Radioactive labeling of proteins in cultured postimplantation mouse embryos I. Influence of the embryo preparation method

Summary Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode’s solution containing [³H]amino acids (mixture) at a concentration of 27 μCi/ml medium. The preparations were: a) yolk sac opened, placenta and blood circulation intact; (b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); c) placenta, yolk sac, and amnion removed (embryo “naked”); d) naked embryos cut randomly into pieces (Day 10 mebryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked mebryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first methods (see above) fulfilled the conditions required at the best. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic Analysis of Cell Proteins).     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Distribution of 3H in rat conceptus cultured in vitro following brief administration of [3H]thymidine

Rat embryos with intact visceral yolk sacs, explanted at 12 1/2 days of gestation, were cultured in vitro for up to 60 min in medium consisting of fetal calf serum, Eagle's MEM, and [3H]thymidine (1.2 kBq ml-1), using the roller bottle method. The total amount of 3H incorporated into the conceptus during the 60-min incubation was 79.2 Bq, and approximately 33, 23, and 44% of this activity was distributed to the embryo, the yolk sac, and the fluid in the exocoelom and amniotic cavity, respectively. The rate of 3H accumulation in conceptuses decreased with time in culture. It appeared that the decrease in the viability of the conceptus was not responsible for this phenomenon. The concentration of 3H in the yolk sac, i.e., 3H activity per gram wet weight, was 2.1 times that in the medium at the end of culture. In contrast, the 3H concentration in the embryo was significantly lower than that in the medium. These findings suggest that the visceral yolk sac of rat conceptuses may act as a barrier to the transport of tritiated thymidine between the medium and embryo.     

Synthesis of steroids in postimplantation mouse embryos cultured in vitro

Steroid and total lipid synthesis have been assessed in postimplantation stage mouse embryos cultured in vitro from the blastocyst to early somite stage. A large increase in acetate incorporation into these compounds is observed during this period. Cholesterol (60–70%), lanosterol (1–15%), and a fraction containing pregnenolone (0–5%) are the major components of the embryo-associated steroid fraction. When embryos are labeled with [³H]pregnenolone, ³H-labeled progesterone, pregnanedione, and a compound identified as acylpregnenolone are produced and secreted into the medium. Production of progesterone and pregnanedione, but not acylpregnenolone, is severely inhibited by the drug cyanoketone (1 μM). Another drug, SU-10603 (10 μM), severely inhibits pregnanedione production, with only a partial repression of progesterone synthesis, and no effect on acylpregnenolone synthesis. Neither drug affects embryonic development. When embryonic tissues were carefully separated and analyzed for their ability to metabolize [³H]pregnenolone it was observed that all tissues (embryo/yolk sac, yolk sac, and trophoblast) can produce progesterone and acylpregnenolone from pregnenolone. Only embryo/yolk sac and yolk sac, but not trophoblast tissue, can produce pregnanedione. The significance of these observations in relation to metabolic communication between the embryo and its mother is discussed.     

In Amnio MRI of Mouse Embryos

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community.     

