Amino acid requirements in the early post-implantation rat conceptus.

Amino acid analysis has been performed on hydrolysates of embryos/fetuses, visceral yolk sacs and ectoplacental cones/placentae from early post-implantation rat conceptuses. The increments in each amino acid between 10.5 and 11.5 days, between 11.5 and 12.5 days, and between 12.5 and 13.5 days, are expressed as percentages of the total amino acid increment. These three profiles are very similar to each other and also strongly resemble the amino acid composition of hydrolysates of rat serum. The results are discussed in the context of the hypothesis that a transudate of plasma is responsible for the amino acid nutrition of the embryo at this stage of gestation, and that inhibition of this pathway can lead to the induction of congenital defects. The results suggest that an inhibition of pinocytosis or lysosomal proteolysis would affect the supply of all protein-derived amino acids to approximately the same extent: there is no indication that the supply of any particular amino acid would be particularly vulnerable.

Multiple antigens in the rat visceral yolk sac induce teratogenic antisera.

Preparative isoelectric focusing was used to fractionate the supernatant from a homogenate of day 19 rat visceral yolk sac. Three fractions, of pI ranges 3.5-5.0, 5.0-7.0, and 7.0-9.0, were isolated and used to immunize rabbits, by four or six weekly injections, each containing 5 mg protein. The resulting antisera were all teratogenic when injected into rats on day 9 of gestation, but widely differing potencies were observed. The most potent antiserum was that against yolk sac components focusing in the pI 7.0-9.0 range: An optimum teratogenic dose of 50 mg protein per kg body weight was observed, and a dose of 100 mg/kg was shown to cause 100% embryonic resorption. Antiserum against the fraction focusing in the pI 3.5-5.0 range was the least teratogenic: A significant incidence of embryonic malformation and death was seen only at doses of 600 mg/kg and above. The two fractions that yielded the more teratogenic antisera were refocused over narrower pH ranges, yielding four subfractions in the pI 5.0-7.0 range and eight subfractions in the pI 7.0-9.0 range. Antisera against each of these 12 fractions were raised in rabbits; most of these antisera were shown to be teratogenic, although of differing potencies. It is concluded that the yolk sac contains many antigens that can elicit antibodies with teratogenic and yolk sac-localizing properties.

Sources of amino acids for protein synthesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac.

Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine and harvested after 24 or 48 h. Hydrolysates of the acid-precipitable fraction of embryo or yolk-sac homogenates were prepared and subjected to paper chromatography. Liquid scintillation counting of the separated amino acids showed that leucine was the only amino acid with above-background radioactivity. This established that radiolabel was not transferred from leucine to other amino acids in the cultured rat conceptus. Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine, as above. After 19 h, some conceptuses were harvested; other conceptuses were rinsed, transferred to culture medium without [3H]leucine, and after a further 24 h of culture the embryos and yolk sacs were harvested. A comparison of the protein-associated radioactivity of embryo and yolk sac before and after culturing for the further 24-h period showed that these structures quantitatively conserve radiolabelled leucine incorporated into their proteins. Further experiments involved culturing the rat conceptus for 24 h as above but in the presence of either [3H]leucine or [3H]leucine-labelled serum proteins. After harvesting the conceptuses, the specific radioactivity of [3H]leucine was determined in the acid-soluble and acid-precipitable fractions prepared from embryo and yolk-sac homogenates. The specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos or yolk sacs from conceptuses grown in the presence of radiolabelled protein was about 120 per cent of that in the culture medium, while that in the acid-precipitable fractions was about 70 per cent of that in the culture medium. By comparison, the specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos and yolk sacs from conceptuses grown in the presence of free [3H]leucine was only 3-4 per cent of that in the culture medium, while that in the acid-precipitable fraction was about 1 per cent of that in the culture medium. If our data on the fate of leucine incorporated into newly synthesized proteins of the early post-implantation rat embryo can be extrapolated to the other amino acids, they suggest that once amino acids have been incorporated into newly synthesized protein in embryo or yolk sac, they are not exchanged to any detectable extent with amino acid pools outside the conceptus. The results also provide independent confirmation of our earlier conclusion that the only significant source of amino acids utilized by the 10th-day embryo is protein taken up by the yolk sac and digested intracellularly.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Sources of amino acids for protein synthesis during early organogenesis in the rat. I. Relative contributions of free amino acids and of proteins.

