The role of the visceral yolk sac in mediating protein utilization by rat embryos cultured in vitro

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 223-234
Author(s):  
Stuart J. Freeman ◽  
Felix Beck ◽  
John B. Lloyd
Keyword(s):  
Serum Proteins ◽  
Yolk Sac ◽  
Rat Serum ◽  
Pregnant Rats ◽  
Digestion Product ◽  
Background Levels ◽  
Visceral Yolk Sac ◽  
Chorioallantoic Placenta

Conceptuses from 9·5-day pregnant rats have been cultured for 48 h in heat-inactivated homologous serum. Embryonic development was normal. The protein contents of embryos and visceral yolk sacs after different periods of culture were recorded. When 125-labelled polyvinylpyrrolidone or [3H]dextran were added to the culture serum, radioactivity was accumulated by the yolk sac, but only background levels were detected in the embryo itself. The amount of radioactivity found in the yolk sac varied with the length of the interval before harvesting during which 125 I-labelled PVP or [3H]dextran was present. When formaldehyde-denatured 125 I-labelled bovine serum albumin was added to the culture serum, little radioactivity accumulated in the yolk sac and only background levels were found in the embryo. Trichloroacetic acid-soluble radioactivity steadily appeared in the culture serum, however. When conceptuses were cultured in glucose- and vitamin-supplemented dialysed serum from rats injected 2 h previously with [3H]leucine, radioactivity was found in both embryos and yolk sacs. The amount of radioactivity in these tissues increased with duration of exposure to 3H-labelled serum proteins. After short exposures little of the yolk sac and embryonic radioactivity was acid-insoluble, but this proportion increased with duration of exposure. These results are interpreted as follows. Intact macromolecules cannot enter the cells of the embryo itself, but are captured by pinocytosis into the cells of the visceral yolk-sac endoderm. Indigestible macromolecules such as 125 I-labelled polyvinylpyrrolidone and [3H]- dextran accumulate within the yolk-sac lysosomes, but proteins are digested there by the lysosomal enzymes. The radiolabelled digestion product of 125 I-labelled bovine albumin is [125 I]iodotyrosine, which cells cannot utilize and so is excreted into the culture serum. The labelled digestion product of the 3H-labelled rat serum proteins is [3H]leucine, which is used for protein synthesis in both embryo and yolk sac. The experiments provide direct evidence for the long-suspected role of the yolk sac in mediating embryonic nutrition in the period of development prior to the establishment of a functional chorioallantoic placenta.

Effect of yolk-sac antibody on rat embryos grown in culture

Development ◽  
1972 ◽  
Vol 27 (3) ◽  
pp. 543-553
Author(s):  
D. A. T. New ◽  
R. L. Brent
Keyword(s):  
Direct Contact ◽  
Culture Medium ◽  
Gamma Globulin ◽  
Yolk Sac ◽  
Amniotic Cavity ◽  
Pregnant Rats ◽  
Rat Embryos ◽  
Visceral Yolk Sac ◽  
Primary Action

Rat embryos, explanted with their embryonic membranes during the early stages of organogenesis ( days gestation), were grown in culture in roller tubes. Yolk-sac antibody (sheep anti rat yolk-sac gamma globulin), known to be teratogenic when injected into pregnant rats, was added to the culture medium. At concentrations of 0·1 mg/ml or more the antibody caused gross retardation of growth and differentiation. Injection of antibody into the amniotic cavity so that it had direct contact with the embryo, or between the amnion and yolk sac so that it was in contact with the mesodermal surface of the yolk sac, had little or no effect on development of the embryo or its membranes. These in vitro experiments indicate that yolk-sac antibody has an effect on development independent of any immunological reaction of the mother, and the primary action is probably on the visceral yolk-sac endoderm.

Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions

2002 ◽  
Vol 92 (2) ◽  
pp. 657-664 ◽  
Author(s):  
C. L. Dumke ◽  
J. Kim ◽  
E. B. Arias ◽  
G. D. Cartee
Keyword(s):  
Glucose Transport ◽  
Serum Proteins ◽  
Muscle Contractions ◽  
Rat Serum ◽  
Serum Free ◽  
Bradykinin Receptor ◽  
Contraction Effect ◽  
Kallikrein Inhibitors

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum ± kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum ± bradykinin receptor-2 inhibitors; or 4) serum-free buffer ± bradykinin. 3- O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum ± kallikrein inhibitors tended ( P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.

