Isolated mouse inner cell mass is unable to reconstruct trophectoderm

The ability of ICM to differentiate into TE is still a controversial issue. Many of authors have showed the reconstruction of TE from isolated ICMs. We showed that immunosurgical method is not 100% efficient and that the original TE cells very often remain on the surface of isolated ICMs. We also found that isolated ICM cells cultured in vitro do not express Cdx2, and that the TE is reconstituted from TE cells which have survived immunosurgery. This indicates that very soon after the formation of TE in the blastocyst, the cells of ICM lose the potency to differentiate into trophectoderm.     

Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos

The developmental potential of the inner cell mass (ICM) of the cultured mouse embryo was determined by testing the ability of the ICM to regenerate trophoblast in vitro. ICM's isolated by immunosurgery from either single or chimeric embryos were able to regenerate trophoblast when they were isolated at 69 hours of culture from the 2-cell stage, but they had lost this capacity by 93 hours of culture. Trophoblast regeneration by isolated ICM's did not appear to require either a critical cell mass at the time of isolation or cell proliferation during regeneration.     

Ability of outside cells from preimplantation mouse embryos to form inner cell mass derivatives

Previous studies have shown that inside cells in the preimplantation mouse embryo do not become committed to the formation of inner cell mass until after blastocyst formation. However, it is not yet clear whether outside cells are also labile late in preimplantation development or whether they become restricted to trophectoderm development at an earlier stage. The present study investigates the potency of outside cells isolated from late morulae just prior to blastocyst formation and shows that some, if not all, outside cells retain the potential to form inner cell mass derivatives in vitro and in vivo. This suggests that trophectoderm cells are not restricted in potential earlier than ICM cells and that all cells of the early embryo may be labile at least until blastulation.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.     

Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst

Mammalian pre-implantation development culminates in the formation of the blastocyst consisting of two distinct cell lineages, approximately a third of the cells comprise the pluripotent inner cell mass (ICM) and the remainder the differentiated trophectoderm (TE). However, the contribution made by these two cell types to the overall energy metabolism of the intact blastocyst has received relatively little attention. In this study, the metabolism of the intact mouse blastocyst and isolated ICMs were determined in terms of total ATP formation (calculated from oxygen consumption and lactate formation), mitochondrial distribution and amino acid turnover to provide an indication of protein synthesis. The TE consumed significantly more oxygen, produced more ATP and contained a greater number of mitochondria than the ICM. Amino acid turnover was significantly greater (p<0.001) in the TE compared with the ICM. Specifically, there was a significant difference in the utilization of aspartate (p=0.020), glutamate (p=0.024), methionine (p=0.037), and serine (p=0.041) between the cells of the ICM and TE. These data suggest that the TE produces approximately 80% of the ATP generated and is responsible for 90% of amino acid turnover compared with the ICM. The major fate of the energy produced by the TE is likely to be the Na(+), K(+)ATPase (sodium pump enzyme) located on the TE basolateral membrane. In conclusion, the pluripotent cells of the ICM display a relatively quiescent metabolism in comparison with that of the TE.     

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.     

Do trophectoderm and inner cell mass cells in the mouse blastocyst maintain discrete lineages?

The extent to which trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst remain distinct during the period from the commencement of cavitation up until 48 h later in culture was investigated. Fluorescent latex microparticles were used to label exclusively all TE cells in nascent blastocysts and the position of labelled progeny in cultured blastocysts was examined by disaggregation, by serial sectioning and by whole-mount analyses. The results indicate that, in most blastocysts (80-90%), TE and ICM lineages are entirely separate during this period while in the remainder lineage crossing is limited usually to only one or two cells of either tissue.     

