Selection of mouse preimplantation embryos carrying exogenous DNA by polymerase chain reaction

A polymerase chain reaction (PCR) system to detect transgenes in mouse preimplantation embryos was employed so that transgenic embryos could be selected before they were transferred to recipient mice. The selection system involves bisection of morulae, selection of the half-morulae containing target sequences within 7 hr, and culture and transfer of the sister half-morulae. PCR analysis of morulae derived from transgenic mice confirmed that the PCR system was reliable. However, five of 41 implanted embryos derived from PCR-positive morulae did not contain the transgenes. Also, one of 28 implanted embryos from PCR-negative morulae were transgenic. The selection system was applied to fertilized mouse eggs into which pSV2-gpt-gE1A DNA was injected. The injected DNA was detected in 30 of 84 morulae derived from the microinjected eggs. All seven implanted embryos developed from PCR-negative morulae had no detectable amount of transgenes, and one of two successfully implanted embryos from PCR-positive morulae was transgenic.     

Sex-related growth rate differences in mouse preimplantation embryos in vivo and in vitro

Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc.     

Isolation of epiblast stem cells from preimplantation mouse embryos

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.     

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.     

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 (Mr 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.     

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.     

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.     

Intercellular connections between germ cells in the developing human ovary

Summary An electron microscopic examination of human fetal ovaries reveals the presence of intercellular bridges between developing germ cells. The bridges are characterized by a band of electron-dense material beneath the lateral limiting membrane, and cell organelles are seen within the confines of these connections. Their general morphology is similar to that described in ovaries of other species. The possible functional significance of these connections is discussed.This work was supported by grants from the Edward G. Schlieder Educational Foundation, New Orleans, Louisiana State University and HD-03288 from the National Institute of Child Health and Human Development.The authors would like to thank Miss Cathy Chase for her technical assistance.     

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.     

Cell allocation to the inner cell mass and the trophectoderm in rat embryos during in vivo preimplantation development

Summary The number of trophectoderm (TE) and inner cell mass (ICM) cells was determined by complementmediated lysis and differential staining in rat embryos collected at different times during in vivo preimplantation development. At 90 h after fertilization, two groups of morulae were discriminated according to the presence or absence of detectable ICM cells, and the analysis of their total cell number indicated that acquisition of a permeability seal between TE cells begins at the 14-cell stage. On the other hand, our data confirmed that blastocoele formation occurs after the fourth cleavage division in the rat. The total cell number increased exponentially with time in blastocysts recovered between 90 h and 127 h but the cell kinetics of TE and ICM cells were different. The proportion of ICM cells consequently varied throughout blastocyst development, with a peak value for expanded blastocysts at 103 h. Finally, a linear-quadratic relationship was found between the numbers of TE and ICM cells when all the embryos with a detectable ICM were analysed together.     

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.     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

Binucleate cells in mouse morulae

Summary Mouse morulae from two strains were examined in whole mounts after dissociation of embryos into single cells and were analysed in serial sections by light and electron microscopy. One or two binucleate cells per embryo were discovered in a statistically significant number of morulae. The frequency of morulae with binucleate cell(s) was higher in older morulae than in younger ones. Binucleate cells were always the outer cells of the embryo. Their ultrastructure did not differ from the ultrastructure of mononucleate cells. It is suggested that cell binuclearity at the morula stage is a possible way to polyploidization of nuclei, resulting in the formation of primary trophoblast giant cells.     

Handling of actinomycin D by preimplantation mouse embryos

A protein isolated from serum is required in the cell suspension for the chemotactic response of normal mouse peritoneal macrophages to complement-derived C5a. Macrophage stimulating protein (MSP) has a molecular weight of 100000 and an isoelectric point of 7.0. It is resistant to changes in pH over a range of 1.3–11, is heat labile especially after partial purification and does not survive proteolytic enzyme attack. Binding to ConA Sepharose suggests that it contains a carbohydrate moiety. Its concentration in normal serum is very low and it is detectable only by virtue of a sensitive bioassay. An upper limit has been estimated at 75 ng/ml; the actual concentration may be considerably lower.     

Comparison of gene expression during preimplantation development between diploid and haploid mouse embryos

Reliable estimation and improvement of the developmental potential of in vitro production (IVP) embryos requires functional criteria of embryo quality. Antiapoptotic and mitogenic effects of insulin-like growth factor I (IGF-I), applied during bovine IVP, were studied. Day 6.5 blastocysts were fixed and processed for TUNEL to detect apoptotic cells, for immunocytochemical detection of proliferating cell nuclear antigen (PCNA), and for propidium iodide (PI) staining to detect all nuclei. Laser scanning confocal microscopy was used to determine apoptotic (TUNEL/PI) and proliferative (PCNA/PI) indices. Addition of IGF-I to the culture but not to the maturation medium increased the morula/blastocyst yield (P = 0.03), but the cleavage rate was not affected. During culture, IGF-I significantly lowered the apoptotic index by decreasing the number of apoptotic cells per embryo and elevated the total cell number of the blastocysts. The frequency of blastocysts with apoptotic cells was not affected. IGF-I increased the proportion of blastocysts with apoptotic cells in the inner cell mass area only by reducing apoptosis in the trophectoderm area. The PCNA index was not affected by IGF-I. A positive correlation observed between apoptotic and PCNA-positive cells was significant in groups stimulated with IGF-I during in vitro culture. Of TUNEL-positive cells, 30%-40% per embryo were also positive for PCNA. This colocalization may indirectly suggest an activation of DNA repair process in TUNEL-positive cells in response to DNA fragmentation. IGF-I reduces apoptosis in bovine IVP embryos. The requirement of IGF-I is more critical during embryo culture than during oocyte maturation. Our data suggest that an assay for TUNEL in conjunction with cell proliferation analysis can provide useful information about the quality of IVP embryos.     

Properties of polar and apolar cells from the 16-cell mouse morula

Summary The surface properties of newly formed, isolated 1/16 mouse blastomeres have been analyzed over the 10–12 h period prior to their division to 2/32 cells. Two populations of cells are formed at the 8- to 16-cell transition and their surface phenotypes vary with their relative position within the morula. Outer cells are polar, relatively non-adhesive and relatively large; inner cells are apolar, adhesive and smaller. The surface phenotypes of both inner and outer 1/16 cells are stable during culture for 11 h in isolation. However, the surface phenotypes can be induced to change by culture in combination with a second 1/16 cell, in a manner that is dependent upon the identity of the second cell. Two aggregated polar cells never flatten completely against each other, and both cells retain a clearly defined polar phenotype for 11–12 h. In aggregates of two apolar cells, cell outlines are lost as a result of intercellular flattening and microvilli are displaced away from areas of cell contact. However, if the two apolar cells are subsequently separated an even distribution of microvilli is restored. In most aggregates of an apolar and a polar cell, the polar cell envelops the apolar cell completely. These results are discussed in the context of the normal fate and potential of each cell type within the morula.