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At least 19 recordsLinked to original sources

PubMed1997

Postimplantation development of mouse blastocysts with two separate inner cell masses.

Blastocysts with double inner cell masses (ICMs) were produced by electrofusion of two blastocysts and transplanted to pseudopregnant recipients. The implanted embryos were either examined histologically (8th day of pregnancy) or dissected and inspected in toto (11th or 12th day). In most cases both ICMs of experimental blastocysts developed into separate egg cylinders. Both cylinders were located in the common yolk sac cavity. Some cylinders were quite normal, but most of them were small and deprived of embryonic membranes. Ectoplacental cones of these cylinders were often oriented laterally or even antimesometrially. The development of cylinders seems to depend upon the position of their ectoplacental cones--cylinders with cones situated antimesometrially were handicapped in development. Among four sets of twin embryos examined on the 11th or 12th day, in one set each of the twins were equally developed, and in the three others one of the twins was more advanced.

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

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. II. Inner cell masses from 3.5- to 4.0-day p.c. blastocysts.

This paper describes the development in culture of inner cell masses isolated immunosurgically from C3H/He mouse blastocysts immediately after collection between 3.5 and 4.0 days p.c. By 24--48 h most of the inner cell masses isolated from half-expanded blastocysts, and about 50% of those from expanded blastocysts, regenerate an outer layer of trophectoderm-like cells and so resemble mini-blastocysts. With further in vitro culture these structures attach to the substratum and give rise to trophoblast-like giant cells, together with clusters of parietal endoderm cells or inner cell masses surrounded by visceral endoderm. Many of the inner cell masses from the remaining expanded blastocysts develop into floating structures with an outer layer of endoderm cells, and by 7 days consist of a large fluid filled cyst surrounding a collapsed vesicle of epithelial cells. Mesodermal cells line the cysts and form numerous blood islands. When mechanically disrupted, and grown as attached sheets of cells, these cystic structures give rise to patches of trophoblast-like giant cells similar to those described in the previous paper. These results suggest that the inner cell mass of normal mouse blastocysts contains cells which are capable of giving rise to trophoblast in culture.

Animals↗

PubMed2003

Metabolic characterization of the bovine blastocyst, inner cell mass, trophectoderm and blastocoel fluid.

The formation of a viable blastocyst is dependent upon the establishment of a correct inner cell mass (ICM):trophectoderm cell ratio but little is known about the metabolism of the two cell populations or about the composition of blastocoel fluid. In this study, the metabolism of intact bovine blastocysts, isolated ICM and trophectoderm was examined in terms of glucose and pyruvate uptake, lactate production, and amino acid consumption or production. The concentration of these nutrients in blastocoel fluid was also determined. The metabolism of glucose, pyruvate and lactate differed significantly between the isolated ICM and trophectoderm. Isolated trophectoderm had a higher pyruvate (P<0.001) and lower glucose (P<0.05) consumption, and higher lactate production (P<0.05) than did ICM. The consumption or production of amino acids by ICM and trophectoderm also differed, with the trophectoderm displaying a higher turnover (the sum of production and consumption). The ICM and trophectoderm both depleted arginine, aspartate and leucine, whereas the production of alanine was consistent. Isolated ICM depleted a further six amino acids, which appeared during trophectoderm culture; the reverse trend was observed for the remaining amino acids. The concentration of lactate in blastocoel fluid was significantly higher than in synthetic oviductal fluid supplemented with amino acids and BSA (SOFaaBSA; P<0.05). However, glucose (P<0.05) and pyruvate (P<0.001) concentrations were both lower. Aspartate, glutamate, glycine, alanine and tryptophan were present at significantly higher concentrations in blastocoel fluid than in SOFaaBSA, whereas threonine and asparagine concentrations were significantly lower. The metabolism of composite blastocysts, obtained by summing the consumption and production profiles of the ICM and trophectoderm, and taking into account their respective number of cells, was higher than that of intact blastocysts, indicating that upon isolation of the two cell populations there may be disruption to paracrine interactions or the onset of culture-induced cellular stress or both.

Amino Acids↗

PubMed1978

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. I. Inner cell masses from 3.5-day p.c. blastocysts incubated for 24 h before immunosurgery.

