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.     

An automated procedure to measure DNA synthesis in preimplantation mouse embryos

This paper describes a sensitive, reproducible, and automated procedure to measure DNA synthesis in preimplantation mouse embryos. Conditions for the DNA synthesis assay have been optimized as follows: (1) 4 μCi/ml³H-thymidine (sp. act. 20 Ci/immole); (2) a labeling period from 2 to 7 hours; (3) a 3-hour preincubation period for blastocysts and from 0 to 7 hours preincubation for 8-cell embryos; and (4) from 1 to 64 embryos per assay. The amount of DNA synthesis per embryo was found to be directly proportional to the number of cells (nuclei) per embryo. The described assay should be useful for future studies on the effect of synthetic and natural compounds on the development of preimplantation mouse embryos, as measured by perturbations in embryonic DNA synthetic activity.     

Scanning microscopy of disaggregated and aggregated preimplantation mouse embryos

Summary Blastomeres isolated from 8-and 16-cell embryos (that is 1/8 and 1/16) show a smooth surface at their point of contact with other blastomeres and a microvillous free surface. Microvilli reappear completely on the smooth surface of 52% of 1/8 embryos and partially on 88% of 1/16 embryos if cultured in vitro for 6 h. When 2-to 8-cell embryos are aggregated to 8-cell embryos and forced apart after 1–3 h, the contact surface of the 8-cell embryos has become smooth. Fixed 8-cell embryos are also able to induce complete disappearance of microvilli on the contact surface of a living 8-cell embryo. Embryos having more than 8 cells do not induce complete disappearance of microvilli on the contact surface of 8-cell embryos. Aggregates of late morulae do not show complete disappearance of microvilli at their contact surfaces but rather a loosening of their peripheral blastomeres.Our results show that isolated 1/8 and 1/16 embryos tend to recover from regionalization, that the process of aggregation of embryos having 8 cells or less is similar to compaction and that embryos having more than 8 cells seem to aggregate by cell sorting. The processes of compaction, adhesion and reassortment are briefly discussed. We submit that blastomere regionalization, which depends on cell to cell contact, may be the spatial basis of embryonic regulation and of the inside-outside normal differentiation of early mouse embryos.     

Lectin-induced blockage of developmental processes in preimplantation mouse embryos in vitro

This study attempts to assess the developmental importance of cell surface glycoconjugates of preimplantation mouse embryos. This was done by incubating early embryos in various lectins and analyzing subsequent development. If specific cell surface glycoconjugates (lectin receptors) are linked to specific developmental processes, such as cell division, compaction, and blastocyst formation, then different lectins should block these different developmental processes. The results show that wheat-germ agglutinin (WGA; N-acetyl-D-glucosamine-specific) at 50 μg/ml prevents the cell division of four-cell embryos. However, this effect of WGA occurs only in embryos with intact zonae pellucidae. Concanavalin A (Con A; α-D-glucose and α-D-mannose-specific) treatment, 20 μg/ml, of four-cell or early eight-cell embryos prevents compaction, the first major change in cell shape in early mouse embryogenesis. Divalent succinly Con A does not affect development, suggesting that the Con A effect is due to crosslinking of cell surface glycoconjugates. Exposure of four-cell or early eight-cell embryos to 10 μg/ml Lotus Tetragonolobus puprureas agglutinin (LTA; α-L-fucose-specific) or 25 μg/ml Limulus polyphemus agglutinin (LPA; sialic acid-specific) allows compaction or development to the morula stage, but blocks blastocyst formation. All lectins tested retard cell division to some extent. Late morulae and early blastocysts are more resistant than earlier stages to all of the lectins studied. This study demonstrates that very low concentrations of these lectins affect different developmental processes, presumably based upon their sugar specificities.     

Cleavage rate of diandric triploid mouse embryos during the preimplantation period.