WHOLE-EMBRYO CULTURE AND THE STUDY OF MAMMALIAN EMBRYOS DURING ORGANOGENESIS

1. Simple and reliable methods are now available for growing rat and mouse embryos in culture at all stages of organogenesis. Primitive-streak embryos can be maintained for up to 5 days in culture while they develop to early foetal stages. Older embryos are maintained for progressively shorter periods and the most advanced stage that can be supported is equivalent to the rat foetus of 15 days' gestation. 2. The rates of protein synthesis and differentiation of the younger embryos in vitro are similar, and of head-fold embryos identical, to those in vivo. After the formation of the limb buds growth is slower, with protein synthesis more retarded than differentiation, resulting in embryos or foetuses that are well formed but smaller than in vivo. This slowing of growth of the older embryos in culture is probably caused by the lack of a functional allantoic placenta. 3. The embryos of some other species, including the guinea-pig, hamster, rabbit and opossum have also been maintained in culture during organogenesis but the results are not yet as good as those for rats and mice. 4. Maximum growth of rat embryos explanted with the visceral yolk sac intact is obtained in undiluted homologous serum, though adequate growth for many studies can be maintained in mixtures of serum with chemically defined tissue-culture media. The best results are obtained in serum prepared from blood centrifuged before clotting has occurred (I.C. serum) and heat-inactivated. The importance of a high concentration of serum in the culture medium may be related to the mechanisms for uptake, transport and digestion of macromolecules by the rodent yolk sac. 5. There is no convincing evidence for a changing rate of oxygen consumption during organogenesis but there is strong evidence for changes in energy metabolism. At the beginning of organogenesis, the embryo shows a high rate of anaerobic glycolysis and of pentose-shunt activity. During the following days these decline while activity of the Krebs' cycle and electron-transport system increases. Anoxia, or exposure of the embryo to carbon monoxide, increases glycolysis and reduces growth rate. 6. The earliest stages of the formation of the heart and blood circulation can be closely observed in culture. The heart rate of the 111/2-day rat embryo is about 160 beats per minute at 38°C, and falls by about 7% per degree for lower temperatures. Several drugs that are cardioactive in the adult also affect the frequency of the heartbeat in the embryo, and the pattern of response suggests that the adrenergic receptors in the embryo develop before the cholinergic receptors. Experiments in which embryo and yolk sac were cultured separately, as well as together, have indicated that haemopoiesis can occur in the embryo only after a migration of stem cells from the yolk sac. 7. Microsurgery has been successfully applied to embryos in culture in studies on morphogenetic movements, heart development, axial rotation, limb-bud regeneration and placenta formation. Biochemical studies of normal morphogenesis have been few, but one has shown a high rate of hyaluronate synthesis by the embryo which may be related to the maintenance and expansion of extracellular spaces and the formation of the neural folds. 8. Embryos are particularly sensitive to teratogenic agents during organogenesis. Teratogens that have been studied on whole embryos in culture include trypan blue, antisera, hyperthermia, anaesthetics, and abnormal concentrations of vitamins, oxygen and glucose. Many of the malformations induced have been similar to those obtained after administration of the same agents in vivo and have demonstrated a direct teratogenic effect on the embryo independent of the maternal metabolism. It is suggested that culture methods may provide a valuable additional screening procedure for new drugs and other potentially embryopathic agents.     

Offspring from Mouse Embryos Developed Using a Simple Incubator-Free Culture System with a Deoxidizing Agent

To culture preimplantation embryos in vitro, water-jacketed CO₂ incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O₂ was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.     

Differential permeability of murine visceral yolk sac to thymidine and to hydroxyurea.

Mouse embryos at the 10–12-somite stage of development were excised from the uterus either with or without the encapsulating visceral yolk sac and were incubated in vitro in 3 × 10^?7M thymidine (methyl-T, 5 μCi/ml) for 30 min or in 4 × 10^?3M hydroxyurea for 45 min with [³H]thymidine present for the last 30 min. Radioautograms of nuclei of neural epithelium enabled an estimate of the effectiveness of the barrier imposed by the visceral yolk sac membrane to the passage of thymidine and hydroxyurea.Labeling of nuclei in the neural epithelium showed that the visceral yolk sac caused a 44% decrease in frequency and a 51% decrease in intensity of label. Hydroxyurea inhibited labeling by 15% in frequency and 37% in intensity irrespective of the presence or absence of visceral yolk sac. These results show that hydroxyurea and the presence of visceral yolk sac independently interfered with labeling of the neural epithelium by thymidine and that visceral yolk sac caused a proportionally greater retardation of label than did hydroxyurea.Nuclei of the endodermal epithelium of the intervening yolk sac, following exposure to hydroxyurea, showed a labeling decrease of 44% in frequency and 77% in intensity. The inhibitory effect of hydroxyurea on yolk sac labeling, however, did not alter yolk sac permeability to hydroxyurea. The results indicate that the visceral yolk sac, by offering no detectable barrier to hydroxyurea, permits prompt teratogenic action of hydroxyurea directly upon the embryo and suggest that the visceral yolk sac is a likely candidate to account for reports that the 8.5-day mouse embryo in situ fails to label with radioisotopic thymidine.     