This paper describes the in vitro development of inner cell masses isolated immunosurgically from mouse blastocysts which had been collected on 3.5 days p.c. and then incubated for 24 h. The inner cell masses continue to grow in culture and develop through a series of stages with increasing complexity of internal organization. By day 1 all of the cultured ICMs have an outer layer of endoderm, and by day 3 some of them have two distinct kinds of inside cells; a columnar epithelial layer and a thin hemisphere of elongated cells. Later, mesodermal cells appear to delaminate from a limited region of the columnar layer, close to where it forms a junction with the thinner cells. By day 5, about 25% of the cultured ICMs have a striking resemblance to normal 7.5-day p.c. C3H embryos, with embryonic ectoderm, extra-embryonic ectoderm and chorion, embryonic and extra-embryonic mesoderm, and visceral endoderm. When mechanically disrupted and grown as attached clumps of cells in a tissue dish, these embryo-like structures give rise to trophoblast-like giant cells. These results suggest that the inner cell mass of 4.5-day p.c. blastocysts contains cells which can give rise to trophoblast derivates in culture.

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PubMed1995

Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts.

Inner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal-stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight-cell block.

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PubMed2005

Granulocyte-macrophage colony-stimulating factor stimulates mouse blastocyst inner cell mass development only when media lack human serum albumin.

The aim of the current study was to examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development and differentiation of preimplantation mouse embryos from different strains and under different culture conditions. Embryos from F1 hybrid mice were cultured in a modified G1 medium lacking amino acids and EDTA (simple G1), human tubal fluid medium (HTF) or in G1/G2 sequential media, supplemented with GM-CSF (0, 2, 4, 8, and 16 ng/ml). Embryos from CF1 mice were subsequently cultured in G1/G2 with (5 mg/ml) or without HSA, in the absence or presence of GM-CSF (2 ng/ml). GM-CSF had no effect at any concentration on F1 embryo development and blastocyst cell numbers, irrespective of the culture media used. Similarly, GM-CSF had no effect on CF1 blastocyst development. However, a stimulatory effect of GM-CSF was evident on total blastocyst cell number and ICM development when CF1 embryos were cultured in the absence of HSA. When HSA was present in the media the beneficial effect of GM-CSF was negated. There was no difference in the number of apoptotic cells in CF1 blastocysts when G1/G2 were supplemented with GM-CSF with or without HSA. These data indicate that there is no beneficial effect of supplementing either simple (simple G1 or HTF) or more complete (G1/G2) media with GM-CSF when protein is present in the medium. However, when culture conditions are suboptimal and non-physiological, i.e. the absence of protein, GM-CSF stimulates development of both total cell numbers and ICM development of CF1 blastocysts.

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PubMed2005

Mouse chimaeras developed from electrofused blastocysts: new evidence for developmental plasticity of the inner cell mass.

Blastocysts obtained from mice differing in pigmentation (albino versus pigmented) and the isoforms of glucose phosphate isomerase (GPI 1A versus 1B) were electrofused and those containing a single chimaeric inner cell mass (ICM) were transferred to the uterus of pseudopregnant recipients. The pups were recovered on the 20(th) day by Caesarian section and fostered by females that had littered on the previous night or 24 h earlier. Altogether nine adult animals and two pups, which died soon after delivery, were available for GPI analysis. Between 9 and 13 organs/tissues were examined and the relative contribution of the GPI 1A and 1B isoforms was estimated using an electrophoretic GPI assay. Eight adult animals were overtly chimaeric and one was chimaeric in some internal tissues only. Eight mice were males: seven were fertile, one was infertile. The ninth adult mouse was a hermaphrodite. The fertile animals produced sperm of one genotype only, i.e. derived either from the albino or from the pigmented component. This is the first report showing that adult chimaeras can be produced from two combined blastocysts, provided that fusion of the adhering trophectoderm cells is first induced and the orientation of blastocysts enables the two ICMs to integrate into a single ICM. Our results suggest that in the preimplantation blastocyst, the organisation of the ICM remains labile thus making it possible for the fused blastocysts to establish new embryonic organisation and to develop into a single organism.

Animals↗

PubMed1987

Production of ovine chimeras by inner cell mass transplantation.

Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.

