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.

An in vitro study of functions of embryonic membranes in the rat

Development ◽  
1972 ◽  
Vol 27 (3) ◽  
pp. 533-542
Author(s):  
G. S. Payne ◽  
E. M. Deuchar
Keyword(s):  
Total Protein ◽  
In Vitro Study ◽  
Axial Rotation ◽  
Yolk Sac ◽  
Amniotic Cavity ◽  
Outer Membranes ◽  
Leucine Uptake ◽  
Visceral Yolk Sac ◽  
Extraembryonic Membranes

Ten-day rat embryos have been cultivated in vitro, with different layers of the extraembryonic membranes removed. The effects of presence or absence of each membrane on the morphology of the embryos, their histodifferentiation and their uptake of leucine into protein have been followed. Explants with all membranes left intact failed to expand fully and to undergo axial rotation of the embryo, but nevertheless showed highest total protein and highest leucine uptake in biochemical estimations and in autoradiographs. Explants with outer membranes removed and the visceral yolk sac left intact showed the most normal morphology and expansion of the extraembryonic cavities when compared with embryos removed from the uterus at 11·5 days” gestation, but they showed less protein and less leucine uptake than the first series. Explants in which the visceral yolk sac was removed underwent little growth or development and had low total protein values and radioactivity counts. The amnion collapsed and the amniotic cavity disappeared. When the amnion was removed there was a greater incidence of death, as well as little or no development, and lower radioactivity counts than in the first two series. It is concluded that the outer membranes and the visceral yolk sac play an important role in the transfer of small metabolites to the embryo, as well as in regulating the volume of the extraembryonic fluids.

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.

Changes in visceral yolk sac ultrastructure after exposure of rat embryos to selected teratogens in vitro

1990 ◽  
Vol 4 (4-5) ◽  
pp. 593-597 ◽  
Author(s):  
G.P. Daston ◽  
J.L. Burns ◽  
M.H. Chestnut
Keyword(s):  
Yolk Sac ◽  
Rat Embryos ◽  
Visceral Yolk Sac

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.

scholarly journals N-Acetylcysteine and α-cyano-4-hydroxycinnamic acid alter protein kinase C (PKC)-δ and PKC-ζ and diminish dysmorphogenesis in rat embryos cultured with high glucose in vitro

2007 ◽  
Vol 192 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Mattias Gäreskog ◽  
Parri Wentzel
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
High Glucose ◽  
Culture Medium ◽  
Hydroxycinnamic Acid ◽  
Oxygen Species ◽  
Rat Embryos ◽  
Protein Kinase C Inhibitors ◽  
Kinase C

Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of α-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-δ and protein kinase C-ζ in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-δ and protein kinase C-ζ. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-δ and protein kinase C-ζ activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-δ and protein kinase C-ζ were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-δ and protein kinase C-ζ.

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.

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