Investigation of the determinative state of the mouse inner cell mass

Development ◽  
1975 ◽  
Vol 33 (4) ◽  
pp. 979-990
Author(s):  
J. Rossant
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
In Utero ◽  
Glucose Phosphate Isomerase ◽  
Trophoblast Cells ◽  
Electrophoretic Variants ◽  
Glucose Phosphate

Inner cell masses (ICMs) were dissected from 3½- and 4½-day blastocysts and cultured in contact with 2½-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 3½ days.

scholarly journals Origin of the ectoplacental cone and secondary giant cells in mouse blastocysts reconstituted from isolated trophoblast and inner cell mass

Development ◽  
1973 ◽  
Vol 30 (3) ◽  
pp. 561-572
Author(s):  
R. L. Gardner ◽  
V. E. Papaioannou ◽  
S. C. Barton
Keyword(s):  
Cell Transformation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Glucose Phosphate Isomerase ◽  
Electrophoretic Variants ◽  
Decidual Tissue ◽  
Glucose Phosphate ◽  
Donor Type ◽  
Ectoplacental Cone

1. Inner cell mass (ICM) and trophoblast tissue were isolated from 3½-day post-coitum mouse blastocysts that were homozygous for different electrophoretic variants of the enzyme glucose phosphate isomerase (GPI). Blastocysts were reconstituted from these tissues, transferred to pseudo-pregnant recipients and allowed to develop to the early somite stage. 2. The embryo plus membranes and trophoblast were dissected and typed separately for GPI. 3. Contamination of trophoblast with maternal decidual tissue was quantified. 4. The trophoblast of the implanted embryos was almost exclusively of the trophoblastdonor GPI type. The embryos plus membranes were mainly of the ICM-donor type but most also showed a substantial proportion of trophoblast-donor type. 5. It is argued that the ICM controls trophoblast proliferation by inhibiting giant cell transformation of adjacent trophoblast cells rather than through making a significant cellular contribution.

Ratio and number of inner cell mass and trophoblast cells of in vitro and in vivo produced porcine embryos

1995 ◽  
Vol 43 (1) ◽  
pp. 304 ◽  
Author(s):  
D. Rath ◽  
H. Niemann ◽  
T. Tao ◽  
M. Boerjan
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells

CSF-1 and mouse preimplantation development in vitro

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1333-1339 ◽  
Author(s):  
P. Bhatnagar ◽  
V.E. Papaioannou ◽  
J.D. Biggers
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Preimplantation Development ◽  
High Rate ◽  
Colony Stimulating Factor ◽  
Trophoblast Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The effects of macrophage colony stimulating factor on the development of the zygote to the blastocyst stage of an outbred strain of mouse have been studied in KSOM, an improved medium that supports a high rate of in vitro development. Macrophage colony stimulating factor accelerates the formation of the blastocyst cavity by day 4 (96 hours post-hCG). It also increases overall embryonic cell number through a differential increase in the number of trophoblast cells, with no significant effect on the number of inner cell mass cells. By day 5 of culture (120 hours post-hCG), colony stimulating factor-treated embryos have about 20 more trophoblast cells than control embryos, an increase of about 30 percent of the total number of cells in a control blastocyst. The maximum response of embryos was obtained at a concentration around 540 U ml-1 colony stimulating factor (identical to 918 Stanley units ml-1), and the cytokine can produce the same effects even if it is present in the medium for only part of the culture period. This in vitro stimulation of preimplantation development with macrophage colony stimulating factor is compatible with continued normal fetal development in vivo.

157 EFFECT OF MELATONIN ON EMBRYO QUALITY OF BOVINE OOCYTES SUBJECTED TO HEAT SHOCK

2016 ◽  
Vol 28 (2) ◽  
pp. 208
Author(s):  
N. G. Alves ◽  
I. J. Ascari ◽  
L. S. A. Camargo ◽  
J. Jasmin ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
Embryo Quality ◽  
Linear Models ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Quality Data ◽  
Trophoblast Cells ◽  
Melatonin Concentration

