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Results for “Inner cell mass (ICM)”

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At least 19 recordsLinked to original sources
PubMed2025

Noninvasive preimplantation genetic testing for aneuploidy using blastocyst spent culture medium may serve as a backup of trophectoderm biopsy in conventional preimplantation genetic testing.

BACKGROUND: To investigate whether the noninvasive preimplantation genetic testing (niPGT) complement conventional preimplantation genetic testing (PGT) in the embryos for aneuploidy. RESULTS: 40 spent culture medium (SCM) samples from routine embryo culture were collected, and half of each SCM (10 µL) sample was used for whole genome amplification, while the other half was stored at -80 °C for 3-6 months. Thirty-six out of 40 fresh SCM samples were successfully amplified and sequenced. Thirty-six paired frozen-thawed SCM samples showed 100% concordance with the freshly amplified SCM samples. Then, SCM and trophectoderm (TE) samples from 149 blastocysts from 51 couples were collected. A 98.0% successful SCM sample amplification rate (146/149) was achieved. For the 146 paired TE biopsy and SCM samples, the overall concordance rate was 82.9% (121/146). Ten embryos with aneuploid TE results but euploid niPGT results were donated. A 70.0% (7/10) true negative rate was achieved by niPGT with respect to the inner cell mass (ICM) results (TE-positive embryos). CONCLUSIONS: These results suggested that SCM stored at -80 °C for 6 months without affecting niPGT results based on NICSInst amplification.

Humans↗
PubMed1976

Patterns of lactic dehydrogenase isozymes in mouse embryos over the implantation period in vivo and in vitro.

Following blastocyst implantation, or outgrowth in vitro, the LDH isozyme pattern changes from that of the maternally inherited B subunit isozyme form (LDH-1) to a pattern dominated by A subunits (Auerbach & Brinster, 1967, 1968). In preimplantation embryos we have observed additional isozyme bands, as yet unidentified. An analysis of the pattern of newly synthesized LDH isozymes and specific activity of LDH in different regions of early postimplantation embryos suggests that there is a sequantial activation of A and B subunits, and that activity first appears in ICM- (inner cell mass) derived tissues and then in trophoblast-derived tissues. In vitro, in the absence of ICM cells, the transition of LDH-isozyme pattern does not occur in outgrowing trophoblast giant cells. This suggests a possible inductive interaction between ICM and trophoblast.

Animals↗
PubMed2025

Generative model for the first cell fate bifurcation in mammalian development.

The first cell fate bifurcation in mammalian development directs cells toward either the trophectoderm (TE) or inner cell mass (ICM) compartments in pre-implantation embryos. This decision is regulated by the subcellular localization of a transcriptional co-activator YAP and takes place over several progressively asynchronous cleavage divisions. As a result of this asynchrony and variable arrangement of blastomeres, reconstructing the dynamics of the TE/ICM cell specification from fixed embryos is extremely challenging. To address this, we developed a live-imaging approach and applied it to measure pairwise dynamics of nuclear YAP and its direct target genes, CDX2 and SOX2, which are key transcription factors of the TE and ICM, respectively. Using these datasets, we constructed a generative model of the first cell fate bifurcation, which reveals the time-dependent statistics of the TE and ICM cell allocation. In addition to making testable predictions for the joint dynamics of the full YAP/CDX2/SOX2 motif, the model revealed the stochastic nature of the induction timing of the key cell fate determinants and identified the features of YAP dynamics that are necessary or sufficient for this induction. Notably, temporal heterogeneity was particularly prominent for SOX2 expression among ICM cells. As heterogeneities within the ICM have been linked to the initiation of the second cell fate decision in the embryo, understanding the origins of this variability is of key significance. The presented approach reveals the dynamics of the first cell fate choice and lays the groundwork for dissecting the next cell fate decisions in mouse development.

Animals↗
PubMed1977

Ultrastructural study of concanavalin-A binding to the surface of preimplantation mouse embryos.

