Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine

Development ◽  
1973 ◽  
Vol 29 (3) ◽  
pp. 601-615
Author(s):  
M. H. L. Snow
Keyword(s):  
Specific Activity ◽  
Inner Cell Mass ◽  
Cell Damage ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Abnormal Development ◽  
Tritium Concentration ◽  
Cell Stage

Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [3H]thymidine. Methyl-T-thymidine and thymidine-6-T(n) were used and both forms found to be lethal at concentrations above 0·1 μCi/ml. Both forms of [3H]Tdr at concentrations between 0·01 and 0·1 μCi/ml caused a highly significant (P < 0·001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [3H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [3H]Tdr damage were found to normally contribute to the inner cell mass. The [3H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [3H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [3H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

2019 ◽  
Vol 31 (1) ◽  
pp. 164
Author(s):  
A. E. Ynsaurralde Rivolta ◽  
M. Suvá ◽  
V. Alberio ◽  
C. Vazquez Echegaray ◽  
A. Guberman ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Monozygotic Twin ◽  
Cell Number ◽  
Control Group ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 125-130 ◽  
Author(s):  
Lei Lei ◽  
Na Guan ◽  
Yan-Ning Xu ◽  
Qing-Hua Zhang ◽  
Jing-Ling Shen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Karyotype Analysis ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lower Number ◽  
Mouse Embryos ◽  
Developmental Pattern ◽  
Cell Stage ◽  
Positive Expression

SummaryPolyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 ± 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 ± 7.2; 18.4 ± 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. β-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

Effect of Li+ on preimplantation mouse embryos

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 51-58
Author(s):  
L. Izquierdo ◽  
M. I. Becker
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Preimplantation Mouse Embryos ◽  
Early Embryos ◽  
Preimplantation Mouse

Two-cell mouse embryos were cultured in vitro for different periods in a medium in which NaCl was partially replaced by LiCl at concentrations ranging from 1 to 30 mm. The relative cell number diminished according to increasing LiCl concentrations but the onset of blastulation was not affected, thus resulting in blastulae with fewer cells than normal and with a reduced or absent inner cell mass. Results are discussed in terms of the possible mechanisms involved and are related with the vegetalization induced by Li+ on early embryos of echinoderms and amphibia.

The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras

Zygote ◽  
2000 ◽  
Vol 8 (3) ◽  
pp. 235-243 ◽  
Author(s):  
Pin-chi Tang ◽  
John D. West
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Advanced Embryo ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Cell Embryo ◽  
Embryo Stage

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid[harr ]8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid[harr ]8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell[harr ]8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid[harr ]8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development 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.

Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos

1990 ◽  
Vol 2 (1) ◽  
pp. 51 ◽  
Author(s):  
GR Somers ◽  
AO Trounson ◽  
LJ Wilton
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Mouse Embryos ◽  
Total Cell Number ◽  
Cell Stage ◽  
Single Blastomere ◽  
Number Of Cells

The allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) was investigated at 6-h intervals from 78 h to 102 h after hCG injection in 3/4 mouse embryos to determine the effect of removal of a single blastomere at the 4-cell stage on early differentiation. The procedures used to produce 3/4 embryos had little effect on embryo development. Embryos that had a single blastomere removed and then re-aggregated (RA embryos) had the same total number of cells as untreated (UT) embryos except at 78 h (P less than 0.05) and 102 h (P less than 0.01) post hCG where there were slightly less cells in RA embryos. Three-quarter embryos always had significantly fewer cells than RA embryos (P less than 0.001), with an average of 74% of the total cell number of RA embryos. As expected, 3/4 embryos always had significantly fewer cells in the ICM and TE compared with RA embryos (P less than 0.001). However, the ICM:TE ratio was also significantly lower in 3/4 embryos compared with RA embryos at 84, 96, and 102 h post hCG, indicating that the allocation of cells to the ICM and TE was disturbed. The ICM:TE ratio of 3/4 embryos could not be manipulated if either an early- or late-dividing blastomere was selectively biopsied at the 4-cell stage; this suggests that the known preferential contribution of an early-dividing blastomere to the ICM is not cell autonomous.

scholarly journals Insulin increases the cell number of the inner cell mass and stimulates morphological development of mouse blastocysts in vitro

Development ◽  
1990 ◽  
Vol 110 (3) ◽  
pp. 963-967 ◽  
Author(s):  
M.B. Harvey ◽  
P.L. Kaye
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Insulin Receptors ◽  
Cell Number ◽  
Morphological Development ◽  
Cell Stage ◽  
Morphological Studies ◽  
Preimplantation Mouse ◽  
Stimulation Of

Previous studies showed that insulin promotes cell proliferation and morphological development of preimplantation mouse embryos. In this report, the receptor responsible for these actions and the cell populations that are affected were investigated. Insulin's 9% stimulation of blastocyst cell number was entirely due to a 23% increase in ICM cell number with an EC50 of 0.54 pM. This and the similar degrees of stimulation of immunosurgically isolated ICMs by both physiological and supraphysiological insulin concentrations suggest that insulin receptors are present on the ICM and respond to exogenous insulin transcytosed through the TE to promote expansion of the ICM cell numbers. In morphological studies, insulin increased the number of blastocysts and decreased the number of morulae by 10% after 54 h culture from 2-cell embryos with EC50s of about 0.95 pM. The equivalence of these EC50s suggests mediation of insulin's stimulation of blastocyst formation via insulin receptors which are functionally expressed around the time of compaction at the 8-cell stage. These results support our hypothesis that insulin has an important role in the regulation of growth during preimplantation development.

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