The aim of this study was to evaluate the effect of different concentrations of melatonin added to in vitro maturation (IVM) medium of oocytes subjected to heat shock on embryo quality. Immature oocytes aspirated from ovaries obtained from a slaughterhouse were selected and randomly allocated in factorial experiment design (3 × 2). Three concentrations of melatonin (0, 10–6, and 10–4 M; M5250, Sigma, St. Louis, MO, USA) were added to the IVM medium and 2 incubation conditions (conventional: 24 h at 38.5°C and 5% CO2; heat shock: 12 h at 41°C followed by 12 h at 38.5°C and 5% CO2) were tested, resulting in treatments: M1 (0 M; 38.5°C; n = 15), M2 (10–6 M; 38.5°C; n = 15), M3 (10–4 M; 38.5°C; n = 15), M4 (0 M; 41°C; n = 15), M5 (10–6 M; 41°C; n = 15), and M6 (10–4 M; 41°C; n = 15). The IVM was performed in a Nunc plate (144444 – Thermo, Fisher Scientific Inc., Pittsburgh, PA, USA) containing 400 µL of TCM-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20 µg mL–1 of FSH (Pluset®, Calier Laboratories, Barcelona, Spain) and 10% oestrus cow serum. Oocytes were IVF in FERT-TALP medium for 20–22 h and incubated at 38.5°C and 5% CO2. After IVF, the presumptive zygotes were denuded and cultured in CR2aa medium supplemented with 2.5% FCS (Nutricell, Campinas, Brazil) in an incubator at 38.5°C under 5% CO2, 5% O2, and 90% N2, and saturated humidity for 8 days. Blastocysts with 8 days post-fertilization from different treatments were fixed in 4% paraformaldehyde in PBS for 1 h and analysed by TUNEL assay (Deadend™ Fluorometric TUNEL System, Promega, Madison, WI, USA) to evaluate embryonic quality. Data were analysed by generalised linear models considering the Poisson distribution and using the Proc Genmod of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA) considering effects of melatonin concentration, incubation conditions, and interaction between the factors. Values shown are the mean ± s.e.m. The interaction between melatonin concentration and incubation conditions was no significant (P > 0.05). The total number of cells was not affected (P > 0.05) by melatonin, but it was decreased (P < 0.05) by heat shock (M1 = 117 ± 6.7a; M2 = 118 ± 4.2a; M3 = 120 ± 6.3a; M4 = 102 ± 6.2b; M5 = 106 ± 5.7b; M6 = 108 ± 8.9b). Melatonin and heat shock did not affect (P > 0.05) the index of embryo apoptotic cells (M1 = 15.3% ± 2.0; M2 = 15.5% ± 1.3; M3 = 13.6% ± 2.0; M4 = 14.9% ± 1.5; M5 = 13.3% ± 1.3; M6 = 13.5% ± 1.2) and the index of trophoblast cells (M1 = 74.6% ± 2.3; M2 = 75.0% ± 1.7; M3 = 75.2% ± 1.9; M4 = 78.4% ± 2.3; M5 = 76.4% ± 3.0; M6 = 75.2% ± 2.6). The melatonin and heat shock affected the index of the inner cell mass (ICM; P < 0.05), and the heat shock reduced the index of the ICM of oocytes not treated with melatonin (M1 = 25.4% ± 2.3a; M2 = 25.0% ± 1.7a; M3 = 24.8% ± 1.8a; M4 = 21.6% ± 2.3b; M5 = 23.6% ± 3.0a; M6 = 24.8% ± 2.6a). In conclusion, melatonin supplementation to the medium IVM of oocytes subjected to heat shock had no effect on blastocyst total cell number, general apoptotic index, or index of the trophoblast cells, but increased index of the ICM. Research was supported by Fapemig, CNPq, Embrapa, and CAPES.

Correlative scanning electron, transmission electron, and light microscopic studies of the in vitro development of mouse embryos on a plastic substrate at the implantation stage

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 23-39
Author(s):  
Matthew A. Gonda ◽  
Yu-Chih Hsu
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Plastic Substrate ◽  
Trophoblast Cells ◽  
Electron Transmission ◽  
Transmission Electron ◽  
Development In Vitro ◽  
Scanning Electron

Correlative scanning electron, transmission electron, and light microscopy were utilized to study the morphogenic events occurring during mouse blastocyst outgrowth and earlyegg- cylinder development in vitro. After hatching and attachment of blastocysts on theplastic surface, the blastocoelic cavity collapses as the mural trophoblasts spread and migrate outward. The inner cell mass is covered with a differentiated endoderm on the blastocoelic cavity side and by the polar trophoblasts on the medium sideat this stage. As the endodermcovered inner cell mass proliferates, being physically restricted from further downward expansion by the plastic coverslip and by lack of space in the collapsed blastocoelic cavity, it migrates upward and protrudes into the culture medium in a breakbetween the polar and mural trophoblast cells. Polar trophoblast cellsapposed to the base of the egg cylinder continue to proliferate forming the ectoplacental cone. Thus, the early egg cylinder lacking a trophoblast barrier begins inverting its growth pattern from towards the culture dish surface to a more upright position. Egg-cylinder development in vitro from the inner cell mass and polar trophoblast cells closely paralleled in vivo. The functional nature of variousembryonic cell types observed in these embryos was revealed by scanning electron microscopy. These studies as well as those of Wiley and Pedersen (1977) suggest that blastocysts can serve as a source of in vitro developing early mouse egg cylinders that appear to resemble their in vivo counterparts and can be used in experimental studies of mouse embryogenesis.