Receptors for Con-A were labelled (using the peroxidase-diaminobenzidine technique) on the plasma membrane of unfertilized and fertilized mouse eggs, cleavage stage embryos, trophoblast and inner cell mass (ICM) of the blastocyst. Embryos were exposed to Con-A concentrations of 10 microgram/ml, 50 microgram/ml, or 1,000 microgram/ml and the lowest concentration was observed to be the most suitable for discerning differences between stages of embryonic development. On the surface of unfertilized and fertilized eggs and 2-cell embryos, reaction product appeared as a thin, discontinuous layer. The surface of 4- and 16-cell stage embryos had a thicker, continuous, although non-uniform, layer of the reaction product. On the surface of the cells of the late morula, and on the trophoblastic cells of the blastocyst, clustering of reaction product was observed. Cells of ICM of intact blastocyst were free of the reaction product, showing that either Con-A and/or peroxidase cannot penetrate tight junctions between trophoblastic cells. Reaction product in the form of a thin, uniform layer covered the free surface of the cells of the ICM after they had been isolated (using immunosurgery) and exposed to 50 microgram/ml of Con-A. The amount and distribution of Con-A receptors is discussed, along with their redistribution and mobility in relation to the agglutinability of preimplantation mouse embryos.

Animals↗
PubMed1977

Inhibition of early postimplantation development of cultured mouse embryos by bromodeoxyuridine.

Mouse embryos were treated with 5-bromodeoxyuridine (BrdUrd) for 24 hours at various preimplantation stages to determine its effect on early postimplantation development. The inhibitory effect of BrdUrd (10(-7) to 10(-6) M) on trophoblast outgrowth and inner cell mass (ICM) development was least severe when embryos were treated at the 2-cell stage and was most severe when they were treated at the morula stage. When embryos were treated at the blastocyst stage, the inhibitory effect on trophoblast outgrowth decreased, but that on ICM development remained severe. The severity of inhibition, particularly that on ICM development, appeared to be related to decreased cell numbers in BrdUrd-treated embryos. However, increasing the cell number by aggregating two ICM's isolated from BrdUrd-treated blastocysts did not increase their chance of survival or of forming two primary germ layers. This indicates that the decrease in cell numbers alone is not the cause of the failure of BrdUrd-treated embryos to develop. The mechanism of BrdUrd inhibition was studied by adding thymidine or deoxycytidine during BrdUrd treatments. A 10-fold excess of thymidine completely protected embryos from the inhibitory effect of BrdUrd. A 10-fold excess of deoxycytidine was less effective. Autoradiography indicated that both thymidine and deoxycytidine protected embryos by interfering with the incorporation of BrdUrd into the DNA.

Animals↗
PubMed1978

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.

Animals↗
PubMed1979

Gap junctional communication in the post-implantation mouse embryo.

We studied the extent of cell-to-cell communication via junctional channels in in vitro-implanted mouse blastocysts by monitoring ionic coupling and the spread of two injected low molecular weight dyes, fluorescein and Lucifer yellow. In the early attached embryos, both trophoblasts and cells of the inner cell mass (ICM) were ionically coupled to one another. Dye injections in either trophoblasts or ICM cells resulted in spread to the entire embryo. As older and more developed embryos were examined, the spread of injected dye was progressively more limited. In the most developed embryos examined, dye injected into a cell in the ICM region resulted in spread throughout the ICM but not into the surrounding trophoblast cells, while dye injected into a trophoblast cell did not spread to any other cell in the embryo. Simultaneous monitoring of ionic coupling and dye injections in embryos of intermediate stages in this transition revealed that the trophoblast and ICM cells were ionically coupled, even across the apparent boundary where no dye was observed to pass. In the latest stage embryos examined in which no injected dye was observed to move out of the ICM, ionic coupling was still observed between the cells of the ICM and the trophoblasts. Furthermore, in the more developed embryos, dye injected into the ICM region frequently was not transferred to all the cells of the ICM, thus suggesting a further compartmentalization of due spread within the ICM. Our observations that ionic coupling is more extensive than the detectable spread of injected dyes may perhaps reflect a reduced number of junctional channels. With fewer channels less dye would pass between cells, so that, together with continuous quenching, the transfer of injected dye would not be detectable. This partial segregation of cell-to-cell communication as indicated by the limited dye spread may parallel specific differentiation processes, in particular that of giant trophoblast, embryonic ectoderm and extraembryonic endoderm differentiation.

