scholarly journals Analysis of gene transcription alterations at the blastocyst stage related to the long-term consequences of in vitro culture in mice

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 271-283 ◽  
Author(s):  
Raúl Fernández-González ◽  
Juan de Dios Hourcade ◽  
Irene López-Vidriero ◽  
Alberto Benguría ◽  
Fernando Rodríguez De Fonseca ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Epigenetic Mechanisms ◽  
Long Term Effects ◽  
Long Term Consequences

We have reported thatin vitroculture (IVC) of preimplantation mouse embryos in the presence of FCS produces long-term effects (LTE) on development, growth and behaviour of the offspring at adult age. To analyse the mechanisms underlying this phenomenon, we have examined development and global alterations in gene expression in the mouse blastocysts produced in the presence of FCS, conditions known to be suboptimal and that generate LTE. Embryos culturedin vitroin KSOM and in KSOM+FCS had a reduced number of cells in the inner cell mass at the blastocyst stage compared within vivoderived embryos; however, only culture in KSOM+FCS leads to a reduction in the number of trophoblast cells. Gene expression levels were measured by comparison among three groups of blastocysts (in vivo, IVC in KSOM and IVC in KSOM+FCS). Different patterns of gene expression and development were found between embryos culturedin vitroorin vivo. Moreover, when we compared the embryos produced in KSOM versus KSOM+FCS, we observed that the presence of FCS affected the expression of 198 genes. Metabolism, proliferation, apoptosis and morphogenetic pathways were the most common processes affected by IVC. However, the presence of FCS during IVC preferentially affected genes associated with certain molecular and biological functions related to epigenetic mechanisms. These results suggest that culture-induced alterations in transcription at the blastocyst stage related to epigenetic mechanisms provide a foundation for understanding the molecular origin at the time of preimplantation development of the long-term consequences of IVC in mammals.

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 43-55
Author(s):  
J. Rossant ◽  
K. M. Vijh
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Parietal Endoderm ◽  
Trophoblast Giant Cells ◽  
Vitro Development ◽  
Ectoplacental Cone

Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6·5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8·5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5·5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives, It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.

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.

scholarly journals Proliferation Failure and Gamma Radiation Sensitivity of Fen1 Null Mutant Mice at the Blastocyst Stage

2003 ◽  
Vol 23 (15) ◽  
pp. 5346-5353 ◽  
Author(s):  
Elisabeth Larsen ◽  
Christine Gran ◽  
Barbro Elisabet Sæther ◽  
Erling Seeberg ◽  
Arne Klungland
Keyword(s):  
Dna Replication ◽  
Gamma Radiation ◽  
Inner Cell Mass ◽  
Excision Repair ◽  
Cell Mass ◽  
Giant Cells ◽  
Radiation Sensitivity ◽  
Blastocyst Stage

ABSTRACT Flap endonuclease 1 (FEN1) has been shown to remove 5′ overhanging flap intermediates during base excision repair and to process the 5′ ends of Okazaki fragments during lagging-strand DNA replication in vitro. To assess the in vivo role of the mammalian enzyme in repair and replication, we used a gene-targeting approach to generate mice lacking a functional Fen1 gene. Heterozygote animals appear normal, whereas complete depletion of FEN1 causes early embryonic lethality. Fen1−/− blastocysts fail to form inner cell mass during cellular outgrowth, and a complete inactivation of DNA synthesis in giant cells of blastocyst outgrowth was observed. Exposure of Fen1−/− blastocysts to gamma radiation caused extensive apoptosis, implying an essential role for FEN1 in the repair of radiation-induced DNA damage in vivo. Our data thus provide in vivo evidence for an essential function of FEN1 in DNA repair, as well as in DNA replication.

scholarly journals Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos

Reproduction ◽  
2007 ◽  
Vol 134 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Gnanaratnam Giritharan ◽  
Said Talbi ◽  
Annemarie Donjacour ◽  
Francesca Di Sebastiano ◽  
Anthony T Dobson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Preimplantation Embryos ◽  
Global Pattern ◽  
Vitro Fertilization ◽  
Trophoblastic Cells

In vitro culture (IVC) of preimplantation mouse embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared gene expression and development of mouse blastocysts produced by in vitro fertilization (IVF) versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC) versus control blastocysts flushed out of the uterus on post coital day 3.5. The global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. It appears that each method of fertilization has a unique pattern of gene expression and development. Embryos cultured in vitro had a reduction in the number of trophoblastic cells (IVF 33.5 cells, IVC 39.9 cells, and 49.6 cells in the in vivo group) and, to a lesser degree, of inner cell mass cells (12.8, 11.7, and 13.8 respectively). The inner cell mass nuclei were larger after culture in vitro (140 μm2, 113 μm2, and 86 μm2 respectively). Although a high number of genes (1912) was statistically different in the IVF cohort when compared with the in vivo control embryos, the magnitude of the changes in gene expression were low and only a minority of genes (29 genes) was changed more than fourfold. Surprisingly, IVF embryos were different from IVC embryos (3058 genes were statistically different, but only three changed more than fourfold). Proliferation, apoptosis, and morphogenetic pathways are the most common pathways altered after IVC. Overall, IVF and embryo culture have a profound effect on gene expression pattern and phenotype of mouse preimplantation embryos.

