Karyotypic normalcy and quasi-normalcy of developmentally totipotent mouse teratocarcinoma cells

Malignant mouse teratocarcinoma cells are, in some cases, able to undergo normal, complete differentiation after injection into blastocysts. Thus far, only three lines—of unrelated origin—have been found (all in this laboratory) to be developmentally totipotent in blastocyst tests. The karyotypes of these lines, and their somatic- and germ-cell derivatives, were investigated by G-banding methods, as a possible clue to their developmental superiority. The first, OTT 6050 (129 strain), is an embryo-derived induced tumor maintained as an ascites transplant line. Its stem cells (from embryoid body “cores”) have 40 chromosomes in the modal class, which comprises two subclasses: one all normal and one with a metacentric chromosome (isochromosome-8). However, mosaic animals from injected blastocysts have only the normal subclass in their teratocarcinomaderived cells; all are of $\text{X}\text{Y}$ male sex chromosome type. Presence of the Y chromosome was verified after transmission through the germ line of two fertile mosaic males, in their F₁ male progeny. The second teratocarcinoma line, 72484-395 (LT strain), is a spontaneous ovarian solid tumor maintained by subcutaneous transplantation. Karyotypes of cells from the tumor, and also of teratocarcinoma-derived cells in mosaic animals, were normal and of $\text{X}\text{X}$ female sex chromosome type. Karyotypes of the F₁ progeny, from tumor-strain germ cells of a fertile mosaic female, were also normal. The third line, NG 2 (129 strain), is a mutant clonal in vitro line deficient in hypoxanthine phosphoribosyltransferase. It originated from an embryo-derived experimental tumor (OTT 5568) that was established in culture (PSA1 line); the culture was then mutagenized and selected for 6-thioguanine resistance. The NG 2 line proved to be quasi-normal, with only two karyotypic anomalies: trisomy of chromosome 6 and $\text{X}\text{O}$ female sex chromosome constitution. Thus, developmental totipotency in all three lines, including one maintained in vitro, is accompanied by karyotypic normalcy or near-normalcy. Other culture lines reported to be aneuploid have not yet given evidence of totipotency. Karyotypic normalcy may therefore have predictive value useful in choosing teratocarcinoma lines with relatively high developmental prospects. This is of importance in identifying those mutant lines that would be promising candidates for introduction, via blastocyst injection, of specific mutant genes into mice.     

Direct visualization, by beta-galactosidase histochemistry, of differentiated normal cells derived from malignant teratocarcinoma in allophenic mice.

Mice obtained from blastocysts injected with malignant teratocarcinoma stem cells may comprise tumor-derived cells in their tissues. Evidence for their presence has hitherto been indirect, i.e., through detection of tissue-specific products of the tumor genotype or of strain-specific enzyme variants in tissue homogenates from healthy mice. Direct visualization and identification of the tumor-derived cells would permit their normalcy and their state of differentiation to be assessed. For this purpose, a histochemical marker is required. The marker chosen was β-galactosidase (BGS), which allows high- vs low-activity cell strains to be distinguished in situ by their differences in staining intensity. BGS has previously been employed for such visualization only in brain [Dewey, M., Gervais, A., and Mintz, B. (1976). Develop. Biol.50, 68–81] and has here been shown to be applicable to other tissues, including kidney, pancreas, and salivary gland. Two unexpected results concerning the marker itself were obtained and affected its application to histochemical comparisons: BGS activity in some tissues of some inbred strains was not concordant with that of brain, on which the existing genotypic classification is based; and some cell types within a tissue varied independently in BGS levels among strains (e.g., exocrine vs endocrine pancreas). BGS visualization clearly disclosed the presence of large numbers of fully differentiated normal cells of the teratocarcinoma strain in tissues, including the Purkinje layer of the cerebellum, the kidney tubules, and the exocrine pancreas of experimental animals. In one individual, the relevant brain region was almost entirely derived from the teratocarcinoma. Yet all tissues were indistinguishable in structure and differentiation from adult controls, and showed no malignant growth. The pattern of cell-strain distribution, which was fine-grained in the brain and patchy in the other tissues named, also resembled that of ordinary allophenic mice produced from blastomere aggregates of two strains. Thus, teratocarcinoma stem cells are here seen to undergo normal histogenesis after they are successfully incorporated into a developing host embryo.     