Development of rat embryos cultured in serum prepared from rats with streptozotocin-induced diabetes

The effects of 40, 50, and 60 mg/kg streptozotocin (SZ) on the body weights and the glucose concentration and the osmolarity of the serum of adult rats were determined. Serum prepared from these SZ-dosed rats was used in embryo culture experiments to investigate effects of diabetic serum on rat embryos during organogenesis. The diabetic serum resulting from each of the tested doses of SZ was teratogenic to 9.5-day rat explants (embryos and their membranes), causing a range of dysmorphic lesions including craniofacial defects, heart defects, and abnormalities of the branchial arches and the otic capsules. Explants cultured in serum prepared from rats dosed with 60 mg/kg SZ also showed abnormal morphology of both the visceral yolk sac and the embryonic blood cells in the yolk sac capillaries. The development of explants repeatedly transferred between control and diabetic serum indicated that the severity of the dysmorphic effect was dependent on the duration of exposure to diabetic serum. The alternation of sera did not in itself appear to be damaging to the embryos. Explants cultured in control serum, control serum with its glucose concentration increased to that of the diabetic serum, or diabetic serum all took up the same amount of glucose from their culture medium; 30% of the embryos from the diabetic serum were abnormal compared to only 4% from the control serum and the control serum plus glucose.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

Cholinephosphophotransferase activities in early rat embryos and their associated placentas.

CDP-Choline:1,2-diglycerolcholinephosphotransferase (EC 2.7.8.2, cholinephosphotransferase) activities were determined in subcellular fractions prepared from rat embryos, placentas, or yolk sacs obtained on the fourteenth day of gestation. It was found that, in all of the tissues studied, cholinephosphotransferase activity (1) copurified with NADPH-cytochrome c reductase activity (EC 1.6.2.4), (2) was maximal around pH 8.0; (3) was stimulated by MgCl₂, exogenous diolein, and cytidine diphosphocholine (CDP-choline); and (4) was highest in homogenates of placentas, lowest in those of embryos, and intermediate in those of yolk sacs. These data substantiate, for the first time, that the early mammalian (rat) embryo, placenta, and yolk sac have the ability to synthesize phospholipids de novo.     

Visceral Endoderm Expression of Yin-Yang1 (YY1) Is Required for VEGFA Maintenance and Yolk Sac Development

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos

BACKGROUND: Proteomic analysis of cultured postimplantation rat embryos is expected to be useful for investigation into embryonic development. Here we analyzed protein expression in cultured postimplantation rat embryos by two-dimensional electrophoresis (2-DE) and mass-spectrometric protein identification. METHODS: Rat embryos were cultured from day 9.5 for 48 h or from day 10.5 for 24 h. Proteins of the embryo proper and yolk sac membrane were isolated by 2-DE and differentially analyzed with a 2-D analysis software. Selected protein spots in the 2-DE gels were identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometric analysis and protein database search. RESULTS: About 800 and 1,000 protein spots were matched through the replicate 2-DE gels each from one embryo in the embryo proper and yolk sac membrane, respectively, and virtually the same protein spots were observed irrespective to the length of culture period. From protein spots specific to the embryo proper (126 spots) and yolk sac membrane (304 spots), proteins involved in tissue-characteristic functions, such as morphogenesis and nutritional transfer, were identified: calponin, cellular retinoic acid binding protein, cofilin, myosin, and stathmin in the embryo proper, and Ash-m, dimerization cofactor of hepatocyte nuclear factor, ERM-binding phosphoprotein, cathepsin, and legumain in the yolk sac membrane. CONCLUSION: Proteomic analysis of cultured postimplantation rat embryos will be a new approach in developmental biology and toxicology at the protein level.     