Rat conceptuses on the 10th day of gestation were cultured for 27 h in whole rat serum. An addition of either [3H]leucine or [3H]leucine-labelled rat serum proteins was made once during the culture period, and the acid-soluble and acid-insoluble radioactivities of embryo and visceral yolk sac measured at harvesting. The extent of radiolabel incorporation into embryonic and yolk-sac proteins increased linearly with the duration of exposure of the conceptus to the radiolabelled leucine or radiolabelled serum proteins, indicating roughly constant rates of incorporation, per unit mass of tissue, throughout the culture period. The incorporation rates, expressed as clearances, were 0.73 and 0.78 microliter/mg tissue protein/h for embryo and yolk sac, respectively, when the source was [3H]leucine; and 1.8 and 1.3 microliters/mg tissue protein/h, for embryo and yolk sac, respectively, when the source was [3H]leucine-labelled serum proteins. It is estimated, from the known leucine and protein concentrations in serum, that protein contributed over 99 per cent of the leucine supplied to the conceptus for its protein synthesis. In parallel experiments, measurements were made on cultures conducted in the presence of an antiserum against rat visceral yolk sac (100 micrograms/ml). Antiserum profoundly inhibited incorporation of radioactivity into embryo and yolk-sac proteins, when the source was 3H-labelled protein, a result consistent with the known ability of the antiserum to inhibit pinocytosis in the yolk sac. Antiserum also decreased incorporation from [3H]leucine in the yolk sac, suggesting that a proportion of the free leucine entering the yolk sac does so by pinocytosis. The failure of antiserum to affect incorporation of [3H]leucine into the embryo probably indicates that leucine can enter the embryo without the mediation of yolk-sac pinocytosis. The primacy of protein, as a source of amino acids for the organogenesis-stage embryo, is consistent with the serious effects, in terms of embryonic death and malformation, that result from the interruption of amino acid supply when either pinocytosis or lysosomal proteolysis in the yolk sac is inhibited.

Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes.

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.

Preparation and developmental toxicity of monoclonal antibodies against rat visceral yolk sac antigens.

Thirty clones producing monoclonal antibodies (MCAs) to rat visceral yolk sac (VYS) antigens have been prepared. These MCAs localized by immunofluorescence in the VYS endoderm in vitro and were tested for developmental toxicity by intraperitioneal injection of ascites fluid into pregnant rats on day 9 of gestation. Five of the hybridomas produced MCAs that induced embryonic death, malformation, and growth retardation; the other MCAs had no developmental toxicity. Five MCAs, three teratogenic and two nonteratogenic, were tested for their ability to inhibit pinocytosis in the isolated day 17-VYS. Only the teratogenic MCAs were inhibitory, providing further evidence for the hypothesis that teratogenic antibodies interfere with the nutritional supply to the embryo.

Carrier-mediated uptake of hexoses by the rat visceral yolk sac.

The rat visceral yolk sac is shown to possess a sodium-independent, phloretin-sensitive, and phlorizin- and ouabain-insensitive transport system for hexoses. The rate of uptake of (3H)2-deoxy-D-glucose was measured in vitro and shown to be greatest on the 12th day, decreasing progressively with increasing gestational age up to the 20th day. Little uptake of 3-O-methyl-D-glucose, alpha-methylglucoside or L-glucose occurred. On uptake by the visceral yolk sac, 2-deoxy-D-glucose was phosphorylated, leading to considerable accumulation of this sugar. Several sugars inhibited 2-deoxy-D-glucose uptake as follows: D-glucose = mannose greater than fructose greater than galactose greater than xylose greater than fucose.

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)

Glucocorticoid and progesterone receptors in yolk sac placenta.

