Preimplantation Stage Mouse Embryos Research Articles

Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos.

Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs.

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

Cdx2 AND Oct4 mRNA in pre-implantation stage mouse embryos and their single cells

In blastocysts, Oct4 is expressed in the inner cell mass and Cdx2 in the trophectoderm. We used duplex RT-LATE-PCR (Reverse Transcription- Linear After the Exponential- Polymerase Chain Reaction) to quantify levels of Cdx2 and Oct4 mRNA in cleavage-stage embryos and single blastomeres to shed light on cell lineages and differentiation.

61. Supplementation with hRad51/54 or Antibodies Against Ku70/86 Increase the Efficiency of Single Stranded DNA-Mediated Genome Alteration in Preimplantation Stage Mouse Embryos

61. Supplementation with hRad51/54 or Antibodies Against Ku70/86 Increase the Efficiency of Single Stranded DNA-Mediated Genome Alteration in Preimplantation Stage Mouse Embryos

P-246: Exposure of thawed cryopreserved human preimplantation embryos to platelet-activating factor improves cell division rate and overall quality

Platelet-activating factor (PAF) plays a significant role in fertility. Preimplantation stage embryos produce PAF (ePAF) and this ePAF is required for development. The amount of ePAF produced by human embryos is related to pregnancy outcomes. PAFs mechanism of action is a receptor-mediated event and its presence has been reported in the developing mouse and human embryo. Exposure of preimplantation stage mouse embryos will result in higher implantation rates, birth rates and birth weights. However, the effect of such treatment on human embryos is not known.

Exposure of Preimplantation Embryos to Platelet-Activating Factor Increases Birth Rate

Platelet-activating factor (PAF) plays a significant role in fertility. Preimplantation stage embryos produce PAF (ePAF) which is required for development. PAF's mechanism of action is receptor-mediated and its presence has been reported in the developing mouse and human embryo. Exposure of preimplantation stage mouse embryos results in higher implantation rates. However, the effect of such treatment on live-birth rates and birth weights has not been reported. Therefore, the objective the study was to determine the effect of exposing preimplantation mouse embryos to PAF on subsequent birth rate and weight. Two-cell stage preimplantation stage mouse embryos exposed to PAF (10(-7) M) for 15 min prior to intraoviductal transfer. Preimplantation stage embryos were recovered from eCG/hCG primed BDF1 female mice. Embryos were exposed to synthetic PAF (10(-7) M) for 15 min. PAF-treated embryos were transferred to the oviducts of pseudopregnant female CD-1 female mice. Superovulated and cultured BDF1 embryos not treated with PAF served as in vitro controls and naturally ovulated embryos with no collection/culture served as in vivo controls. Embryos were permitted to develop to term (18-21 days). The number of pups born per litter and litter weights subsequently were recorded. A total of 160 BDF1 mouse embryos were collected, treated, and transferred (20 per CD-1 recipient) as described. There was a significant (P < 0.05) increase in the number of pups born to the PAF treatment group (56/80; 70%) as compared to the control group (44/80; 55%). There was also a significant difference (P < 0.05) in litter birth weights between the PAF (1.31 g/litter) and controls groups (1.25 g/litter). There was a significant difference (P < 0.05) in birth weights between the PAF treatment group and the in vivo group (1.51 g/litter). There was a significant difference in birth weights between the in vitro-control and in vivo groups (1.51 g/litter). There were no observational malformaties to pups born in any group. Brief exposure of preimplantation stage embryos to PAF will result in a significant increase of delivery rates (pups/litter) as well as birth weights. However, the increase of birth weight was significantly below that found naturally. Additional studies are warranted to elucidate the mechanism of PAF's action in the preimplantation stage embryo and subsequent uterine development.

Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos.

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).

Increased frequencies of gene and chromosome mutations after X-irradiation in mouse embryonal carcinoma cells transfected with the bcl-2 gene.

Preimplantation stage mouse embryos are known to be highly sensitive to the killing effect of DNA damaging agents such as radiation. Interestingly, however, this stage of development is well protected from radiation induction of malformation and carcinogenesis in postnatal life. In recent years, it has become clear that the stem cells of preimplantation stage embryos undergo extensive apoptosis after DNA damage. It has been postulated that this apoptosis is likely to be responsible for the resistance to malformation, by excluding cells carrying deleterious DNA damage. We have tested the possible role of apoptosis in elimination of gene and chromosome mutations in undifferentiated mouse embryonal carcinoma cell line, F9, transfected with human bcl‐2 cDNA. The colony radiosensitivity of F9 cells was not affected by overexpression of the bcl‐2 gene, but the apoptotic cell death was suppressed, as examined by DNA ladder assay and Hoechst staining. This suppression was accompanied by an increase in the frequencies of hprt mutation and micronucleus formation after X‐irradiation. These results support the idea that maintenance of genomic integrity during early development is likely to be executed by apoptotic elimination of cells at risk.