The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one-cell-stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used 1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, 2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and 3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications

Cells subjected to the events occurring before, during, and after freezing and thawing are exposed to major changes in the osmotic pressure of the surrounding medium; i.e., the osmolalities can exceed 30. An important question in understanding the mechanisms of injury is whether cells respond as ideal osmometers to these strongly anisotonic solutions. Mouse and bovine embryos from eight-cell to blastocyst stage were used to investigate the question. They were found to behave as ideal osmometers at room temperature over a wide range of tonicities; i.e., from four times isotonic to almost 1/3 times isotonic, ideality being defined by a Boyle-van't Hoff equation. Embryo volumes increased from 40 to 200% of isotonic over this range and survivals of mouse embryos were unaffected. However, outside this range the membrane apparently becomes leaky and the survival of mouse embryos drops sharply. Osmolalities rise to high values during freezing and the paper develops the thermodynamic equations to show how computed cell volumes as a function of subzero temperature can be translated into the Boyle-van't Hoff format of cell volume as a function of the reciprocal of osmolality.     

生殖细胞及早期胚胎基因组印记的表观调控

基因组印记是一种区别父母等位基因的表观遗传过程,可导致父源和母源基因特异性表达。印记是在配子发生过程中全基因组表观重编程时获得的,且在早期胚胎发育过程中得以维持。因此,在全基因组重编程过程中,对印记的识别和维持十分重要。本文概述了原始生殖细胞的印记清除、双亲原始生殖细胞的印记获得以及早期胚胎发育过程中印记维持的相关过程,并对在印记区域内保护印记基因免受全基因组DNA去甲基化的表观遗传因子的相关作用机制进行了讨论。     

Increased survival of preimplantation mouse embryos in medium with recombinant cytokine LIF

We studied the effects of cytokine LIF on in vitro development of 2-cell mouse embryos to the late blastocyst stage. LIF at 10 ng/ml enhanced the blastocyst formation and hatching from zona pellucida. When blastocysts were cultivated in a medium with LIF for a longer time, the trophoblast adhesive properties and proliferative activity were enhanced. In the presence of this cytokine, the trophoblast cells were attached to the substrate surface and fulfill the function of a sublayer for growth of the inner cell mass colonies with a high activity of endogenous alkaline phosphatase. Expression of LIF was detected in the oviduct and uterus epithelial tissues from day 1 until day 4 of pregnancy, thus suggesting its involvement in early development. According to the data of cultivation, cytokine LIF enhanced the adhesive properties and functional activity of the trophoblast cells, which is essential for implantation of blastocysts in the uterus.     

Fate of microinjected genes in preimplantation mouse embryos.

The state of genes microinjected into mouse embryos was followed from the one-cell to the blastocyst stage using the polymerase chain reaction (PCR). Microinjected DNA was detected in all one-, two-, and four-cell injected embryos and in 44% of morula and 26% of blastocysts. Head-to-tail ligation of microinjected genes, a common feature of stably integrated transgene arrays, was detected in all embryos after injection of microinjected genes and occurred irrespective of the structure at the ends of the injected genes. Sensitivity of microinjected DNA to a methylation-dependent restriction endonuclease Dpn I was lost in all embryos by the two-cell stage (24 hr), indicating a change in DNA methylation, independent of transgene integration. Dissociation of blastomeres prior to compaction revealed a mosaic distribution of the microinjected DNA within the embryo and supports the notion that injected genes form a limited number of arrays, which segregate independently until they integrate into the genome or are degraded.     

The effects of aphidicolin and α-amanitin on DNA synthesis in preimplantation mouse embryos

The effects of aphidicolin and α-amanitin on DNA synthesis by preimplantation mouse embryos were studied. It was found that both blastocyst and 8-cell embryos showed marked inhibition of ³H-thymidine incorporation into DNA by aphidicolin at concentrations of 20–50 μg/ml. However, aphidicolin did not inhibit the conversion of morula embryos to blastocyst embryos, although aphidicolin-treated blastocysts lost their blastocoel and collapsed into a compact form after prolonged exposure to the drug. Both 8-cell and blastocyst embryos were found to be susceptible to inhibition of DNA synthesis by α-amanitin.     