Comparative Study on the Ability of Various Teratocarcinomas to Form Chimeric Mouse Embryos

Various embryonal carcinoma cells of different origins were compared as to the ability to form chimeric blastocysts by means of aggregating with normal 8-cell stage mouse embryos. The teratocarcinoma lines examined were OTT6050 and five newly established ones including a spontaneous testicular teratocarcinoma STT-2. The present results have revealed that distinct differences existed in the ability of colonizing blastocysts among teratocarcinomas and also among embryonal carcinoma cell lines.
Since STT-2 stem cells were found to be incorporated into blastocysts most efficiently, further development of the blastocysts were examined in utero. It was found that STT-2 stem cells could be incorporated into the fetuses up to the 7-to 28-somite stages. This is the first case to demonstrate that testicular teratocarcinoma cells with the male germ cell origin have the developmental potency to participate into mouse embryogenesis.     

Fate of Friend leukaemia cells and lymphoma cells injected into mouse blastocysts

Our aim was to verify whether differentiated murine cells grafted into mouse blastocysts would follow the same fate as teratocarcinoma cells which lose their malignant character and participate in the development of the host embryo yielding chimaeric animals. Two combinations of host embryo-cancer cells were used. Animals were examined after birth or by pregnancy interruption at various stages of development. More than 350 animals obtained from cancer-cells-injected embryos failed to show direct evidence of loss of malignancy.     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

Vitrification of in vitro produced ovine embryos at various developmental stages using two methods

This study was conducted to evaluate the effects of developmental stage of in vitro produced (IVP) ovine embryos and the type of vitrification procedure used on embryo cryotolerance.The IVP embryos were vitrified at five different developmental stages: 4-, 8- and 16-cell, morula, and blastocyst. For each stage, half of the embryos were vitrified in either 30 μl 3.4 M glycerol + 4.6 M ethylene glycol in straw (method 1) or in <0.1 μl 2.7 M ethylene glycol + 2.1 M Me₂SO + 0.5 M sucrose placed on the inner surface of a straw (method 2) of vitrification solution, based on two different procedures. After warming embryo viability was determined by assessing the rates of re-expansion, survival, and blastocyst formation. The quality of surviving embryos was evaluated by their hatching rate and blastocyst cell numbers. In both vitrification methods, embryo survival progressively increased as the developmental stage progressed. In method 1 few of the early cleavage stage embryos (4-, 8- and 16-cell) could reach to the blastocyst stage following warming. There was no significant difference in blastocyst cell numbers (total, ICM, and trophectoderm cells) or hatching rate of blastocysts derived from vitrified embryos at different developmental stages. The number of dead cells in vitrified blastocysts in method 1 was higher than for non-vitrified blastocysts (P < 0.05). The number of apoptotic cells in vitrified blastocysts was higher than for non-vitrified counterparts (P < 0.05). In conclusion, both the developmental stage of IVP ovine embryos and the method of vitrification have a significant effect on the viability and developmental competence of sheep embryos.     

Incorporation of teratocarcinoma stem cells into blastocysts by aggregation with cleavage-stage embryos

Embryonal carcinoma cells from the in vitro teratocarcinoma cell line PSA-1 were combined with normal, eight-cell stage, embryonic cells of the strain $\text{SWR}\text{J}$. The aggregates compacted and formed apparently normal blastocysts within 48 hr. Glucose phosphate isomerase (GPI) assays of the blastocysts revealed the presence of both PSA-1 and $\text{SWR}\text{J}$ GPI isozymes. Inner cell masses isolated from the blastocysts by immunosurgery expressed predominantly the PSA-1 GPI type.     

Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis

Key message

Extended antipodal life-span.

Abstract

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.     

Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor

Murine embryonic stem (ES) cells can be maintained as stem cells in vitro only in the presence of feeder cells or a soluble factor produced by a number of cell lines. We have previously demonstrated that leukemia inhibitory factor (LIF) is the molecule which prevents ES cell differentiation in culture. In this report we demonstrate that recombinant LIF can substitute for feeder cells in maintaining the full developmental potential of ES cells. The totipotent D3 ES cell line, previously isolated and maintained on growth-arrested primary embryo fibroblasts, was transferred to media supplemented with 1000 U/ml (10 ng/ml) recombinant LIF. In the presence of LIF the ES cells were maintained for over 2 months as undifferentiated cells in the absence of any feeder cells. When injected into blastocysts the ES cells which had been maintained in LIF-supplemented media efficiently formed germ-line chimeras.     

Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells

Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 with FBA (27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0. 05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Factors affecting the success rate of porcine embryo vitrification by the Open Pulled Straw method

The objectives of this study were: (1) to evaluate the influence of porcine embryo developmental stage on in vitro embryo development after vitrification, (2) to study the efficiency of the one-step dilution procedure, compared with conventional warming, for vitrified embryos at different stages of development, and (3) to determine the influence of the embryo donor on the in vitro survival of vitrified embryos at morulae and blastocyst stages. Two to four cell embryos, morulae and blastocysts were collected by laparotomy from weaned crossbred sows (n=55). Vitrification and conventional warming were performed using the OPS procedure with Superfine Open Pulled Straws (SOPS). For one-step dilution, embryos were placed in 800 microl TCM199-HEPES containing 20% of new born calf serum and 0.13 M sucrose for 5 min. To evaluate development, two to four cell embryos, morulae and blastocysts were cultured in vitro for 120, 48 and 24h, respectively. Some fresh embryos from each developmental stage were not vitrified and cultured as controls. Embryos were morphologically evaluated for their developmental capacity during the in vitro culture by stereomicroscopy. The total cell number of embryos was assessed by Hoechst-33342 staining and fluorescence microscope observation. There was a significant effect of the stage of development on the in vitro survival, perihatching rate and the number of cells of embryos after vitrification and warming (Experiment 1; p<0.001). The survival and perihatching rates of two to four cell embryos were lower than those obtained for morulae and blastocysts (p<0.001). No differences (p>0.05) in survival rates were found between vitrified and fresh blastocysts. The warming procedure did not affect the development and total cell number of vitrified two to four cell embryos, morulae or blastocysts (Experiment 2). However, donor had a significant effect (p<0.001) on the in vitro development and the number of cells of morulae and blastocysts after vitrification and warming (Experiment 3). In conclusion, the embryo developmental stage and the embryo donor were important factors that affected the development of porcine embryos after OPS-vitrification and warming. OPS-vitrification and the one-step dilution are efficient procedures to be used with intact porcine morulae and blastocysts.     

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.     

The effect on preimplantation embryo development of non-specific inflammation localized outside the reproductive tract

The aim of this study was to evaluate the possible effect of non-specific acute inflammation localized outside the reproductive tract on the quality of preimplantation embryos. In fertilized female mice two experimental models of inflammation were used—trinitrobenzene sulfonic acid colitis and carrageenan paw oedema. Inflammation was induced during the cleavage period of embryo development and embryos were collected at 92 h post hormonal synchronization. Stereomicroscopical evaluation of in vivo derived embryos showed that the presence of inflammation in the maternal body did not affect their basic developmental abilities, i.e. there were no significant differences in the proportion of early blastocysts, morulas, slowly developing embryos and degenerates between embryonic pools obtained from mothers with induced inflammation and control mothers. In the next step, non-degenerated embryos from all mothers were cultured in vitro under standard conditions for another 24 h, and the average cell number (fluorescence DNA staining) and the incidence of cell death (fluorescence viability staining combined with TUNEL assay) were evaluated. The majority of cultured embryos reached expanded blastocyst stage. There were no significant differences in the average cell numbers of blastocysts, but blastocysts derived from mothers with induced inflammation showed a significantly higher incidence of dead cells in both experiments. The majority of dead cells were of apoptotic origin. These results show that non-specific inflammation localized outside the reproductive tract has no detrimental effect on the preimplantation embryo growth; however it can affect the embryo quality.     

Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer

Nuclear transfer (NT) is associated with epigenetic reprogramming of donor cells. Expression of certain genes in these cells might facilitate their expression in the NT embryo. This research was aimed to investigate the effect of constitutive expression of OCT4 in bovine somatic cells used for NT on the developmental potential of derived cloned embryos as well as in the expression of pluripotency markers in the Day-7 resulting embryos. Cloned blastocysts were generated from five cell lines that expressed OCT4. Pools of blastocysts were screened to detect OCT4, SOX2, and NANOG by qPCR. In vitro-fertilized time-matched blastocysts were used as controls. The development potential was assessed on the basis of blastocysts rate; grading and total cell counts at Day 7. OCT4 expression in the cell lines positively correlates with blastocysts rate (r?=?0.92; p?=?0.02), number of grade I blastocysts (r?=?0.96; p?=?0.01), and total cell number (r?=?0.98; p?=?0.002). The high expression of OCT4 in the cell line did not improve the final outcome of cloning. Somatic expression of OCT4 lead to increased expression of OCT4 and SOX2 in cloned grade I blastocysts; however, there was a bigger variability in OCT4 and SOX2 (p?=?0.03; p?=?0.02) expression in the embryos generated from cells expressing highest levels of OCT4. Probably the higher variability in OCT4 expression in cloned embryos is due to incorrect reprogramming and incapability of the oocyte to correct for higher OCT4 levels. For that reason, we concluded that OCT4 expression in somatic cells is not a good prognosis marker for selecting cell lines.     

In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN₂) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN₂ has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.     

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo.

Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.