Metabolism in preimplantation mouse embryos

The ability of preimplantation mouse embryos to utilize glucose oxidatively is controlled, in part at least, at the level of glycolysis. Various experimental observations are reviewed that indicate the regulatory mechanism in delayed implanting blastocysts involves the classic negative allosteric feedback of high levels of ATP on phosphofructokinase while the situation in 2-cell embryos appears to be more complicated. That is, in addition to the usual negative effect of ATP and citrate on phosphofructokinase, there appears to be a modification of hexokinase that prevents phosphorylation of adequate amounts of glucose and results in low levels of fructose-6-phosphate at the 2-cell stage and consequently there is a failure to release the inhibition of phosphofructokinase even if ATP and citrate levels decrease. Although both types of embryos have limited glycolytic activity, they do have adequate capacity for citric acid cycle activity and oxidative phosphorylation, and are able to maintain a high ATP : ADP. It is argued, therefore, that the reduced levels of macromolecular synthesis characteristic of 2-cell and delayed implanting blastocysts are not due to restricted energy substrates or regulatory controls on glycolysis and a subsequent low energy state. On the contrary, it seems that the reduction in oxidative utilization of glucose in these situations is a result of diminished energy demand because of the low level of synthetic activity. The potential significance of this relationship between energy production and utilization in terms of potential regulatory mechanisms in preimplantation embryos is discussed.     

Alkaline phosphatase in preimplantation mouse embryos

This report deals with alkaline phosphatase in preimplantation mouse embryos. The enzyme activity is cytochemically demonstrated by an azo dye coupling method and biochemically determined by measuring phosphate liberated from β-glycerophosphate. The cytochemical procedure reveals alkaline phosphatase beginning suddenly in late 4-cell embryos. With the biochemical procedure, in spite of the large samples used, no activity is detected until the 8-cell stage when the activity rises abruptly, though less abruptly than the cell number. These results, which suggest the initiation of enzyme activity, are discussed and compared with those obtained by the Gomori-Takamatsu method on the same material.     

Nucleic acid synthesis in preimplantation mouse embryos

Summary Mouse embryos were collected at the 2-cell stage, cultured in vitro in the presence of³H deoxyuridine or uridine for 6 or 4 h and autoradiographed.Deoxyuridine is actively incorporated into the DNA of cleaving mouse embryos indicating the existence of thymidylate synthetase activity at least at the 4-cell stage and presumably already before this.RNAase treatment of embryos squashed on slides shows a weak but obvious incorporation of uridine into DNA of cleaving mouse embryos, from the 4-cell stage onwards; this incorporation is totally inhibited by hydroxyurea. The reduction of ribonucleotides to deoxyribonucleotides is a metabolic pathway already required for cleavage, as shown by hydroxyurea experiments.The second polar pody, known to incorporate thymidine, is unable to incorporate either deoxyuridine or uridine.     

Stage-specific insulin binding in mouse preimplantation embryos

Stage-specific insulin binding in the developing mouse embryo was demonstrated by an indirect immunofluorescence technique using an antibody against insulin. Concentration-dependent fluorescence labeling was observed in the morula and blastocyst stages of development, whereas no reactivity was seen in unfertilized oocytes or in 2-, 4-, and 8-cell embryos. The possible significance of these observations is discussed. This represents the first report of stage-specific insulin binding during mammalian preimplantation development.     

Sex-specific gene expression in preimplantation mouse embryos

The 3.5-day-old blastocyst-stage mouse embryo consists of two tissues and contains approximately 60 cells. This tiny structure has now been observed to express nearly 600 genes in a sex-specific fashion, including at least one gene (Rhox/Pem) expressed only in females from their paternal X chromosome.     

Lectin-mediated agglutination of preimplantation mouse embryos

Plant lectins were used to monitor qualitative changes in carbohydrate-containing receptors during preimplantation mouse development. Beginning at the morula stage, an age-related decline was observed in agglutination of early mouse embryos by concanavalin A (ConA). In contrast, wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) agglutinated embryos strongly throughout preimplantation development.     

DNA polymerase activity in preimplantation mouse embryos.

