An alternative method of deriving embryonic stem cell-like clones by aggregation of diploid cells with tetraploid embryos
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We previously showed that increasing the cell number of host tetraploid (4n) embryos by aggregating multiple 4n embryos at two to four-cell stages can improve the birthrate of mice from embryonic stem cells (ES mice). In the present study, we assessed whether in vitro aged blastocysts (e.g., E4.5 or E5.5), where their cell number also increased with development, can be used as hosts for generating ES mice. As expected, the cell number of in vitro aged 4n blastocysts increased with development, i.e., 26.5+/-2.4, 49.6+/-8.4, and 84.9+/-20.9 cells for E3.5, E4.5, and E5.5 respectively. Three independent ES cell lines were injected into 4n aged blastocysts, and their developmental ability was compared with that of E3.5 4n blastocysts commonly used for this procedure. We found that the birthrate of ES mice derived from E4.5 blastocysts were comparable with those of mice generated from E3.5 blastocysts. On the other hand, the birthrates decreased when E5.5 blastocysts were used. These results suggest that not only the cell number but also developmental age is important for producing ES mice. We also discuss a comparison of the present findings with those of our previous study, where ES mice were generated using an aggregation method employing the same ES cell lines.
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The goal of this study was to compare mouse embryo development in a defined synthetic medium (human tubal fluid) against the same medium supplemented with a defined synthetic serum (SS), co-culture on human tubal epithelium (TECC), and culture on human fibronectin (FN) with and without SS. After 48 h, TECC, SS and FN + SS cultures demonstrated accelerated development with > 70% achieving > or = 8-cell stage. After 72 h, these culture conditions also significantly increased the proportion of embryos reaching the blastocyst stage but only TECC significantly increased the number of hatching blastocysts. Nuclei of the trophectoderm of unhatched and hatched blastocysts were stained with propidium iodide before fixing and labelling both the trophectoderm and inner cell mass with bisbenzimide. Blastocysts from the TECC contained a significantly higher total cell number (TCN) and trophectoderm and inner cell mass cell numbers than all other groups. These findings indicate equivalent improvements in mouse embryo development to the blastocyst stage in response to TECC, SS and FN and an enhanced number of cells and rate of hatching found only with TECC.
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The pattern of cell proliferation of early rat embryos in vivo was examined. Naturally mated female rats were sacrificed every 2 to 3 h between 0000 h on Day 1 (day of estrus) and 2200 h on Day 5 of pregnancy. Embryos were obtained by flushing the oviducts and uterus with M2 medium. Embryonic stages were morphologically categorized into 12 stages, i.e. 1-cell, 2-cell, 3-cell, 4-cell, 5-7 cell, 8-cell, 8-cell-morula, morula, early blastocyst, blastocyst, large expanded blastocyst, and hatched blastocyst. The 2-cell stage lasted 31 h and was longer than any other stage. Duration of the 1-, 4-, 5-7-and 8-cell stage was 23, 11, 5, and 4 h, respectively. Embryonic shape at the 4-cell stage was either square or rhomboidal, and embryos at the 8-cell stage were either circular or rectangular. After the 8-cell stage, an increase in cell numbers was more dependent on time than the progression of developmental stages; thus, the number of blastomeres in each embryonic stage after the 8-cell stage varied considerably. Understanding the chronology of rat embryonic development in vivo will provide the basic knowledge that is required for manipulating the rat embryo.
