Characterization of a 280-kD protein restricted to the coated pits of the renal brush border and the epithelial cells of the yolk sac. Teratogenic effect of the specific monoclonal antibodies.

Intermicrovillar areas and apical vesicles characterized by an extensive clathrin coat can be identified in some epithelial cell types. We describe a 280-kD protein, characteristic of these areas in the proximal tubule brush border and epithelial cells of the visceral yolk sac. When injected to 9-d pregnant rats, mAbs to the 280-kD protein regularly induced fetal resorption and/or malformations. Antibodies to a 330-kD protein that is also coated-pit-restricted had no effect. Our observations point to a key function for p280 and suggest that immunity to specific constituents of the receptor-mediated endocytotic system may be involved in the induction of fetal abnormalities.

Location and identification of the collagen found in the 14.5-d rat embryo visceral yolk sac.

The collagens associated with 14.5-d rat visceral yolk sacs were localized and identified by a variety of procedures. Morphological examination showed that both the visceral epithelium and mesothelium rested upon thin basement membranes, whereas the majority of the extracellular matrix consisted of a stroma containing occasional cells and abundant banded fibrils. Immunohistochemistry at the electron microscope level showed that the basement membranes specifically cross-reacted with antibodies directed against mouse basement membrane components, whereas the stroma specifically cross-reacted with antibodies directed against rat type I collagen. Extractions of acellular visceral yolk sacs and subsequent analyses showed that type I collagen components were prevalent. Furthermore, in vitro biosynthetic studies showed only the presence of type I procollagen components (or their conversion products) and alpha-fetoprotein. These findings, taken together with our previous studies on the 14.5-d rat parietal yolk sac, provide us with protein markers for studying the origin of cells in rat parietovisceral yolk sac carcinomas.

Insulin-gene expression in extrafetal membranes of rats.

We quantified fetal rat extrapancreatic insulin-gene expression by measuring mRNA in the yolk sac and placenta. Yolk sac makes a significant contribution to the total fetal insulin stores. The placenta contains a much smaller amount of insulin mRNA. Yolk sac insulin mRNA is barely detectable at 16 days gestation but increases markedly to a maximum at 21 days, 1 day before birth. In contrast to the pancreatic 550-nucleotide (n) insulin mRNA, yolk sac has a 720-n mRNA. However, on removal of the terminal poly(A), both transcripts produce a 440-n RNA, the size predicted for a fully processed insulin mRNA.

Somatomedin inhibitors from diabetic rat serum alter growth and development of mouse embryos in culture.

Offspring of diabetic humans and laboratory animals have been shown to have a higher incidence of congenital malformations with attendant growth retardation. These defects have been attributed to alterations in the intrauterine environment and specifically to changes in maternal serum factors, e.g., glucose and ketone bodies. Our investigation examines the potential teratogenicity of a low-molecular-weight (940) serum fraction with demonstrated somatomedin inhibitory activity isolated by column chromatography from streptozocin-induced diabetic rats. Mouse embryos were exposed to control or the inhibitor fraction at concentrations of 0.25-0.6% vol/vol (0.9-3.0 micrograms protein/ml culture medium) in whole embryo culture and evaluated for the presence of malformations and growth retardation. Embryos exposed to inhibitor during the period of neurulation (3-5 somites) exhibited neural tube and craniofacial defects, whereas those exposed during early limb bud stages (18-19 somites) exhibited abnormalities of the forebrain and face. In addition, both stages were growth retarded. Control fractions produced no abnormalities. These results demonstrate a potential role for somatomedin inhibitors in diabetic embryopathy and suggest that factors other than hyperglycemia and hyperketonemia may contribute to the higher incidence of malformations among infants of diabetic mothers.

Erythroid progenitor cells and stimulating factors during murine embryonic and fetal development.