scholarly journals The 'GO' system--a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage

Reproduction ◽  
2003 ◽  
pp. 161-169 ◽  
Author(s):  
GA Thouas ◽  
GM Jones ◽  
AO Trounson
Keyword(s):  
Culture System ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Control Culture ◽  
High Density ◽  
Preimplantation Embryos ◽  
Mouse Zygotes

A novel system of in vitro culture termed the 'glass oviduct' or 'GO' culture system is described. Mouse zygotes were cultured in pairs to the blastocyst stage in open-ended 1 microl glass capillaries. 'GO' culture supported the development of significantly more hatching or hatched blastocysts than did a standard microdroplet (10 zygotes per 20 microl) control culture (48.3 versus 3.3%, respectively). 'GO' bslastocysts contained significantly larger populations of cells (92+/-3 versus 75+/-3), and inner cell mass (25+/-1 versus 21+/-1) and trophectoderm (68+/-2 versus 53+/-3) subpopulations, compared with microdroplet-derived blastocysts. Before blastulation, 'GO'-derived morulae were found to contain significantly more cells than microdroplet-derived morulae (27+/-0.7 versus 14+/-0.5). After implantation, 'GO' blastocysts formed fetuses at a similar rate to microdroplet-derived blastocysts (55 versus 62%), but at a lower rate than blastocysts derived in vivo (80%). 'GO'- and microdroplet-derived fetuses were similar in wet weight to each other (0.412 and 0.415 g, respectively) but were heavier than fetuses derived from flushed blastocysts (0.390 g). An additional experiment investigated whether the beneficial effect of 'GO' culture was due to the significantly increased embryo density. Proportions of hatching or hatched blastocysts after 'GO' culture (50%) were higher than after standard microdroplet culture (7.6%), but were not different from culture in high embryo density microdroplets (20 zygotes per 10 microl; 42%). 'GO' blastocysts contained more cells (79.6+/-2.1) than did standard microdroplet-derived blastocysts (68.7+/-2.0), but were similar to high density microdroplet-derived blastocysts (85.8+/-2.7). Similarly, 'GO' blastocysts contained more trophectoderm cells (62.2+/-2.0) than did standard microdroplet-derived blastocysts (52.7+/-1.7), but were similar to the high density microdroplet blastocysts (68.8+/-2.5). Numbers of inner cell mass cells ('GO', standard microdroplet and high density microdroplet culture) were not different from each other (17.4+/-0.5, 16+/-0.5 and 17+/-0.4, respectively). In conclusion, the 'GO' culture system represents an alternative method to the microdroplet system for small numbers of preimplantation embryos, without detriment to implantation potential.

scholarly journals The role of Trp53 in the mouse embryonic response to DNA damage

2019 ◽  
Vol 25 (7) ◽  
pp. 397-407
Author(s):  
Yvonne Wilson ◽  
Ian D Morris ◽  
Susan J Kimber ◽  
Daniel R Brison
Keyword(s):  
Dna Damage ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Stage ◽  
X Irradiation

Abstract Apoptosis occurs primarily in the blastocyst inner cell mass, cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation-related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo-derived blastocysts were compared to control embryos X-irradiated at the two-cell stage and cultured to Day 5. An analysis of DNA by comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured two-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild-type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices that alter apoptosis in human embryos and/or select embryos for transfer, which potentially lack a functional Trp53 gene, need to be carefully considered.

scholarly journals Loss of RBBP4 results in defective inner cell mass, severe apoptosis, hyperacetylated histones and preimplantation lethality in mice†

2020 ◽  
Vol 103 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Xiaosu Miao ◽  
Tieqi Sun ◽  
Holly Barletta ◽  
Jesse Mager ◽  
Wei Cui
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lineage Specification ◽  
Mouse Embryo Development ◽  
Evolutionarily Conserved ◽  
Early Mouse

Abstract Retinoblastoma-binding protein 4 (RBBP4) (also known as chromatin-remodeling factor RBAP48) is an evolutionarily conserved protein that has been involved in various biological processes. Although a variety of functions have been attributed to RBBP4 in vitro, mammalian RBBP4 has not been studied in vivo. Here we report that RBBP4 is essential during early mouse embryo development. Although Rbbp4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts cannot hatch from the zona or can hatch but then arrest without further development. We find that while there is no change in proliferation or levels of reactive oxygen species, both apoptosis and histone acetylation are significantly increased in mutant blastocysts. Analysis of lineage specification reveals that while the trophoblast is properly specified, both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification. In summary, these findings demonstrate the essential role of RBBP4 during early mammalian embryogenesis.

Sign in / Sign up

Export Citation Format

Share Document