Development of isolated sheep inner cell masses/embryonic discs in vitro

Ovine inner cell masses (ICMs)/embryonic discs cultured in vitro, in conditions copying those in which mouse embryonic stem cells (ESCs) arise from mouse blastocysts, give rise to ectodermal colonies. Day 10–11 ICMs/epiblasts produce ectodermal colonies sufficiently often (55–60%) for it to be considered worthwhile trying to generate presumed ESCs from them. Younger ICMs can only be taken into account if culture conditions can be improved so that ICM/ectodermal cells are more numerous. Older embryonic discs (12–13 day) are inconvenient because of the problem of endoderm overgrowing ectoderm. Secondary cultures of ectodermal colonies form epithelial or mesenchymal cells, which can be passaged at least seven times (50 days).This study was financed by the State Council for Scientific Research (grant no 5.5701.91.02 to J.A.M) and by funds from Edison Biotechnology Institute, Ohio University     

Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses

The effects of two metabolic inhibitors, methyl palmoxirate (MP) and amino-oxyacetate (AOA), on mouse preimplantation embryo development and cell number, and inner cell mass (ICM) cell metabolism have been examined. Two-cell embryos were cultured in media supplemented with either MP, which inhibits fatty acid oxidation, or AOA, which inhibits the transamination of glutamate into α-ketoglutarate. Embryos were scored for development daily. On day 5, expanded blastocysts were differentially labeled with fluorochromes to visualize TE and ICM cell nuclei, or the ICMs isolated by immunosurgery and their energy metabolism determined using microfluorometric methods. Embryos exposed to the two inhibitors developed into fully expanded blastocysts, although cell numbers of both the TE and ICM cells were significantly reduced compared to controls. The uptake of glucose in the presence of 1 mM MP or AOA did not differ from the controls, but less glucose was accountable for by lactate production. MP significantly reduced lactate production. In the presence of 4 mM AOA, the amount of glucose oxidized and the amount of lactate formed by ICMs were significantly reduced. The results indicate that the fuels used by isolated mouse ICMs vary in response to substrate availability and that fatty acids may be a potential energy source. © 1996 Wiley-Liss, Inc.     

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12-16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.     

Rabbit blastocyst: Allocation of cells to the inner cell mass and trophectoderm

The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be a sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P<0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P<0.05), with increasing proportions of ICM cells over time (P<0.05). One-cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P<0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one-cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P<0.05). However, in vitro culture of morula-stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days, respectively, similar to those for embryos developing in vivo at each of the three recovery times. These data establish for the first time the number and proportion of cells allocated to the ICM of the rabbit embryo developing in vivo or under defined conditions in vitro. © 1995 Wiley-Liss, Inc.     

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.     

Experimental induction of two inner cell masses in mouse embryos by vinblastine treatment in vitro

Induction of artificial fission of the inner cell mass in an in vitro embryonal culture system was attempted. Mouse blastocysts were collected from uteri on day 3 of gestation and exposed to vinblastine sulfate after removal of zona pellucida. Embryos in the control group had a single inner cell mass on the trophectoderm and developed to the postblastocyst stage. On the other hand, the inner cell masses of the embryos in experimental groups subdivided into two or more. The present results, therefore, revealed that the vinblastine treatment at the blastocyst stage induced fission of the inner cell mass in mouse embryos. Further studies are planned in improved culture conditions to determine whether each inner cell mass subdivision develops into independent embryos.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.     

Glucose metabolism in the trophectoderm and inner cell mass of the rabbit embryo

The metabolism of glucose in the intact Day-6 and -7 post coitum (p.c.) rabbit blastocyst and in the separated trophectoderm and inner cell mass (ICM) of the Day-7 p.c. embryo was investigated. At Day-6 p.c., glucose traversed the trophectoderm with a half-time of 39 +/- 9.3 min, and was metabolized to CO2 at a rate of 25.5 +/- 1.6 nmol.cm-2.h-1. Neither the Na+ ionophore, amphotericin B, nor cyclic AMP had an effect on glucose metabolism to CO2. Lactate production by the Day-6 blastocyst was largely independent of glucose. At Day-7 p.c. in the intact embryo, CO2 production from glucose significantly decreased to 7.76 +/- 2.8 nmol.cm-2.h-1. Per unit surface area, the metabolism of glucose to CO2 was similar in the separated Day-7 p.c. trophectoderm and ICM. We conclude that the rabbit blastocyst is not highly dependent on glucose, and that the ICM does not utilize glucose as a metabolite to a greater extent than does the trophectoderm, at least in the Day-7 p.c. embryo.