The effect of teratogenic antiserum on yolk-sac function in rat embryos cultured in vitro

Development ◽  
1982 ◽  
Vol 71 (1) ◽  
pp. 63-74
Author(s):  
Stuart J. Freeman ◽  
Robert L. Brent ◽  
John B. Lloyd
Keyword(s):  
Embryonic Development ◽  
Yolk Sac ◽  
Rabbit Serum ◽  
Identical Result ◽  
Normal Rabbit Serum ◽  
Pregnant Rats ◽  
Homologous Serum ◽  
Visceral Yolk Sac ◽  
Parallel Control

The teratogenicity of rabbit anti-rat visceral yolk-sac antiserum, injected into pregnant rats at either 8·5 or 9·5 days of gestation, has been confirmed. Normal rabbit serum was found not to be teratogenic. When conceptuses from 9·5-day pregnant rats were cultured for 48 h in heat-denatured homologous serum, to which antiserum was added for the final (or the penultimate) 6 h of culture, embryonic development was normal. The protein contents of embryos and yolk sacs (at harvesting) were however decreased. When antiserum was present in cultures for the final 6 h, pinocytosis by the yolk sac, as measured by the uptake of 125I-labelled polyvinylpyrrolidone (PVP), was decreased to an extent related to the concentration of antiserum in the culture medium and to a minimum level of about 40%. The presence of antiserum in cultures for the penultimate 6 h only, with 125I-labelled PVP present for the final 6 h only, produced an identical result. No uptake of radioactivity into the embryo was observed, in either the absence or presence of antiserum. When conceptuses were cultured for the final 6 h in vitamin- and glucose-supplemented dialysed homologous serum whose proteins were [3H]leucine-labelled, the presence of antiserum for either the final or penultimate 6 h again resulted in a decrease in the uptake of radioactivity by conceptuses. Uptake of radioactivity into yolk sac and embryo was decreased by the same amount, indicating that proteolysis in yolk-sac lysosomes was not inhibited. In parallel control experiments in which normal rabbit serum replaced rabbit anti-rat visceral yolk-sac antiserum, no effects on embryonic development, on protein contents of yolk sacs and embryos at harvesting, or on the uptake of radioactivity by conceptuses were observed. These results are interpreted as providing evidence that teratogenic antibodies decrease pinocytosis of protein by visceral yolk sac at the early organogenesis stage and consequentially decrease the availability of amino acids and thus protein synthesis in both yolk sac and embryo. It is proposed that this effect constitutes the mechanism of action of teratogenic antisera.

scholarly journals Inhibition of proteolysis in rat yolk sac as a cause of teratogenesis. Effects of leupeptin in vitro and in vivo

Development ◽  
1983 ◽  
Vol 78 (1) ◽  
pp. 183-193
Author(s):  
Stuart J. Freeman ◽  
John B. Lloyd
Keyword(s):  
Amino Acids ◽  
Protein Content ◽  
Culture Medium ◽  
Specific Inhibitor ◽  
Yolk Sac ◽  
Cysteine Proteinases ◽  
Rat Serum ◽  
Pregnant Rats ◽  
Stage Of Development

Conceptuses from 9·5-day pregnant rats were cultured for 48 h in heat-inactivated homologous serum to which leupeptin, a specific inhibitor of the lysosomal cysteine-proteinases, was added for the final or the penultimate 6h. The presence of leupeptin (25 µg/ml or above) increased the protein content of yolk sacs at harvesting to approximately twice the control value. The protein content of the embryo at harvesting was lower than that of controls. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. The presence of leupeptin did not affect the rate of uptake of the radiolabelled macromolecule by the yolk sac, nor facilitate its entry into the embryo. When formaldehyde-denatured 125I-labelled bovine serum albumin was added to the culture medium for the final 6 h of culture, little radioactivity was found in the yolk sac at harvesting, and barely any was found in the embryo. Trichloroacetic acid-soluble radioactivity was found in the culture serum. The presence of leupeptin sharply increased the levels of radioactivity in the yolk sac (but not the embryo) and sharply decreased the acid-soluble radioactivity of the culture medium. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in both yolk sac and embryo at harvesting. The presence of leupeptin increased the amount found in the yolk sac and decreased that found in the embryo. These results are interpreted as follows. Leupeptin enters the lysosomes of the yolk sac, inhibiting their cysteine proteinases. The digestion of proteins pinocytosed by the yolk sac is consequently inhibited, resulting in the accumulation of protein by the yolk sac and a decreased flow of amino acids to the embryo. Leupeptin (50 mg/kg), injected into pregnant rats at either 8·5 days or 9·5 days of gestation, induced congenital malformation in the offspring. It is proposed that leupeptin exerts its teratogenic action by inhibiting proteolysis in the lysosomes of the yolk sac, and so depriving the developing embryo of its supply of amino acids at a critical stage of development.