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Mouse preimplantation embryonic cleavage rate is dependent upon the presence or absence of the Preimplantation-embryo-development (Ped) gene; which is linked to the Qa-2 subregion of the H-2 complex. Expression of Qa-2 antigens by fast developing mouse embryos correlates with Ped gene pheno-type: Qa-2a. It is not known if the Ped gene (Qa-2a) participates in cell differentiation in the preimplantation mouse blastocyst. Therefore, the study objective was to determine the differentiation of cells to the inner cell mass (ICM) and trophectoderm (TE) in Qa-2a positive (Ped ) and Qa-2a negative (Ped −) mouse blastocysts. One-cell stage embryos were recovered from the excised oviducts of PMSG (5 IU) and hCG (5 IU) primed virgin female (3–4 weeks) BALB/cByJ (Qa-2a: Ped −) and BALB/cJ (Qa-2a: Ped ) mice mated to fertile males (12 weeks). Embryos were collected, 14 hr after hCG, and cultured in modified α-MEM, to the hatched blastocyst stage in an atmosphere of 5% CO2 in air, 95% relative humidity at 37°C. Cell differentiation was determined by differential staining (bis-benzimide and propidium iodide) and fluorescence microscopy. Data were analyzed by Students t-test. There was no significant difference in total cell number between BALB/cJ (mean 139) and BALB/cByJ (mean 143) embryos. A significant difference (p < 0.001) was found in the number of cells differentiating to the ICM between BALB/cJ (mean 59.0) and BALB/cByJ (mean 29.0) mouse embryos. The number of cells differentiating to the TE, between BALB/cJ (mean 80.0) and BALB/cByJ (mean 114) embryos, approached significance (p = 0.062). The results suggest that the Ped gene (Qa-2a) may have an influential role in preimplantation blastocyst cell differentiation. Additional studies are warranted to further elucidate the role of the Ped gene in preimplantation embryo development and blastocyst formation.
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This study was to explore the physiological function of rapamycin(RAPA),the specific inhibitor of mammalian target of rapamycin(mTOR),during early development of mouse embryos derived fromin vitro fertilization(IVF).Chemically defined medium with different concentrations of RAPA(the control group:0ng/mL;the treatment groups:0.1,1,10 and 100ng/mL)was used for embryo culture,and the in vitro cleavage rate,formation rate and quality of blastocysts(the number of inner cell mass(ICM)cell,trophectoderm(TE)cell and total cell)of the embryos were examined.Results were as follows.After 24 hours of embryos cultured,the cleavage rate was found no significant difference when RAPA added from the pronuclear stage embryos,but after 90-96hours of embryos cultured,the blastocyst rate of 100ng/mL group was significantly lower than that of the other groups(including the control group)(P0.05)when RAPA added both from the pronuclear stage embryos and 2-cell stage embryos.The result of blastocyst cell counting showed,the 100ng/mL group was found obviously decreased compared to the control group(P0.05)on the number of ICM cell,TE cell and total cells,which indicated that the quality of blastocyst development was affected.Our research suggests the early development of in vitro mouse embryo was restrained by RAPA at the beginning concentration of 100ng/mL,and the mTOR signal pathway within the maintenance of mouse blastocyst formation is essential.
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【Objective】 The study was to explore whether hESCs can survive and contribute to different tissues in the mouse embryos,which will provide the basis for the feasibility study of interspecific chimeras using injection of hESCs into mouse blastocysts.【Method】 154 mouse blastocysts of 3.5 days postcoitum(dpc) were randomly divided into 3 groups:experiment group(injection of hESCs),sham group(needle only pierced the zona pellucida and trophoblast cells,but not injected hESCs and solutions) and control(not injection).The rate of hatched blastocyst was calculated after embryo cultured 24 h in vitro.3.5 dpc ICR mouse blastocysts were injected with EGFP-hESCs and allowed to culture 3,24,48,69,77,94 and 116 h in vitro,or were transferred to the uterine horns of pseudopregnant recipient mice.By fluorescence microscope observation,the EGFP-hESCs contribution pattern of survival,migration,proliferation,and differentiation was investigated.【Result】 24 h after injection,the blastocyst hatched rate of the experiment group and the sham group were 73.6% and 77.1%,respectively,which was significantly greater than that of the control(38.3%)(P0.01).At this stage,engrafted cells were localized to the ICM or around ICM of 88.9%(32/36)embryonic chimeras.48 h after culture in vitro,the positive EGFP cells were further decreased.Only 3 to 4 EGFP positive cells scattered out of the ICM in one embryo 116 h after culture in vitro.For chimeric embryos development in vivo,43 hESCs-injected blastocysts were implanted into the uterus of 6 pseudopregnant foster mice and harvested 22 formed deciduaes,of which 17 contained embryos.14 of these embryos were morphous normal and did not contain any EGFP-positive hESCs-137 derivatives.【Conclusion】 It is very difficult for hESCs to chimeras with mouse blastocysts.
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Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.
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