Animals↗
PubMed1975

Long term culture of cells derived from mouse blastocysts.

The development of mouse blastocysts in primary culture has been followed for up to two months. The trophectoderm layer of the blastocyst gives rise to a monolayer of trophoblast cells; cells resembling both ectoplacental cone cells and primary giant cells are observed. The former can transform to giant cells, presumably secondary trophoblast, after several days in culture. Giant trophoblast cells are evident in the culture for much longer than the normal gestation period. Under the culture conditions described, the proportion of blastocysts showing substantial inner cell mass (ICM) proliferation in vitro is higher than that noted in previous studies. The ICM clumps develop into either egg cylinder-like structures, or, more commonly, into spherical, fluid-filled vesicles. The vesicles, which resemble yolk sac morphologically and biochemically [10, 11], continue to enlarge in size during several weeks of culture. The vesicles are attached to the underlying trophoblast monolayers by a stalk. Cells appear to migrate from this stalk out along the culture dish. The result after two to four weeks of culture is the appearance of a mixed monolayer containing a variety of different cell types. Secondary cultures of blastocyst cells have been continuously maintained in vitro for more than one year. Four lines of cells, all developing from the same pool of blastocysts, have been monitored for morphological, growth and biochemical properties, as well as chromosome number. Each line contained two or more morphologically distinct cell types, clearly indicated by cloning studies after eight months of culture. Doubling times and saturation densities among the four lines differed, as did biochemical properties. Although none of the cell lines resembled trophoblast biochemically after 7.5 months in culture, one line, MB4, possessed a number of biochemical properties in common with midgestation yolk sac. After a further five months of culture, some enzymes in the four lines were relatively unchanged; in other cases, notably with alkaline phosphatase, a sharp drop in enzyme activity was observed. One cell line, MB2, and specifically one of the cell types in this line, produced a yellow-orange pigment with a spectrum resembling that of a heme protein. After 7.5 months of culture, two of the four lines, MB21 and MB31, contained large numbers of cells with a diploid number of chromosomes. However, by 12.5 months in culture, the large majority of metaphases in all four cell lines possessed a hypotetraploid chromosome number. In a number of studies carried out to date, none of the cell lines generated tumors when injected into syngeneic hosts.

Animals↗
PubMed1978

Correlation between cytological and morphogenetic effects of acute X-irradiation of preimplantation mouse embryos cultured in vitro.

Inhibition of cavitation by acute X-irradiation of 2-cell stage eggs was due to the early killing of the cells. Eggs in which cavitation did take place had a smaller number of cells due mainly to mitotic delay, leading to lack of inner cell mass (ICM) and its derivatives in further development. A similar effect is responsible for the inhibition of ICM formation after irradiation of morulae.

Animals↗
PubMed1975

Investigation of the determinative state of the mouse inner cell mass. I. Aggregation of isolated inner cell masses with morulae.

Inner cell masses (ICMs) were dissected from 3 1/2- and 4 1/2-day blastocysts and cultured in contact with 2 1/2-day morulae. Blastocysts and morulae were homozygous for different electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Aggregation of ICMs and morulae was observed, and such aggregates were able to form blastocysts in vitro and morphologically normal foetuses in utero. GPI analysis of these conceptuses revealed that most were chimaeric. However, donor ICM-type isozyme was only detected in the embryonic and extra-embryonic fractions of the chimaeras and never in the trophoblastic fraction. Thus, ICM cells appear unable to form trophoblast derivatives even when exposed to 'outside' conditions as experienced by developing trophoblast cells. This is evidence that ICM cells, although not overtly differentiated, are determined by 3 1/2 days.

Animals↗
PubMed1975

Investigation of the determinative state of the mouse inner cell mass. II. The fate of isolated inner cell masses transferred to the oviduct.