Improved Development of “Rat Egg-Cylinders” <Emphasis Type="Italic">in vitro</Emphasis> as a Result of Fusion of the Heart Primordia

EMBRYOS at the stage of formation of the primitive streak (“egg-cylinders”) are highly susceptible to teratogenic agents in vivo because of the numerous complex and closely integrated events occurring at this time¹. If an in vitro system is used for the study of teratogenesis or other aspects of embryogenesis, then development should be as near normal as possible. In a study by New and Daniel of explanted rat egg-cylinders (7.5 and 8.5 days old) cultured in homologous serum, development was usually retarded by the formation of a double heart resulting from a failure of the paired heart primordia to fuse². These embryos rarely developed a yolk sac circulation and none turned (the process of axial rotation by which the embryo adopts the characteristic foetal position). Embryos with double hearts had also been obtained previously by Goss who applied pressure between the heart primordia to prevent their fusion³. New and Daniel suggested that in their embryos in vitro the failure of the heart primordia to fuse might be an effect of mechanical stresses resulting from the abnormal shape of the yolk sac. Attempts which I have made to remove these stresses by altering the shape of the yolk sac have resulted in even more irregular development or early death of the embryo. By using “true” serum⁴ or plasma instead of serum as the nutrient medium, however, more normal development in vitro has now been achieved.     

Effects of embryotrophic factors on the embryogenesis and organogenesis of mouse embryos in vitro

In order to study embryogenesis and organogenesis in vitro, two cell mouse embryos were cultured with alpha-MEM supplemented 10% FCS and embryotrophic factors (ETFs). The ETFs were separated from the conditioned medium of a SKG-II-SF cell line derived from a human uterine cervical epidermoid carcinoma. IL-1 beta, IL-6, IL-8, EGF, GH, PDGF-AB, basic FGF, VEGF were also detected in the conditioned media of this cell line. Using ETFs and a 10% FCS supplemented culture medium, 23% of the mouse two cell stage embryos developed to the bilaminar disc stage, 13% to the trilaminar germ disc stage, 9% were observed with blood islets in the yolk sac, and the heart beat was noted in 7% (28 embryos) of the embryos. Furthermore, primordial organs, such as the liver, heart, kidney, notochord, retina-like structure, etc. were observed. Usually, structures associated with the primordial streak stage (bilaminar germ disc embryo) developed in vitro using ETFs from two cell stage embryos. These closely resemble structures found at the same stage in the normal embryo in vivo. After the primordial streak stage, the cultured embryos showed no resemblance to the same stage in normal embryos. None of the external appearances of these embryos appeared normal. On the other hand, trilaminar disc stage embryos never developed when using only a 10% FCS supplemented culture system.     

Inhibition of Glycosphingolipid Biosynthesis Does Not Impair Growth or Morphogenesis of the Postimplantation Mouse Embryo

Abstract: Whole embryo culture (WEC) of organogenesis-stage mouse embryos was adapted for glycosphingolipid (GSL) metabolic studies to evaluate the hypothesis that de novo GSL biosynthesis is a prerequisite for growth and morphogenesis of the early postimplantation embryo. WEC supports the growth and development of postimplantation mouse embryos to stages that are indistinguishable from those achieved in vivo. N -Butyldeoxygalactonojirimycin ( N B-DGJ) is an N -alkylated imino sugar that specifically inhibits biosynthesis of all glucosylceramide-based GSLs. N B-DGJ inhibited glucosylceramide and lactosylceramide biosynthesis nearly completely and inhibited ganglioside biosynthesis ∼90% in both the embryo and visceral yolk sac. N B-DGJ also significantly reduced total ganglioside content in both the embryo and visceral yolk sac as estimated by the cholera toxin immunooverlay technique. A shift in expression from the structurally simple to the structurally complex gangliosides was also observed in N B-DGJ-treated embryos and yolk sacs. Despite causing major changes in GSL biosynthesis and composition, N B-DGJ had no effect on embryo viability, growth, or morphology. The findings suggest that de novo GSL biosynthesis may not be a prerequisite for the growth and morphogenesis of the organogenesis-stage mouse embryo.     