DNA polymerase activity was measured in mouse embryos at stages before implantation to determine whether it increases in proportion to the amount of DNA synthesis, as it does in populations of differentiated mammalian cells, or remains constant, as it does in early sea urchin embryos. Total enzyme activity was found to be relatively unchanged following fertilization and in the first few cleavage stages. However, between the 12- and 120-cell (blastocyst) stage, the amount of activity increased by several-fold. These results indicate that the relationship between amount of DNA polymerase activity and DNA synthesis in mouse embryos exhibits two phases: in the early cleavage phase it is similar to that in sea urchin embryos, whereas, in the blastocyst phase, it is similar to that in differentiated mammalian cells.     

Sequence-dependent DNA replication in preimplantation mouse embryos.

Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha-beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.     

A method of culturing preimplantation mouse embryos

A modification was proposed for the method of cultivation of preimplantation mouse embryos which does not require mineral oil and strict maintenance of CO2 content of gaseous phase.     

Decompaction and recompaction of mouse preimplantation embryos

Summary Early (non-compacted) and late (compacted) 8-cell embryos were observed after few hours of culture in vitro. The former embryos underwent compaction and the latter embryos were found decompacted. Cell counting suggested that decompaction preceded fourth cleavage division of any blastomere and lasted until the blastomeres divided.About one third of mouse morulae, which had about twenty cells, were found non-compacted upon obtaining from females. After few hours of culture in vitro these embryos underwent recompaction and cavitation. Increasing the contributions of mitosis-arrested and cytokinesisarrested cells within the morulae by culture with nocodazole and cytochalasin B respectively, did not delay recompaction.The data show that periods of decompaction and recompaction alternate in preimplantation development.     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

Glycine transport in mouse eggs and preimplantation embryos

In pre-compaction embryos glycine was taken up by the glycine-specific gly-system, which is concentrative, weakly exchangeable and dependent on Na+. After compaction glycine uptake increased, apparently due to the expression of the A-transport system and its reactivity with glycine. Studies of the metabolic fate of carbon from glycine indicated conversion to serine and alanine. These changes are interpreted to show that glycine could provide carbon for intermediary energy metabolism, resulting in CO2, as well as for macromolecular synthesis.     

Spatial patterns of gene expression in preimplantation mouse embryos

The distribution of total polyadenylated RNA and mRNAs from the beta-actin, fibronectin, and cytokeratin Endo A genes was examined in preimplantation mouse embryos using in situ hybridization of riboprobes to RNA in sections of embryos. Polyadenylated RNA was found in the cytoplasm of all cells of blastocyst-stage embryos, whereas the specific mRNAs displayed three distinct patterns of expression: uniform throughout the embryo (beta-actin), enriched in the inner cell mass (fibronectin), and enriched in the trophectoderm (Endo A). In eight-cell embryos, the polyadenylated RNA was more concentrated in nuclei than in the cytoplasm (as noted previously), although this was not the case in blastocysts, nor was it true for the specific mRNAs that were examined. These experiments demonstrate that there is localized gene expression in the early mouse embryo, which correlates with the formation of the trophectoderm and the inner cell mass.     

Cell allocation in half- and quadruple-sized preimplantation mouse embryos

The size of preimplantation mouse embryos was experimentally manipulated in order to examine the consequences for the allocation of cells to the two primary tissues, trophectoderm and inner cell mass (ICM). Half embryos were produced by the mechanical lysis of one cell at the two-cell stage and quadruple embryos by the aggregation of four whole eight-cell embryos. Such procedures are shown not only to alter the absolute number of cells that are assigned to the trophectoderm and ICM, but also to disturb significantly the proportions of these two tissues in the blastocyst. The proportion of trophectoderm is directly related to the surface area of the morula, as is predicted by a purely epigenetic scheme for cell allocation.     

Glycine and Na+ transport in preimplantation mouse embryos

The dependence of glycine uptake on glycine and Na+ concentrations was determined and compared in 2-cell embryos and blastocysts. In both, uptake was by a combination of a Na+-independent non-saturable route and a Na+-dependent saturable system with Km values of 62 microM and 274 microM for 2-cell embryos and blastocysts respectively. Na+-dependent saturable uptake by embryos of both stages had a Hill coefficient of 2 for Na+, indicating co-transport of 2 Na+ with each glycine molecule. These results suggest that glycine enters 2-cell embryos by the gly-system, and blastocysts by a similarly Na+-dependent system with characteristics resembling the A-system.