Effect of short interspersed element sequences on the integration and expression of a reporter gene in the preimplantation-stage mouse embryos.

Based on the assumption that foreign DNA sequences may have increased chance of integration into the host genome if they are flanked by high copy-numbered genomic sequences such as SINEs (short interspersed elements), we investigated the integration frequency of Lac Z reporter gene flanked by a fused B1/B2 in an in vivo system using pronuclear microinjection technique in the mouse. The SINE-flanked DNA showed a 4-fold increased integration frequency of the reporter gene than the control DNA (63% vs. 16%). Moreover, the level of beta-galactosidase expression, estimated from the X-Gal staining intensity in transgenic embryos, was greatly higher in SINE-carrying DNA. These results suggest that the SINE sequences can serve a very useful tool in improving the efficiency of current transgenic animal technology.

Immune interferon gamma inhibits translational mobility of a plasma membrane protein in preimplantation stage mouse embryos: A T-helper 1 mechanism for immunologic reproductive failure

OBJECTIVE: Immune interferon gamma adversely affects mouse embryo development and has been proposed as a mediator of reproductive failure involving T-helper 1 immunity. We hypothesized that one mechanism by which interferon gamma could exert an adverse effect on embryos was by altering plasma membrane organization and transmembrane protein mobility. STUDY DESIGN: The fluorescence photobleaching recovery technique was used to measure the effect of the T-helper 1 cytokine interferon gamma on the translational mobility of a specific embryonic surface glycoprotein recognized by the monoclonal antibody S75. RESULTS: Compared with controls interferon gamma significantly decreased the fractional mobility of fluorescein isothiocyanate S75 in one- and two-cell mouse embryos. CONCLUSION: Interferon gamma may alter plasma membrane domains or cytoskeletal organization in early-stage embryos. By restricting plasma membrane protein mobility interferon gamma could effect T-helper 1 - mediated reproductive failure. (A M J O BSTET G YNECOL 1996;174:282-7.)

Factors affecting cellular mosaicism in the expression of a lacZ transgene in two-cell stage mouse embryos.

In the present study, we have analysed the expression pattern of a lacZ transgene (CMZ12) in preimplantation stage mouse embryos. The transgene is expressed at the two-cell stage, where it shows cellular mosaicism due to variable expressivity. The variable gene expression indicates a partial penetrance of the transgene. The extent of variation in expression is influenced by the genetic background of the oocyte. DBA/2 and CFLP genetic backgrounds promote high expression of the transgene, while Balb/c, C57BL/6, DDK, and F1 (C57BL/6 x CBA) genetic backgrounds give none or very little lacZ activity. In vitro culture of one-cell embryos to the two-cell stage induces the expression of lacZ in all strain backgrounds tested. The variation in CMZ12 expression is a transient phenomenon and does not affect later stage activity of the transgene. Nuclear transfer experiments and DNA methylation analysis suggests that a heritable modification of the transgene locus has not occurred.

Presence of the adenovirus E1A-like activity in preimplantation stage mouse embryos.

The presence of the adenovirus E1A-like activity in embryonal carcinoma stem cells has been reported. We now show that preimplantation stage mouse embryonic cells allow transcription of the E1A-dependent E2A gene when infected with E1A-deleted mutant dl312, indicating the presence of the E1A-like activity in morulae and blastocysts. Moreover, such activity seems to decrease or disappear at about the time of implantation.

Presence of the adenovirus E1A-like activity in preimplantation stage mouse embryos.

The presence of the adenovirus E1A-like activity in embryonal carcinoma stem cells has been reported. We now show that preimplantation stage mouse embryonic cells allow transcription of the E1A-dependent E2A gene when infected with E1A-deleted mutant dl312, indicating the presence of the E1A-like activity in morulae and blastocysts. Moreover, such activity seems to decrease or disappear at about the time of implantation.

Factors affecting the survival of mouse embryos cryopreserved by vitrification

Preimplantation stage mouse embryos have been used to examine the response of a simple multicellular system to cryopreservation by the complete vitrification of the suspension. Successful vitrification requires the use of a solution of cryoprotectants that is sufficiently concentrated to supercool and solidify into a glass at practicable cooling rates. Factors that influence the survival of embryos include the concentration and composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution. High rates of survival are obtained when embryos are dehydrated prior to vitrification in solutions composed of saline plus multimolar concentrations of either mixtures of permeating cryoprotectants (e.g. dimethyl sulphoxide-acetamide-propylene glycol) or single permeating cryoprotectants (propylene glycol or glycerol). Full permeation of cryoprotectants into the cells is not necessary and may lead to chemical toxicity and osmotic injury. Partial permeation and osmotic shrinkage concentrates the endogenous cytoplasmic macromolecules and greatly increases the likelihood of intracellular vitrification. Vitrification is a practical approach for embryo cryopreservation and offers new opportunities to examine fundamental aspects of cryoprotection and cryoinjury in the absence of freezing.