Inner cell masses (ICMs) were dissected from 3 1/2- and 4 1/2-day mouse blastocysts and inserted into empty zonae before transfer to the oviducts of pseudopregnant mice. The ICMs survived in the oviduct for at least 2 days with little evidence of reduction in cell number. After 1 day, isolated 3 1/2-day ICMs were still viable, since they could form chimaeras when injected into 3 1/2-day blastocysts. However, no evidence of trophoblast formation could be detected in any of the isolated ICMs, despite exposure of some of their cells to 'outside' conditions. This is further evidence that ICM cells, although not overtly differentiated, are determined by 3 1/2 days. Although no trophoblast was formed, 3 1/2-day ICMs formed an outer endoderm layer after 1 day in the oviduct, as judged by light and electron microscopical evidence. It is suggested that cell position may be important in endoderm differentiation.

Animals↗
PubMed1975

Effects of bromodeoxyuridine, cytosine arabinoside and Colcemid upon in vitro development of mouse blastocysts.

Mouse blastocysts in culture have been treated with increasing concentrations of cytosine arabinoside, bromodeoxyuridine or Colcemid. Concentrations of all three antimetabolites have been found which interfere with neither hatching of the blastocysts from their zona pellucidae nor subsequent attachment of the blastocysts to the culture dish, but which eventually result in death of the inner cell mass (ICM) and its derivatives. The effect upon the ICM is selective at these antimetabolite concentrations since many or, in some cases, all trophoblast cells continue to survive, and by a number of criteria, undergo normal patterns of differentiation and development.

Animals↗
PubMed1977

Evaluation of the technique of immunosurgery for the isolation of inner cell masses from mouse blastocysts.

Inner cell masses (ICMs) immunosurgically-isolated from 31/2-day mouse blastocysts were examined for trophoblast cell contamination and developmental capacity. Blastocysts were preincubated in rabbit anti-mouse antiserum, washed thoroughly and then incubated in complement. The ICMs were then easily dissected by drawing through a fine pipette. Various experiments confirmed that the trophectoderm had been completely removed by this treatment. Firstly, the ICMs did not bind a fluorescein-conjugated antibody directed against rabbit IgG, indicating the absence of cells exposed to the rabbit antiserum during the immunosurgical procedure. Secondly, ICMs dissected from blastocysts preincubated in a suspension of melanin granules did not include any of the trophoblast cells that had phagocytosed the granules. And, thirdly, the protein synthetic profile of these ICMs was similar to microsurgically dissected ICMs, and in particular, trophoblast specific spots were absent. The developmental capacity of immunosurgically-isolated ICMs was tested by injecting them into blastocysts and transferring to the uterus of 2 1/2-day pseudopregnant recipients. Extensive chimaerism was detected in the majority of implants, 5-6 days after transfer, but only in ICM-derived tissues. This demonstrates both the lack of trophoblast cell contamination and functional viability of these ICMs.

Animals↗
PubMed1978

Localization of trophoblast-defined surface antigens during early mouse embryogenesis.

The binding pattern of a rabbit antiserum raised against mouse-ectoplacental-cone trophoblast on component cell populations in the pre-implantation and early post-implantation mouse embryo has been examined at the electron-microscope level using an immunoperoxidase-labelling technique. Binding was not detectable on the 1-cell stage, appeared at low levels at the 8-cell stage and was heavy on the trophectoderm and its trophoblast giant cell and extra-embryonic ectoderm descendants in the post-implantation embryo. In contrast, immunosurgically isolated 3 1/2-day inner cell masses (ICM) showed only slight labelling, whilst ICM derivatives in the 7 1/2-day embryo were unlabelled. The results indicate that the antiserum may be identifying a trophoblast-specific surface determinant(s), which appears with the differentiation of the trohectoderm and is maintained on some of the cell populations derived from this tissue at least until the early post-implantation stages.

Animals↗
PubMed1978

Isolation and development of the inner cell mass after exposure of mouse embryos to calcium ionophore A23187.