scholarly journals Evidence that protein ingested by the rat visceral yolk sac yields amino acids for synthesis of embryonic protein

Development ◽  
1983 ◽  
Vol 73 (1) ◽  
pp. 307-315
Author(s):  
Stuart J. Freeman ◽  
John B. Lloyd
Keyword(s):  
Amino Acids ◽  
Free Amino ◽  
Polyacrylamide Gel Electrophoresis ◽  
Yolk Sac ◽  
Proteolytic Digestion ◽  
Pregnant Rats ◽  
Short Period ◽  
Visceral Yolk Sac ◽  
Rat Reticulocytes

[3H]Leucine-labelled haemoglobin was prepared from rat reticulocytes incubated in the presence of [3H]leucine. Conceptuses from 9·5-day pregnant rats were incubated in vitro for 48 h, with [3H]leucinelabelled haemoglobin present for the final 12, 8, 4, 2 or 0·5 hours. Radioactivity accumulated in visceral yolk sac and in embryonic tissue. When exposure to labelled haemoglobin was for only a short period before harvesting, all the radioactivity found in the embryo and most of that found in the visceral yolk sac was trichloroacetic acid-soluble (i.e. associated with free amino acid rather than with protein). After longer exposures the proportion of radioactivity that was acid-soluble decreased to minimum values of about 20 %. SDS-polyacrylamide gel electrophoresis of the protein-associated radioactivity in visceral yolk sac and embryo was performed. After exposure to labelled haemoglobin for 1 h only prior to harvesting, the yolk sac contained a single peak of radioactivity coincident in mobility with haemoglobin. The embryo contained no protein-associated radioactivity. After exposure to labelled haemoglobin for 12 h, many protein bands in both yolk sac and embryo were radiolabelled. Thus a single radiolabelled protein pinocytically captured by the visceral yolk sac can give rise to the presence of many labelled proteins in embryo and visceral yolk sac. These results indicate that the source protein underwent proteolytic digestion and that the amino acids generated were re-utilized for protein synthesis in both embryonic and visceral yolk-sac cells.

Apolipoprotein expression by murine visceral yolk sac endoderm

Development ◽  
1984 ◽  
Vol 81 (1) ◽  
pp. 143-152
Author(s):  
Wei-Kang Shi ◽  
John K. Heath
Keyword(s):  
Culture Supernatant ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Yolk Sac ◽  
Apolipoprotein Ai ◽  
Visceral Endoderm ◽  
Ldl Particles ◽  
Visceral Yolk Sac ◽  
Specific Localization

Apolipoprotein expression was examined in the postimplantation mouse embryo. Antibodies directed against murine Apolipoprotein AI and human low-density lipoprotein (LDL) particles specifically immunoprecipitated metabolically labelled radioactive apolipoproteins from the culture supernatant of 10·5 days post coitum (days p.c.) yolk sac visceral endoderm cultured in vitro. No evidence for apolipoprotein expression by other embryonic or extraembryonic tissues at this stage was obtained. Immunohistochemical staining at sectioned 10·5 days p.c. embryos with anti-Apolipoprotein AI antibodies revealed specific localization of immunoreactive material in the yolk sac visceral endoderm. We conclude that the yolk sac visceral endoderm is a source of lipoproteins during postimplantation embryonic development.

scholarly journals Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2436-2443 ◽  
Author(s):  
MC Yoder ◽  
VE Papaioannou ◽  
PP Breitfeld ◽  
DA Williams
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Visceral Endoderm ◽  
Hematopoietic Progenitor

Abstract The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

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