Murine embryonic and fetal yolk-sacs, peripheral blood, and livers were assayed for hemopoietic multipotential and progenitor cell content between days 6 and 13 of gestation. Multipotential cells (Mix-CFC), erythroid-committed progenitor cells (BFU-E), and nonerythroid progenitor cells (predominantly GM-CFC) were assayed by their ability to form hemopoietic colonies in vitro when stimulated by pokeweed-mitogen-stimulated spleen-cell-conditioned media (as a source of Multi-CSF) and either human or murine erythropoietin. Late erythroid progenitor cells (CFU-E) were stimulated to form colonies by erythropoietin. Mix-CFC, BFU-E, and nonerythroid cells were first detected on day 8 in yolk-sacs, day 9 in peripheral blood, and day 11 in liver. Maximum absolute numbers of yolk-sac Mix-CFC (182), BFU-E (331), and non-erythroid CFC (1358) occurred at 11 days of gestation. The maximum frequency of peripheral blood mix-CFC (24/10(5) cells) and BFU-E (55/10(5) cells) occurred at ten days of gestation. The absolute numbers of hepatic Mix-CFC, BFU-E, nonerythroid CFC, and CFU-E increased exponentially from 11 to 13 days' gestation. CFU-E were first detected at nine days in peripheral blood, at ten days in yolk-sac, and 11 days in liver and at all ages were equally responsive to erythropoietin. The maximum frequency (151/10(5) cells) of CFU-E in the peripheral blood and the maximum number per yolk-sac (1699) both occurred on day 11 of gestation. In confirmation of previous studies, yolk-sac fluid was found to contain a macrophage colony-stimulating activity. In addition, an activity capable of stimulating fetal liver CFU-E was also detected in yolk-sac fluid. However, no activity (Multi-CSF) capable of stimulating Mix-CFC or BFU-E was detected in either yolk-sac fluid or fetal plasma.

Organization of 5'-proximal c-myb exons in chicken DNA. Implications for c-myb tissue-specific transcription.

The organization of 5'-proximal c-myb exons in chicken DNA has been established by restriction enzyme mapping and nucleotide sequencing. Hybridization studies performed with cDNA probes revealed that yolk sac and thymic c-myb RNAs differ in their 5'-termini. A comparison of the genomic c-myb sequence with that of cDNAs isolated from normal thymic and lymphoma avian cells suggests that different promoter regions are used to initiate c-myb transcription in hematopoietic cells of different origins.

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

Glycogen metabolism in the rat visceral yolk sac. I. Glycogen content and gestational age.

The glycogen content of the rat visceral yolk sac was determined between 13.5 and 20.5 days of gestation by the best available colorimetric method. The concentration of glycogen in the tissue increased ten-fold between 13.5 and 18.5 days, to reach a value similar to that for mammalian muscle, but then decreased by 50 per cent between 18.5 and 20.5 days. Determination of the iodine-iodide spectra and fractionation of the glycogen particles by a novel sodium citrate centrifugation method indicated broad similarities between the structures of glycogen particles, isolated by a mild phenol-water method, from the yolk sac and the liver of the rat. However, the proportion of 'high'-molecular-weight glycogen in the yolk sac increases between 18.5 and 20.5 days, as a result of the preferential loss of 'low'-molecular-weight glycogen, so that at term the proportion approaches that found in liver glycogen.

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.

Epidermal growth factor receptors in rat placenta, amnion and yolk sac: characteristics of specific binding are dependent on gestational age.

Studies of [125I]-EGF binding to the rat placenta, amnion and yolk sac were carried out on days 14, 17 and 20 of gestation. In the placenta EGF binding was detectable on all 3 days; in the amnion EGF binding was undetectably low on day 14 but was present on days 17 and 20, while in the yolk sac EGF binding was undetectably low on all 3 days. Although Scatchard analysis of EGF binding to placental tissue raised the possibility of high and low affinity receptors, a statistical analysis of the ligand binding data was consistent with the presence of only one type of EGF receptor. The overall affinity of the receptors did not change with stage of gestation. However, the concentration of EGF receptors was lower in placental tissue on day 17 than on days 14 or 20 of gestation; the receptor concentrations were similar on days 14 and 20. It is suggested that EGF binding to the placenta, amnion, and yolk sac may reflect the levels of cell proliferation in those tissues in the latter part of gestation.

The distribution of rabbit alpha-foetoprotein (RAFP) and its molecular variants throughout gestation.

The levels of rabbit alpha-foetoprotein (RAFP) in foetal serum, neonatal serum and amniotic fluid during gestation have been evaluated by quantitative radial immunodiffusion. RAFP attained its peak concentration on the 24th day of pregnancy in foetal serum, while in amniotic fluid peaks were observed on ;the 14th and 26th day. Temporal effects on the heterogeneity of RAFP were analysed by concanavalin A affinity chromatography and agarose electrophoresis. The proportion of concanavalin A non-reactive RAFP decreased during pregnancy in foetal serum, amniotic fluid and yolk sac extracts and these findings were confirmed by crossed affino-immunoelectrophoresis. On ordinary crossed immunoelectrophoresis 14-day foetal serum migrated as a single peak, but by 24 days a second peak was evident. No similar change was detected in amniotic fluid, yolk sac or liver extract. The findings are compared with those from similar studies on other species and a model is proposed to explain the observed heterogeneity of RAFP.

Water-soluble lipoproteins from yolk granules in sea urchin eggs. II. Chemical composition.