Comparison of polysaccharide synthesis between preimplantation stage mouse embryos and F9 embryonal carcinoma cells

Polysaccharide synthesis was compared between preimplantation stage mouse embryos and F9 embryonal carcinoma cells. When cells were labelled with [ 3H]fucose, elution profiles of the pronase digest from a Sephacryl S-200 column were similar for the two cell types and both had characteristic large polysaccharides of the same size (M r 20–500K), although embryos contained less large polysaccharide than F9 cells, and embryonic polysaccharides were less acidic, suggesting poor sialosylation. A significant proportion of [ 3H]mannose was incorporated into complex-type glycopeptides in F9 cells, whereas in the case of embryonic cells most glycopeptides remained as high mannose-type. The most striking difference was observed when cells were labelled with galactose. In the case of embryonic cells, 90 % of the radioactivity was incorporated into glycogen, whereas only 0.1 % of the incorporated galactose occurred in this molecule in F9 cells. These observations indicate that carbohydrate metabolism of morula-stage embryos is very different from that of F9 cells.

Reproduction in a population of chimeric mice: relationship of chromosomal sex to functional germ cells and proportions of chimeric components in several tissues.

When preimplantation stage mouse embryos are aggregated to form chimeras, 50% should have a single (XX/XX, XY/XY) and 50% a mixed (XX/XY) chromosomal sex. The XX/XY chimeras develop as males or females (with a preponderance of males) rather than as hermaphrodites. The purpose of this report is to provide additional and comprehensive information on sex ratios, progeny tests, karyotype analyses, and estimates of chimeric proportions in tissues from a population of chimeras derived from two inbred mouse strains. BALB/cByJ ↔ C57BL/6J chimeras were produced following aggregation of 8–12-celled embryos. Of 30 chimeras produced, 18 were males and 12 were females. From karyotype analyses (and breeding analyses of single chromosomal sex chimeras), we concluded that 7 were of single chromosomal sex (4 males, 3 females), 12 were of mixed chromosomal sex (8 males, 4 females), and 11 were of uncertain chromosomal composition (6 males, 5 females). Many in this latter category had a low proportion (or absence) of one genotype in their tissues. Our results support the contention that XX/XY females must be predominantly of the female genotype; otherwise, XX/XYs develop as males. Progeny tests revealed that 22 of 29 chimeras produced germ cells of only one genotype. No apparent germ cell reversals were discovered in the sex ratios of progeny of the XX/XY chimeras. Great fluctuations were observed in the frequencies of genotypes in successive litters of single chromosomal sex chimeras. Analyses of chimerism in white blood cells, coat colors, and the ovaries and testes revealed great variability in the proportions of the two genotypic components among and within animals.

SSEA-3: A rat monoclonal antibody against preimplantation-stage mouse embryos

SSEA-3: A rat monoclonal antibody against preimplantation-stage mouse embryos

Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1).

A monoclonal antibody derived by fusion of mouse myeloma cells with spleen cells from a mouse immunized with F9 teratocarcinoma cells is described. This antibody, which reacts with embryonal carcinoma cells of mouse and human origin and with some preimplantation stage mouse embryos, defines an embryonic stage-specific antigen. This stage-specific antigen (SSEA-1) is first detected on blastomeres of 8-cell stage embryos. Trophectodermal cells are transitorily positive; however, each cell in the inner cell mass eventually expresses this antigen.

Phytohemagglutinin-mediated blastomere aggregation and development of allophenic mice

Phytohemagglutinin (PHA) causes cells from two or more preimplantation-stage mouse embryos (after removal of the zona pellucida) to adhere rapidly and firmly upon a few minutes' exposure in vitro. Adhesion is successful at room temperature, and the cells remain agglutinated after transfer to room-temperature culture medium without PHA. The incubated aggregates continue their development. After surgical transfer to pseudopregnant recipients, aggregates comprising two different cellular genotypic populations can give rise to viable allophenic mice with both cellular genotypes. PHA treatment is thus a simple substitute for temperature-induced (37°C) aggregation of mouse blastomeres in vitro, and it provides a new and efficient tool for experimental studies of mammalian development.