The parietal yolk sac (PYS) of the rat fetus at the 14th day of gestation contains glucocorticoid as well as progesterone receptors; both are present in the trophoblast cell layer. Following heat activation the receptors are capable of binding to deoxyribonucleic acid- (DNA-)cellulose. Glucocorticoid receptors, but not progesterone receptors, are also present in the visceral yolk sac (VYS) at the 14th day of gestation. Greater amounts (some 250 femtomoles/mg cytosol protein) of a glucocorticoid receptor are present in the VYS on the 17th day of gestation. The Kd is approximately 4 X 10(-9) M; following activation it also binds to DNA-cellulose. The elution pattern of the activated VYS receptor from diethylaminoethyl-(DEAE-)Sephadex, however, is similar to that found with kidney and colon rather than that of liver (i.e., it resembles corticosteroid binder IB rather than binder II) indicating a possible role in transport. Although the receptors are separate entities, progesterone competes as effectively as corticosterone for binding to the glucocorticoid receptors in both the PYS and and VYS, thus raising the question of the possible effect of changes in progesterone concentrations on the functioning of glucocorticoids during development.

Variation of DNA polymerase activities of rat giant trophoblast cells in mid-gestation.

The activities of the DNA polymerases alpha, beta, and gamma were determined in rat giant trophoblast cells during mid-gestation. Trophoblasts at this period of time have ceased to divide but continue to carry out DNA endoreduplication resulting in polyploidy. DNA polymerase-alpha activity in extracts was found to drop sharply from a high level at Day 11 to 1/10 of that level at Day 12 and to continue at a constant level thereafter. A similar pattern of activity was observed for polymerase-gamma, however, in this case the drop represented 50% of the activity at 11 days. Polymerase-beta showed no significant change in activity during this period of development. Endoreduplication (polyploidy) continued during this period as measured by a linear increase in chromosomal DNA content. The sharp drop in alpha-polymerase activity from Day 11 to Day 12 appears to result from the loss of a protein(s) which specifically stimulate(s) alpha-polymerase.

Materno-fetal transport of creatine in the rat.

The transfer of 14C-creatine to the rat fetus was studied following continuous i.v. infusion into the mother. In the presence of a relatively constant maternal plasma 14C-creatine concentration, creatine was accumulated by the chorioallantoic placenta and visceral yolk sac to concentrations higher than that found in maternal or fetal plasma. The ability of the extraembryonic membranes to accumulate creatine changed during gestation; nevertheless, these membranes concentrated creatine against a gradient throughout the period studied (14-22 days of gestation). Neither 14C-creatine nor 14C-urea were concentrated in the placentae or fetal plasma when compared to maternal plasma. Simultaneous infusion of beta-guanidinopropionic acid with 14C-creatine reduced both movement and accumulation of creatine into the fetoplacental unit. It is concluded that the accumulation of creatine by the chorioallantoic placenta and by the visceral yolk sac is an active process with creatine diffusing down its concentration gradient into the fetal circulation.

Morphologic alterations in the parietal yolk-sac of the rat from the 12th to the 19th day of gestation.

The rat parietal yolk-sac and its adherent epithelial cells were examined at various stages of gestation using an en face technique. Specimens were observed a both the light and electron microscopic level. Diastase pretreatment and PAS-staining were used to determine the presence of glycogen. As early as the 12th day of gestation the cytoplasm of the parietal yolk-sac cells contained numerous ribosomes and mitochondria and a large amount of endoplasmic reticulum. The glycogen content of the epithelial cells increased from the 12th day of gestation and accumulated in large quantities by the 16th day. By the 17th day many cells exhibited variable degrees of degeneration. Cellular elements of degenerating cells appeared to be trapped within Reichert's membrane. Contrary to the reports of other investigators, the present study indicates that the capsular portion of the parietal yolk-sac consisting of Reichert's membrane and its adherent epithelial cells remained intact until at least the 18th day of gestation. Some of the unique characteristics of the parietal yolk-sac provide experimental models to study the effects of environmental factors on (1) the synthesis of basement membranes, (2) the ageing of cells and (3) the correlation of these histologic changes with the functions of the parietal yolk-sac.