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.

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.

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.

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.

Abnormal embryonic development induced by antibodies to rat visceral yolk-sac endoderm: Isolation of the antigen and localization to microvillar membrane

1985 ◽  
Vol 107 (2) ◽  
pp. 432-441 ◽  
Author(s):  
Christopher C.K. Leung ◽  
Cheng Lee ◽  
Boonlert Cheewatrakoolpong ◽  
David Hilton
Keyword(s):  
Embryonic Development ◽  
Yolk Sac ◽  
Visceral Yolk Sac

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.

Release of bioactive ACTH by perifused human placenta at early and late gestation

1993 ◽  
Vol 136 (2) ◽  
pp. 345-353 ◽  
Author(s):  
B. J. Waddell ◽  
P. J. Burton
Keyword(s):  
Human Placenta ◽  
Late Gestation ◽  
Rabbit Serum ◽  
Maximal Response ◽  
Normal Rabbit Serum ◽  
Apparent Difference ◽  
Adrenocortical Cells ◽  
Adult Rats ◽  
Constant Rate

ABSTRACT This study assessed whether bioactive ACTH is released by the human placenta during perifusion in vitro at early and late gestation. Human placental villous fragments from early (8–12 weeks) and late (38–40 weeks) gestation were perifused at a constant rate for 6·5 h. To assess ACTH-like bioactivity released by this tissue, the perifusion effluent was redirected through adjacent chambers containing freshly dispersed adrenocortical cells obtained from adult rats. Baseline secretion of corticosterone by these adrenocortical cells averaged 95±26 (s.e.m.) fmol/min, and this increased at least fivefold (P <0·01, two-way ANOVA) in response to placental effluent at early and late gestation. The magnitude of this increase, expressed as a percentage of the maximal response to a subsequent stimulus with ACTH(1–24), was similar for placentas obtained at early (41 ± 12% of maximal response) and late (42 ± 17%) gestation. Immunoreactive (I)-ACTH was readily detectable in placental effluent from all preparations (5·5±2·3 fmol/min per g tissue), and there was no apparent difference with stage of gestation. To determine whether all of the ACTH-like bioactivity released by the placenta was attributable to I-ACTH, a second series of placental/adrenal perifusions was conducted. In these, I-ACTH was selectively removed from placental effluent by immunoneutralization, and the residual bioactivity measured. Immunoneutralization involved preincubation of placental effluent with ACTH antiserum (1:100), and preincubation with normal rabbit serum (NRS) served as a control. Preincubation with ACTH antiserum, but not with NRS, resulted in a marked reduction in ACTH-like bioactivity present in placental effluent at both early (P <0·01, paired t-test) and late (P <0·05) gestation. This inhibition was significantly more effective (P <0·05, unpaired t-test) at early than at late gestation. Overall, these data establish that the human placenta can release bioactive ACTH-like activity at both early and late gestation, and that much, but not all, of this bioactivity is directly attributable to I-ACTH. These findings clearly demonstrate a potential role for placental ACTH in directly influencing the maternal and/or fetal hypothalamic-pituitary-adrenal axes during human pregnancy. Journal of Endocrinology (1993) 136, 345–353

Partial purification of antigens collected during in vitro cultivation of the larval stages of Taenia pisiformis

Parasitology ◽  
1976 ◽  
Vol 72 (3) ◽  
pp. 269-279 ◽  
Author(s):  
M. D. Rickard ◽  
J. C. Katiyar
Keyword(s):  
Protective Immunity ◽  
Culture Medium ◽  
Serum Proteins ◽  
Skin Tests ◽  
Rabbit Serum ◽  
Partial Purification ◽  
In Vitro Cultivation ◽  
Normal Rabbit Serum ◽  
Skin Reactions

SummaryLarvae of Taenia pisiformis were cultured in vitro in medium containing 2·5, 5 or 20% (v/v) of normal rabbit serum (NRS). Greatest development occurred in 20% NRS, and the potency of antigens collected in medium from each culture tested by intradermal (i/d) skin tests in infected rabbits paralleled the in vitro growth rate of larvae. ‘Culture’ antigens from 5% NRS stimulated good immunity in rabbits to a challenge infection with T. pisiformis eggs, although they were poorly reactive in skin tests.T. pisiformis larvae were also cultured in 10% (v/v) of nitrates of serum reduced to one-half of its volume by passage through 300 000 MW cut oif (XM300F) or 100000 MW cut off (XM100F) ultrafiltration membranes. Larvae cultured using XM300F had growth rates comparable with those cultured in 20% NRS, and the antigens released into the culture medium had equal potency in i/d tests and in stimulating protective immunity in rabbits. Larvae did not develop in XM100F orproduce skin-reactive or protective antigens.Crude ‘culture’ antigen from cultures in 20% NRS was separated into 4 fractions by nitration on Sephadex G200. All of these fractions gave i/d skin reactions in infected rabbits. Fraction 3 (F3) was the most active, but was shown by acrylamide gel electrophoresis and immunoelectrophoresis to be highly contaminated with rabbit serum proteins. F3 was separated into fractions on DEAE-Sephadex A50, and the third fraction from this was as active as the original culture medium in i/d skin tests, but had only 5% of the original protein concentration. Electrophoresis demonstrated few serum contaminants, and 2 indistinct protein bands that were not present in a similar fraction of NRS.Neither Sephadex G200 F3 nor DEAE-Sephadex F3 stimulated protective immunity in rabbits, suggesting that antigens stimulating immunity against the establishment of T. pisiformis in rabbits and those provoking cell-mediated immune reactions may be different.

scholarly journals IMMUNE RESPONSE OF RABBITS TO INJECTION OF PLASMODIUM KNOWLESI

1939 ◽  
Vol 70 (2) ◽  
pp. 131-139 ◽  
Author(s):  
Monroe D. Eaton ◽  
L. T. Coggeshall
Keyword(s):  
Immune Response ◽  
Plasmodium Knowlesi ◽  
Conclusive Evidence ◽  
Red Cells ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Passive Protection ◽  
Normal Monkey ◽  
Protective Antibodies

Specific complement-fixing antibodies are produced in the serum of rabbits in response to injections of living or dead Plasmodium knowlesi. Sera from rabbits receiving injections of either parasitized or normal monkey erythrocytes are parasiticidal in vitro for P. knowlesi. Because absorption of parasiticidal rabbit sera with normal monkey erythrocytes abolishes the parasiticidal effect, it is concluded that the effect is largely due to an antibody to the red cells. Normal rabbit serum is not parasiticidal. Experiments on passive protection in monkey malaria with serum from rabbits which have received intraperitoneal injections of living or dead P. knowlesi yield no conclusive evidence that protective antibodies are formed.

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