Yolk Sac Dysfunction Research Articles

Proteomic analysis of indium embryotoxicity in cultured postimplantation rat embryos

Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24 h in the presence of 50 μM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

Nutritional studies of the embryo during early organogenesis with normal embryos and embryos exhibiting yolk sac dysfunction

In 1961 we reported that heterologous kidney antiserum when injected into pregnant rats resulted in wide spectrum of congenital malformations. Further studies identified that it was the IgG component of the antiserum that was teratogenic and that complement was not necessary to produce the teratogenic effect. Labeled antibody studies demonstrated that the kidney antiserum localized in the kidney and in the visceral yolk sac (VYS) and parietal yolk sac placentas. Preparation of yolk sac (YS) antiserum proved to be more potent than the kidney antiserum. Adsorption studies with VYS and parietal yolk sac antiserum revealed that the site of the teratogenic process was located in the VYS. In vitro embryo culture experiments demonstrated that direct injection of the teratogenic antibody into the amniotic or YS cavity did not injure the embryo, thus indicating that the teratogenic antibody had to come in contact with the absorptive surface of the VYS. Collaboration with Dr. John Lloyd demonstrated that teratogenic antibody interfered with the process of pinocytosis and the delivery of amino acids (AA) to the developing embryo. Our studies into the nature of the source of AA for the embryo indicated that in some instances >95% of the AA present in the developing embryo were derived from maternal proteins and the remainder from free AA in the maternal serum. We also demonstrated that embryonic methionine was derived primarily from the digestion of maternal serum proteins but that more of the methionine was diverted from the synthesis of embryonic proteins, supporting the view that it has important functions other than the synthesis of proteins. All these studies focus on the role of the YS in human development and whether human YS dysfunction may play a role in the pathogenesis of congenital malformations. Further studies on the delivery of AA to the embryo are warranted to determine whether certain AA are in short supply in maternal serum and place the embryo at risk if nutritional alterations in the maternal environment occurs. Furthermore, the YS may be an organ whose role might offer opportunities for pregnancy control. (J Pediatr 1998;132:S6-S16.)

Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.

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.

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 4h incubation period and to proceed at a rate of ∼2.5 μl/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.

Effects of presensitization of antiheart serum on the rat embryonic heart

After sensitization by rabbit anti-rat-heart serum (AHS), Wistar rats were injected with 9 ml/kg of AHS on day 9 of gestation. The incidence of malformations was 31.5% and that of cardiovascular malformations was 21.8%. The main malformations were microphthalmia, ventricular septal defect, pulmonary stenosis, and general edema. No antinuclear antibody was detected in the maternal sera by the fluorescent antibody technique. No complete heart block was found in the fetuses by electrocardiography. The fluorescent antibody technique demonstrated localization of rat IgG and rat C3 on the maternal myocardium, but a light-microscopic study revealed no myocarditis or endocarditis in either the maternal or the fetal heart. The types of malformation were similar to those observed after a single administration of antikidney serum, which presumably involves yolk sac dysfunction. These results show that both the existence of the immune complexes of antibodies against AHS and the administration of AHS on day 9 are necessary to cause yolk sac dysfunction or other teratogenic changes.

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)

An in vitro study of teratogenicity in the rat due to antibody-induced yolk sac dysfunction : Identification of the yolk sac antigen involved.

An antiserum was prepared in rabbit against rat visceral yolk sac endoderm. The initial injection was of a ConA-Sepharose purified fraction of endoderm, and subsequent injections were of whole endoderm. The antiserum was found to be a potent rat teratogen in vivo, the most common defects observed being anophthalmia and hydrocephaly.Using rat whole embryo culture, the antiserum was demonstrated to induce dysmorphogenesis and growth retardation in a concentration dependent manner. The most frequent abnormalities were of the optic primordia, suggesting a similar embryonic response in vitro to that observed in vivo.In further culture experiments, the antiserum was shown to inhibit macromolecule (125I-labelled PVP) uptake by the visceral yolk sac, an essential process in embryonic nutrition. This effect of impaired yolk sac-mediated nutrition confirms previous observations using anti-whole yolk sac antiserum (Freeman et al. 1982), and it is proposed as the primary cause of teratogenesis.In order to identify the yolk sac antigen(s) involved in the teratogenic response, yolk sac endoderm peptides were separated by PAGE and electrophoretically transferred to nitrocellulose for analysis. With an enzyme linked immunoassay, the antiserum was observed to cross-react with a single 30 kd peptide, demonstrated by a ConA-binding technique to be a glycopeptide. Control serum showed no evidence of cross-reaction with yolk sac peptides.

