithine in the macrophage, buth inhibited in the yolk sac.     

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