Characterization of antisera against mouse teratocarcinoma OTT6050: Molecular species recognized on embryoid bodies,preimplantation embryos,and sperm

Antisera raised in rabbits by hyperimmunization with small embryoid bodies of the transplantable teratocarcinoma OTT6050 recognize several distinct antigenic protein species on the surfaces of cells of the immunogen. Some of these antigens were found on the cells of preimplantation mouse embryos, on cells from parietal yolk sac carcinoma, and on mouse sperm. These antigens have been distinguished by polyacrylamide electrophoresis of the immune precipitates from detergent extracts of lactoperoxidase-iodinated cells. The intact embryoid bodies from the ascitic form of the OTT6050 teratocarcinoma exhibited five major protein bands (approximate MW 150K, 115K, 82K, 48K, and 12K), one band that ran at the dye front of the gels (R_f ≥1) and one minor band (approximate MW 22K). Two different rabbit antisera recognized an essentially identical pattern of antigens which, however, varied on the different cell types tested. Antisera were also elicited in syngeneic male mice using glutaraldehyde-fixed or irradiated OTT6050 embryoid bodies. The isoantisera had very poor titers in comparison to the absorbed xenoantisera, as assessed by complement-mediated cytotoxic activity against the immunizing cell types. Complement-mediated cytotoxicity could also be demonstrated using parietal yolk sac carcinoma cells, preimplantation mouse embryos from all cleavage stages, blastocysts, and immunosurgically isolated inner cell masses, as targets. The complexity of the antisera generated by intact embryoid bodies described here indicates that these structures bear multiple antigenic specificities not present on adult somatic cells, some of which are stage-specific embryonic polypeptides.     

Beta-Actin Is Involved in Modulating Erythropoiesis during Development by Fine-Tuning Gata2 Expression Levels

The functions of actin family members during development are poorly understood. To investigate the role of beta-actin in mammalian development, a beta-actin knockout mouse model was used. Homozygous beta-actin knockout mice are lethal at embryonic day (E)10.5. At E10.25 beta-actin knockout embryos are growth retarded and display a pale yolk sac and embryo proper that is suggestive of altered erythropoiesis. Here we report that lack of beta-actin resulted in a block of primitive and definitive hematopoietic development. Reduced levels of Gata2, were associated to this phenotype. Consistently, ChIP analysis revealed multiple binding sites for beta-actin in the Gata2 promoter. Gata2 mRNA levels were almost completely rescued by expression of an erythroid lineage restricted ROSA26-promotor based GATA2 transgene. As a result, erythroid differentiation was restored and the knockout embryos showed significant improvement in yolk sac and embryo vascularization. These results provide new molecular insights for a novel function of beta-actin in erythropoiesis by modulating the expression levels of Gata2 in vivo.     

Nitroxide radicals protect cultured rat embryos and yolk sacs from diabetic-induced damage

BACKGROUND: Diabetic teratogenicity relates, partly, to embryonic oxidative stress and the extent of the embryonic damage can apparently be reduced by antioxidants. We investigated the effects of superoxide dismutase-mimics nitroxides, 2,2,6,6-tetramethyl piperidine-N-oxyl (TPL) as an effective antioxidant, on diabetes-induced embryopathy. METHODS: Embryos (10.5 day old) and their yolk sacs from Sabra female rats were cultured for 28 h in the absence or in the presence of nitroxides at 0.05-0.4 mM in control, diabetic subteratogenic, or diabetic teratogenic media, and monitored for growth retardation and congenital anomalies. The oxidant/antioxidant status was examined by oxygen radical absorbance capacity and lipid peroxidation assays, whereas the yolk sac function was evaluated by endocytosis assay. RESULTS: Diabetic culture medium inhibited embryonic and yolk sac growth, induced a high rate of NTDs, reduced yolk sac endocytosis and embryonic antioxidant capacity, and increased lipid peroxidation. These effects were more prominent in the embryos with NTD compared to those without NTD. TPL added to diabetic teratogenic medium improved embryonic and yolk sac growth, reduced the rate of NTDs, and improved yolk sac function. The oxidant/antioxidant status of embryos was also improved. TPL at 1 mM did not damage the embryos cultured in control medium. CONCLUSIONS: In diabetic culture medium, oxidative damage is higher in the malformed rat embryos compared to those without anomalies; the nitroxide provides protection against diabetes-induced teratogenicity in a dose-dependent manner. The yolk sac damage, apparently caused by the same mechanism, might be an additional contributor to the embryonic damage observed in diabetes.