Spatio-temporal localization of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos

We report for the first time the detection of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos. Cholera toxin β (CTβ), which binds to the raft-enriched ganglioside GM1, was selected to label rafts. In a novel application a Qdot reagent was used to detect CTβ labeling. This is the first reported use of nanocrystals in mammalian embryo imaging. Comparative membrane labeling with CTβ and lipophilic membrane dyes containing saturated or unsaturated aliphatic tails showed that the detection of GM1 in mouse oocytes and embryo membranes was consistent with the identification of cholesterol- and sphingolipid-enriched rafts in the cell membrane. Distribution of the GM1 was compared with the known distribution of non-raft membrane components, and disruption of membrane rafts with detergents confirmed the cholesterol dependence of GM1 on lipid raft labeling. Complementary functional studies showed that cholesterol depletion using methyl-β-cyclodextrin inhibited preimplantation development in culture. Our results show that the membranes of the mouse oocyte and zygote are rich in lipid rafts, with heterogeneous and stage-dependent distribution. In dividing embryos, the rafts were clearly associated with the cleavage furrow. At the morula stage, rafts were also apically enriched in each blastomere. In blastocysts, rafts were detectable in the trophectoderm layer, but could not be detected in the inner cell mass without prior fixation and permeabilization of the embryo. Lipid rafts and their associated proteins are, therefore, spatio-temporally positioned to a play a critical role in preimplantation developmental events.     

Effects of uranium poisoning on cultured preimplantation embryos

The toxic effect of uranium in cultured preimplantation embryos of the mouse is presented. Embryos were obtained from hybrid females CBA×C57 BL following induction of superovulation and were incubated in M16 cultured medium. Two different experiments were performed. In one, embryos in a one-cell stage were placed in culture media with final concentrations of uranyl nitrate of 104 and 208 μg/mL during 120 h in the same dish. In the other experiment, embryos in a one-cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104, and 208 μg/mL. At 24 h, those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions with uranyl nitrate were added. The percentage of embryos in two-cell stage, morula, early blastocyst, expanded blastocyst, and hatched blastocyst were recorded at 24, 72, 96 and 120 h of culture. The results obtained showed that concentrations as from 26 μg U/mL induced the delay of embryo development and the impairment of blastomere proliferation. The toxic effect of uranium increased in those experiments in which the embryos were transferred to a new medium. This embryo-culture system appears to be appropriate to evaluate the toxic effect of uranium on embryos removed from maternal influences and represents a suitable test system for environmental pollutants.     

Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation

Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.     

Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period.

The lack of a paternal genome in parthenogenetic embryos clearly limits their postimplantation development, but apparently not their preimplantation development, since morphologically normal blastocysts can be formed. The cleavage rate of these embryos during the preimplantation period gives a better indication of the influence of their genetic constitution than blastocyst formation. Conflicting results from previous studies prompted us to use a more suitable method of following the development of haploid and diploid parthenogenetic embryos during this period. Two classes of parthenogenetic embryos were analysed following the activation of oocytes in vitro with 7% ethanol: 1) single pronuclear (haploid) embryos and 2) two pronuclear (diploid) embryos. Each group was then transferred separately during the afternoon to the oviducts of recipients on the 1st day of pseudopregnancy. Control (diploid) 1-cell fertilised embryos were isolated in the morning of finding a vaginal plug, and transferred to pseudopregnant recipients at approximately the same time of the day as the parthenogenones. Embryos were isolated at various times after the HCG injection to induce ovulation, from each of the three groups studied. Total cell counts were made of each embryo, and the log mean values were plotted against time. The gradient of the lines indicated that 1) the cell doubling time of the diploid parthenogenones was 12.25 +/- 0.34 h, and was not significantly different from the value obtained for the control group (12.74 +/- 1.17 h), and that 2) the cell doubling time of the haploid parthenogenones (15.25 +/- 0.99 h) was slower than that of the diploid parthenogenones and the control diploid group.(ABSTRACT TRUNCATED AT 250 WORDS)