The chemical composition of yolk lipoproteins (YLP-1, 2, and 3) was determined. YLP-1, 2, and 3 were quite similar as regards the chemical composition of lipids, proteins, and carbohydrate moieties. Each lipoprotein has an average dry weight composition of lipids (55--72%) and apo-lipoproteins (28--45%) containing protein, hexose, hexosamine, and sialic acid. In each lipoprotein, triacylglycerol is a major lipid component (70--83%), followed by phospholipid (8--16%), cholesterol (free and esterified, 8--10%), and free fatty acid (3--4%). Phosphatidylcholine and phosphatidylserine account for 68--74% and 16--24% of the phospholipids, respectively. The fatty acid compositions of total lipids from each lipoprotein are quite similar, with a high degree of unsaturation (63--65%). The carbohydrate content of apolipoprotiens from each lipoprotein is remarkably high (27--31% of apo-lipoproteins) and their composition is very simple: mannose and glucosamine are major constituents in the polysaccharide moiety of each lipoprotein and sialic acid is all in the N-glycolyl form. The amino acid compositions of apo-lipoproteins are quite similar in YLP-1, 2, and 3, with high contents of aspartic acid, glutamic acid, threonine, serine, and leucine. Furthermore, a small amount of glycolipids is present in the yolk lipoproteins. They were separated into six components on TLC. All of them are resorcinol-positive, indicating the presence of sialoglycolipids.

Transplacental pharmacokinetics and teratogenicity of a single dose of retinol (vitamin A) during organogenesis in the mouse.

Pregnant mice received 10 or 100 mg retinol/kg body wt. by gavage on day 11 of gestation (plug day = day 0). One group of animals was used for a pharmacokinetic study. At various times after dosing, plasma and tissue samples were collected and analyzed by HPLC for retinyl esters, retinol, 13-cis- and all-trans-retinoic acid and 13-cis-4-oxo and all-trans-4-oxoretinoic acid. In the other group the fetuses were removed on day 18 and examined for malformations. After 10 mg/kg retinol, no teratogenic effect was observed. The pharmacokinetic investigation revealed a moderate increase of retinyl esters, retinol and all-trans-retinoic acid in plasma, embryonic tissue, placenta, yolk sac membranes and extraembryonic fluid. A high incidence of severe fetal malformations occurred after 100 mg/kg retinol. These malformations included limb defects (81% of fetuses) and cleft palate (55% of fetuses) which are characteristically found after administration of a single teratogenic dose of an active retinoid on day 11 of gestation. The concentration-time profile of retinoids after 100 mg/kg on day 11 showed a pronounced increase of retinyl esters and retinol in all compartments including the embryo and a massive generation of the polar metabolites all-trans-retinoic acid and all-trans-4-oxoretinoic acid. These polar metabolites were found in the embryo with peak concentrations of 327 +/- 115 and 143 +/- 20.7 ng/g (mean +/- SE) wet tissue, respectively. It is likely that all-trans-retinoic acid and all-trans-4-oxoretinoic acid, both well-known teratogens, largely contributed to the teratogenic outcome. The in-vivo oxidation of retinol may be an important factor in the teratogenic activity of high doses of vitamin A.

Catecholamines in the yolk sac epithelium of the rat.

Catecholamines were found histochemically in the visceral yolk sac of the rat from embryonic day (ED) 10, i.e. before the amines become detectable in peripheral or central neurons of the fetus. Formaldehyde-induced fluorescence was confined to the apical part of the yolk sac epithelial cells. The specificity of histofluorescence has been confirmed by borohydride reduction, microspectrofluorimetry revealing an emission peak at 480 nm and administration of reserpine. The catecholamines present were identified by mass fragmentography using N,O-trifluoroacetyl derivatives. At ED 13 both dopamine and norepinephrine were present, while only dopamine was detected at ED 18 1/2. Maternal circulation or the epithelial cells themselves appear as possible sources of these catecholamines. The occurrence of amines in the yolk sac epithelium may reflect an intracellular role of these compounds, a barrier function of the epithelium or a step in a transport to the fetus where the amines might assume regulatory functions.

Morphology and chemical analysis of the sheep conceptus from the 13th to the 19th day of pregnancy.