Compacted morulae and blastocysts were obtained from CBA, BALB/c and CFLP strains of mice. The embryos were incubated in medium containing 2 X 10(-5) M or 2 X 10(-6) M ionophore A23187. With 2 X 10(-6) M ionophore, morulae survived for up to 12 h showing slight decompaction. Normal development resumed when the morulae were explanted to fresh medium. There was no detectable effect on blastocysts. With 2 X 10(-5) M ionophore, morulae survived for about 20 min and then extensive cell death occurred after this time. With blastocysts however, selective lysis of trophectoderm cells occurred after approximately 30 min following their swelling and vesiculation but the inner cell mass cells (ICM) remained apparently intact and viable. Nearly 80% of the early blastocysts obtained 87 h post-ovulation and all of the late blastocysts used after 12 h in culture (99 h blastocysts) showed this response. Individual fluid accumulating cells were detected in a few isolated ICMs after their overnight culture in vitro, especially in those obtained from early blastocysts, but the majority of the ICMs did not have these cells. All aggregates of three to five ICMs, except one which reformed into a blastocyst, developed as embryoid bodies after 2 days in culture and these survived for up to 10 days; in some cases they developed into cystic embryoid bodies or attached to the culture dish displaying a variety of cell types. The development of the isolated ICMs in vivo was judged to be normal after their transfer to intact host blastocysts as these developed as chimaeric embryos to term.

Animals↗
PubMed1978

Time of commitment of inside cells isolated from preimplantation mouse embryos.

Groups of inside cells (ICs) and inner cell masses (ICMs) were isolated from individual mouse embryos between the late morula and 3 1/2-day expanded blastocyst stages using a modified immunosurgical procedure, and their purity and developmental potential were assessed in vitro. Several different techniques failed to detect the presence of viable contaminating outside cells on ICs isolated from any of the stages studied. The numbers of inside cells isolated from the earlier stages, counted in air-dried preparations, were considerably higher than previous estimates from serial sections; whereas the numbers isolated from expanded blastocysts were in reasonable agreement. Thus the proportion of inside cells recovered by immunosurgery decreases over this period of development. In view of the evidence that inside cells divide at a faster rate than outside cells at these stages, it is argued that there may be an outward movement of inside cells capable of forming trophectoderm, during expansion of the blastocyst. ICs and ICMs in vitro were observed to develop in one of two distinct ways according to the stage at which they were isolated. ICs from late morulae and some early cavitating blastocysts formed blastocyst-like vesicles over a period of 24--36 h in culture. The presence of trophectoderm cells in these vesicles was confirmed by the persistence of giant cells after ectopic transfer. In contrast ICs from a minority of early cavitating blastocysts, and all ICMs from 3 1/2-day expanded blastocysts did not form vesicles, but proliferated endoderm-like cells. Thus at least some inside cells do not appear to lose the capacity to form trophectoderm and do not become committed to an ICM fate until after the initial formation of the blastocoel cavity.

Animals↗
PubMed1979

Investigation of the lethal yellow Ay/Ay embryo using mouse chimaeras.

Chimaeric combinations of normal and mutant embryonic tissues were used to investigate the lethal effect of the yellow gene. The homozygous mutant embryos could not be identified before implantation. Therefore, embryos from both intercross matings and control backcross matings were used to provide inner cell masses (ICMs) for injection into genetically marked blastocysts of the CFLP random bred stock. All conceptuses obtained from reimplanted blastocysts were analysed at mid-gestation for the presence of donor isozyme of glucose phosphate isomerase. A similar proportion of chimaeras were found in the experimental and control series, indicating rescue of the lethal Ay/Ay ICM tissue. The reciprocal experiment also produced a similar proportion of chimaeras but there was a 25% postimplantational loss of injected embryos evidenced by empty decidual swellings. The results suggest that the yellow mutation primarily affects the trophectoderm which cannot be rescued by a normal ICM, whereas Ay/Ay ICM is capable of survival in a chimaera at least until mid-gestation.

Animals↗
PubMed1979

The effect of prolonged decompaction on the development of the preimplantation mouse embryo.

A rabbit antiserum to a mouse embryonal carcinoma cell line blocks compaction of cleaving mouse embryos. Cell division is not affected up to the 32-cell stage but intracellular junctions fail to develop. Removal of the antibody at this stage permits compaction to occur and a normal blastocyst develops. Prolonged decompaction beyond the 32-cell embryo results in an increasing proportion of malformed blastocysts in which trophectodermal cells predominate and functional inner cell mass (ICM) cells are reduced or absent. The relationship of compaction to the generation of ICM and trophectoderm lineages in the intact embryo is discussed.

Animals↗