scholarly journals Mcl-1 deficiency results in peri-implantation embryonic lethality

2000 ◽  
Vol 14 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Julie L. Rinkenberger ◽  
Susan Horning ◽  
Barbara Klocke ◽  
Kevin Roth ◽  
Stanley J. Korsmeyer
Keyword(s):  
Family Member ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
In Utero ◽  
Embryonic Lethality ◽  
Preimplantation Development

We disrupted the Mcl-1 locus in murine ES cells to determine the developmental roles of this Bcl-2 family member. Deletion of Mcl-1 resulted in peri-implantation embryonic lethality. Mcl-1−/− embryos do not implant in utero, but could be recovered at E3.5–4.0. Null blastocysts failed to hatch or attach in vitro, indicating a trophectoderm defect, although the inner cell mass could grow in culture. Of note, Mcl-1−/−blastocysts showed no evidence of increased apoptosis, but exhibited a delay in maturation beyond the precompaction stage. This model indicates that Mcl-1 is essential for preimplantation development and implantation, and suggests that it has a function beyond regulating apoptosis.

Lineage analysis of inner cell mass and trophectoderm using microsurgically reconstituted mouse blastocysts

Development ◽  
1982 ◽  
Vol 68 (1) ◽  
pp. 199-209
Author(s):  
Virginia E. Papaioannou
Keyword(s):  
Enzyme Assay ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Glucose Phosphate Isomerase ◽  
Mouse Blastocyst ◽  
Cell Lineages ◽  
Glucose Phosphate ◽  
Parietal Endoderm ◽  
First Time ◽  
Lineage Analysis

The fate of mouse blastocyst tissues was examined following reconstitution of blastocystsfrom isolated inner cell mass (ICM) and trophectoderm differing for electrophoretic variantsat the glucose phosphate isomerase (GPI-1) locus. A modified microsurgical method was usedand a more sensitive enzyme assay allowed finer dissection of developing chimaeric con-ceptuses. In seven of nine cases, the extraembryonic ectoderm or the later ectoderm of thechorion was entirely of the blastocyst trophectoderm enzyme type, providing the first ditectevidence that this tissue can be wholly derived from the trophectoderm. The two exceptionscould represent contamination of the ICM with trophectoderm or might indicate somedevelopmental lability of ICM cells. In addition, the results confirm the cell lineages of othertissues of the 7·5- to 9·5-day pc embryo and, for the first time, directly demonstrate the ICMorigin of the parietal endoderm.

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

Development ◽  
1975 ◽  
Vol 34 (2) ◽  
pp. 467-484
Author(s):  
Michael I. Sherman ◽  
Sui Bi Atienza
Keyword(s):  
Cytosine Arabinoside ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells ◽  
Culture Dish ◽  
In Vitro Development ◽  
Vitro Development

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.

scholarly journals Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 207-218 ◽  
Author(s):  
Anna Piliszek ◽  
Jacek A Modliński ◽  
Kazimiera Pyśniak ◽  
Jolanta Karasiewicz
Keyword(s):  
Cell Fusion ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Hybrid Cells ◽  
Cellular Processes ◽  
Glucose Phosphate ◽  
High Contribution ◽  
Foetal Membranes

Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts’ outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions. To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney. We conclude that somatic cells (FFs) can proliferate in earlyembryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.

276 EXPRESSION OF CD9 AND ALPHA6-INTEGRIN IN PORCINE BLASTOCYSTS

2009 ◽  
Vol 21 (1) ◽  
pp. 235 ◽  
Author(s):  
M. D. Goissis ◽  
F. R. O. de Barros ◽  
M. G. Marques ◽  
C. M. Mendes ◽  
M. P. Milazzotto ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Rna Extraction ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Trophoblast Cells ◽  
Pcr Products ◽  
Nucleotide Database ◽  
First Time

Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers is necessary for validation of a pluripotent cell line. Not all markers observed in ESC from other species are characterized in swine embryos. The objective of this study was to characterize CD9 and α6-integrin expression in porcine blastocysts and to derive porcine ESC using Matrigel. In vitro or in vivo porcine blastocysts were submitted to total RNA extraction for RT-PCR, fixation for immunocytochemistry or immunosurgery for culture of inner cell mass. Expression of Oct-4, CD9, and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass (ICM) and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. This study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts. Financial support: Fapesp 05/57314-0.

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