Between the 13th and 19th day of pregnancy the sheep conceptus developed into a structure showing considerable differentiation and all the extraembryonic membranes were established. Both length and dried weight of the embryo increased exponentially during this period. A highly significant regression of dried weight on length of embryos was found but measurement of the additional variable, width, did not improve the accuracy of estimating weight from the embryo's dimensions. The mass of the extraembryonic membranes also increased greatly. The dried weight of the trophoblast increased 90-fold over this period; that of the yolk sac increased 17-fold from day 15 to day 19. The protein content of each of the structures making up the sheep conceptus approached 50% of dried weight, which is similar to the proportion in adult soft tissues. The contribution of glycogen to dried weight was low in the sheep embryo and embryonic membranes when compared with estimates in the mouse blastocyst. However, at about the time of implantation the level of this polymer in the embryo was high compared with that in adult soft tissues and approached the level found in adult muscle. Concentrations of DNA and RNA in the sheep conceptus are much higher than the levels in most adult soft tissues and probably reflect higher synthetic rates and a smaller cytoplasmic volume in the embryonic cells.

Unsaturated fatty acid compositional changes and desaturation during the embryonic development of the chicken (Gallus domesticus).

An investigation has been made to correlate the activities of the delta 9- and delta 6-long chain fatty acid desaturation systems with the increased levels of oleic and arachidonic acids in the liver relative to the yolk sac membrane of the chick embryo during the last week of development. The membrane exhibited high levels of both stearic and linoleic acid desaturation in the early stages of yolk lipid mobilization, the activities of both enzyme systems decreasing with the approach of hatching. Stearic acid desaturation in the liver also decreased with the approach of hatching, but linoleic acid desaturation increased. The observed levels of desaturation in the yolk sac membrane are capable of making a considerable contribution to the accumulations of mono- and polyunsaturated fatty acids in the embryonic liver, the requirement for which does not appear to be satisfied by the yolk lipids. With the approach of hatching and the functional regression of the yolk sac membrane, the role is taken over by the embryonic tissues.

Comparison of the accumulation of 125I and 144Ce in the growing oocytes of the Japanese quail.

Japanese quail were doubly-labeled with 125I (carrier free, as sodium iodide) and 144Ce (.015 micromol/100 g, as trichloride). by 1 hr after administration, the growing oocytes and the ova had accumulated 29% of the 125I and 21% of the 144Ce. By 18 hr, the accumulations were 30% of the 125I and 79% of the 144Ce. Lanthanum (15 micromol/100 g, as trichloride), given iv 5 min before the radionuclides, resulted in 1-hr accumulations of 4% for 125I and 23% for 144Ce. On fractionation of the oocyte yolk, 86% of the 125I present in the yolk was found in the low density fraction and 69% of the 144Ce remained with the phosvitin fraction. These results were consistent with the suggestion of Schjeide and Prahlad (1977) that, in plasma, iodide becomes associated with the calcium-containing vitellogenin complex.

Reactivity of visceral yolk sac endoderm with lectins: changes related to age and species.

The expression of receptors for lectins reacting with extraembryonal endoderm was compared between mouse and rat at different stages of gestation. Fluorescein-conjugated Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA), Sophora japonica agglutinin (SJA), peanut agglutinin (PNA), Bandeiraea simplicifolia agglutinin 1 (BSA-1) and gold-conjugated DBA and PNA were used. It was shown that the rat visceral endoderm does not express the receptors for HPA, while the mouse tissue does. Other lectins react with the visceral endoderm of both species but the reactivity disappears at different days of gestation. The control of expression of receptors for the lectins suggests that they may be involved in the recognition system important during differentiation.

Expression of c-onc genes: c-fos transcripts accumulate to high levels during development of mouse placenta, yolk sac and amnion.

The c-onc genes c-fos and c-fms are expressed at high levels specifically in mouse extra-embryonal tissues. Here, we report the results of a detailed analysis of expression of these genes within the developing placenta and extra-embryonal membranes (i.e., yolk sac and amnion). (i) The c-fos gene is expressed at relatively high, but nearly constant levels in the undissected placenta throughout gestation. (ii) The level of c-fos transcripts is greater than or equal to 15-fold higher in the separated outer portion of the midgestation placenta (primarily undifferentiated fetus-derived cytotrophoblast maternal decidua) relative to the inner moiety (predominantly differentiated syncytiotrophoblast). (iii) In the inner placenta and in the extra-embryonal membranes c-fos transcripts accumulate as gestation proceeds. The abundance of c-fos transcripts in the micro-surgically isolated 18th day amnion reaches a level which is two orders of magnitude greater than that in midgestation fetuses, and is thus close to the level of v-fos transcripts in virus-transformed cells. (iv) The distribution of c-fos transcripts within the developing extra-embryonal tissues is markedly different from that of the c-fms gene. It is suggested that the c-fos and c-fms proteins may participate in differentiation, growth or transport processes occurring in mouse extra-embryonal tissues.