Animals↗

PubMed2007

Evaluation of a laser technique to isolate the inner cell mass of murine blastocysts.

hESCs (human embryonic stem cells) are pluripotent cells derived from the ICM (inner cell mass) of blastocysts that can be used to derive several kinds of cells of the human body for the treatment of some previously untreated diseases. In considering the future use of hESCs in regenerative medicine and cell-therapy programmes, several research centres have begun projects involving the derivation of hESC lines using spare human embryos from IVF (in vitro fertilization) cycles. In some stem-cell banks, such as ours, the law also permits us to obtain these cell lines. The low availability of spare IVF human embryos, and the low rate of success in the derivation of hESC lines, give these embryos a great research value that limits experiments with new techniques. The use of murine embryos would be a good model with which to do research to discover the best methodologies to use in order to derive new hESC lines. The aim of the present study was to evaluate a new method of isolation of the ICM and derivation of ESC lines in a murine blastocyst model using laser drilling to eliminate the trophectoderm cells and compare it with the usual control method consisting of culturing the whole murine blastocyst. We also tested the adhesion and growth of primary colonies of mESCs (murine ESCs) over two different growth surfaces, namely an MEF (inactive murine fibroblastic feeder layer) or gelatin-coated dishes, in order to achieve the best culture conditions for future derivation of human stem-cell lines for application in human transplantation.

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PubMed2001

Quantitative grading of a human blastocyst: optimal inner cell mass size and shape.

OBJECTIVE: To investigate the predictive value of quantitative measurements of blastocyst morphology on subsequent implantation rates after transfer. DESIGN: Prospective observational study. SETTING: Private assisted reproductive technology center. PATIENT(S): One hundred seventy-four IVF patients receiving transfers of expanded blastocyst-stage embryos on day 5 (n = 112) or day 6 (n = 62) after oocyte retrieval. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Blastocyst diameter, number of trophectoderm cells, inner cell mass (ICM) size, ICM shape, and implantation and pregnancy rates. RESULT(S): Blastocyst diameter and trophectoderm cell numbers were unrelated to implantation rates. Day 5 expanded blastocysts with ICMs of >4,500 microm(2) implanted at a higher rate than did those with smaller ICMs (55% vs. 31%). Day 5 expanded blastocysts with slightly oval ICMs implanted at a higher rate (58%) compared with those with either rounder ICMs (7%) or more elongated ICMs (33%). Implantation rates were highest (71%) for embryos with both optimal ICM size and shape. Pregnancy rates were higher for day 5 transfers of optimally shaped ICMs compared with day 5 transfers of optimally sized ICMs. CONCLUSION(S): Quantitative measurements of the inner cell mass are highly indicative of blastocyst implantation potential. Blastocysts with relatively large and/or slightly oval ICMs are more likely to implant than other blastocysts.

Adult↗

PubMed1995

Glutamine transport by mouse inner cell masses.

Mouse blastocysts take up glutamine by specific transport systems. Glutamine is an important precursor for macromolecular synthesis and a potential alternative fuel to glucose. This study compared glutamine uptake in blastocysts and isolated inner cell masses and characterized the major participating systems in the latter. Inner cell masses take up glutamine by facilitated transport systems. The identity of these was investigated using substrate competition and kinetic studies. Na(+)-dependent uptake of 13 mumol glutamine l-1 was inhibited by 60% by 1 mmol tryptophan l-1, 25% by 1 mmol 2-amino-2-norbornanecarboxylic acid l-1 and 50% by 1 mmol lysine l-1. Furthermore, 1 mmol 2-methyl(amino)isobutyric acid (MeAIB) l-1 inhibited uptake by 29%. Kinetic analysis of MeAIB-resistant uptake revealed a predominant Na(+)-dependent facilitated uptake system with Km and Vmax values of 434 +/- 72 mumol l-1 and 237 +/- 38 fmol per inner cell mass per 10 min, respectively. The inhibition of Na(+)-dependent uptake by tryptophan, lysine and the analogue 2-amino-2-norbornanecarboxylic acid suggests that most uptake of glutamine by inner cell masses occurs via the same system that predominates in whole blastocysts, Bo,+. The period of assay was so brief that significant participation of the inner cell mass in whole blastocyst uptake was precluded showing that system Bo,+ is expressed by both the trophectoderm and inner cell mass components of the blastocyst. However, MeAIB inhibited uptake by inner cell masses but not by blastocysts. This MeAIB-sensitive uptake had a Km value of 4.3 +/- 1.7 mmol l-1 and a Vmax value of 451 +/- 119 fmol per inner cell mass per 10 min. These characteristics suggest the first embryonic appearance of system A, which is a common Na(+)-dependent transporter in many somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1993

Embryonic stem cells derived from morulae, inner cell mass, and blastocysts of mink: comparisons of their pluripotencies.