Cardiovascular Anomalies Induced by Anti‐Tissue Sera and Immunological Cross‐Reaction

AbstractWistar rats were administered rabbit anti‐tissue serum alone or in combination with sub‐teratogenic doses of anti‐kidney serum (AKS) on day 9 of gestation. The results showed that the incidence of cardiovascular malformations was highest (46.7%) when a small dose (1 ml/kg) of AKS given on day 9 was followed by 9 ml/kg of anti‐heart serum (AHS) on day 10. The most common cardiovascular malformations were ventricular septal defects associated with aortic arch anomalies. The spectrum of malformations did not vary with the injected sera. Immunodiffusion studies indicated that the kidney, umbilical cord and yolk sac tissues have at least one common antigen. Between the AHS and the kidney antigen, a slight precipitin band was also demonstrated. These results suggest that the synergistic effects of antisera depend on the degree of immunological cross‐reaction with the yolk sac, and that the mechanisms of teratogenicity by the combined administration is caused mainly by yolk sac dysfunction.

Effects of sensitization of hemolytic streptococcal M-protein fraction on embryonic heart in the rat.

After sensitization by M-protein fraction (MP) of hemolytic streptococci for two generations, Wistar rats were injected with 9 ml/kg of MP on day 9 of gestation (plug day = day 0). The incidence of malformations was about 12% whether anti-MP antibody was positive or not in the maternal sera. The primary malformations were ventricular septal defect, anophthalmia, and microphthalmia. Light microscopic study revealed no myocarditis nor endocarditis in either the maternal or fetal heart. No antinuclear antibody was detected in the maternal sera by the fluorescent antibody technique. No complete heart block was found in the fetus by electrocardiogram. These results were similar to previous findings using a single injection of MP, and therefore further demonstration that the principal factor in the teratogenesis induced by MP administration in the rat is not an inflammation in the fetal organs caused by an autoimmune mechanism but rather yolk sac dysfunction.

Production of congenital malformations using tissue antisera: XV. Reichert's membrane and visceral yolk sac antisera

Rabbit anti-rat Reichert's membrane (RM) serum, when injected into pregnant rats on the ninth day of gestation, produced embryolethality and stunting but a surprisingly low incidence of malformations. Immunofluorescent studies revealed that the maternally injected RM antiserum localizes in RM and in the basement membrane of some maternal kidney tubules but not in the maternal glomerulus or in the cytoplasm of the visceral yolk sac (VYS) cells as had teratogenic VYS and kidney antisera. When antiserum against rat RM was injected into mice, localization occurred in the glomerular basement membranes, thus confirming the findings of Pierce and co-workers. When teratogenic term VYS antiserum is adsorbed with RM, it still localizes in the VYS, does not localize in RM, and is still teratogenic. When this same antiserum is adsorbed with VYS, the antiserum is no longer teratogenic. It appears that the localization of antibodies in the VYS cells is the major contributor to the production of congenital malformations because localization of antisera in RM alone was associated with a very low incidence of teratogenesis. Since both structures participate in a multitude of biochemical processes, which include adsorption, transport, catabolism, and anabolism of nutrients, nonnutrients, and waste products, there are many plausible hypotheses to explain the nature of the parietal and visceral yolk sac dysfunction.

The Morphology of Immunologically Induced Hydrocephalus in the Newborn Rat

Anti-rat-kidney serum injected into a pregnant rat on the 9th day of gestation, before the appearance of the neural plate, resulted in megalocephalic hydrocephalus in the offspring. The present study shows that there is no obstruction to the flow of the CSF from the ventricles to the venous sinuses, seen with tracer studies and light microscopy, and that there is evidence of an increase in CSF pressure within the ventricular system. Although it is known that these teratogenic antisera localize in the yolk sac and produce yolk sac dysfunction, the mechanism of induction of hydrocephalus is yet to be clarified. Hydrocephalus has been induced immunologically in fetal rats with heterologous nephrotoxic antiserum (rabbit or sheep anti-rat kidney serum [RARKS or SARKS]) (8), anti-rat placental serum (6), and anti-rat yolk serum (3). Prenatally induced immunological hydrocephalus has not been recorded as a human disease. We report here the first morphological study of ARKS-induced hydrocephalus in the brain of newborn rats. ARKS injected into pregnant rats results in the birth of offspring presenting with a wide variety of anomalies, the most common being hydrocephalus (4). The highest incidence of hydrocephalus was produced by an intravenous (I.V.) or intraperitoneal (I.P.) injection of ARKS on the 9th day of gestation (5).