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the ICM with both Xs active; 70-100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical "cystic" mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation.

Animals↗

PubMed1979

Investigation of the fate of 4-5 day post-coitum mouse inner cell mass cells by blastocyst injection.

Two distinct patterns of chimaerism were found in conceptuses produced by injecting dissociated 4.5-day inner cell mass cells into genetically dissimilar blastocysts. Pattern 1: donor cells were found in the endoderm layer of the visceral yolk sac, but not in the adjacent mesoderm layer of this organ or in the foetus itself. Pattern 2: donor cells were found in the mesoderm layer of the visceral yolk sac and/or foetus, but never in the yolk-sac endoderm as well. Primitive endoderm cells of donor inner cell masses are responsible for the first pattern and primitive ectoderm cells for the second. These results, together with those of previous studies, suggest that the entire foetus, including its endodermal components, is formed from the primitive ectoderm, and that primitive endoderm forms only extra-embryonic endoderm of the conceptus.

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PubMed2001

Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts.

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 +/- 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 +/- 5.48 and 217.8 +/- 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.

Journal Article↗

PubMed1982

Development of preimplantation mouse embryos in vivo and in vitro.

The culture conditions for the development in vitro of (C57BL/6 x CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85% of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2: 5% CO2: 90% N2. The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

Animals↗

PubMed2001

Evaluation of mouse preimplantation embryos cultured in media enriched with insulin-like growth factors I and II, epidermal growth factor and tumor necrosis factor alpha.

Culture of preimplantation embryos is complex and requires strictly defined culture media to sustain their viability and quality. In the current study, an effort was made to evaluate comprehensively the quality of mice embryos, grown in media enriched with IGF I, IGF II, EGF and TNFalpha. For that purpose, critically chosen and thoroughly described, complex morphological methods based on contrast-phase, fluorescent and confocal microscopy were used. The study evaluated blastulation and hatching rates, total blastocyst cells, inner cell mass cell numbers (differential staining) as well as identified embryo cells with positive reactions for necrosis or apoptosis (TUNEL). The critical evaluation of the effects of the studied cytokines allowed for simultaneous, meticulous assessment of the applied study methods. Significantly more blastocysts were found in culture media enriched with IGF-I, IGF II and EGF. Significantly more hatched blastocysts were found in media with IGF-I and IGF II. Additionally, IGF I and II increased inner cell mass and total blastocyst cell numbers. Very few cells with necrosis and apoptosis were found in the culture media enriched with IGF I, IGF II and EGF. TNFalpha produced negative effects. The observed effects were dose-dependent.

Animals↗

PubMed2002

The developmental potential of the inner cell mass of blastocysts that were derived from mouse ES cells using nuclear transfer technology.

The present study examined the causes of the low developmental potential of enucleated oocytes that have received ES cells and consequent postnatal death of the young. The inner cell masses (ICM) of nuclear-transferred blastocysts or diploid blastocysts were injected into tetraploid blastocysts (group B) or nuclear-transferred tetraploid blastocysts (group C), respectively. The developmental potential of these groups was compared with tetraploid blastocysts injected with ICM of diploid blastocysts (group A). The potential of reconstituted blastocysts to develop into live young in group B increased slightly (5%) but was significantly lower than that in group A (45%). The rate of postnatal death of young in group B did not decrease. The implantation rate of reconstituted blastocysts in group C was very low and no live fetuses were obtained. The results of the present study indicate that the inferior potential of both ICM and trophectoderm cells of nuclear-transferred blastocysts underlies the low developmental rate of nuclear-transferred oocytes receiving ES cells and the higher rate of postnatal death of ES cell-derived young.

Animals↗