Development of gynogenetic and parthenogenetic inner cell mass and trophectoderm tissues in reconstituted blastocysts in the mouse.

Development ◽  
1985 ◽  
Vol 90 (1) ◽  
pp. 267-285
Author(s):  
Sheila C. Barton ◽  
Catharine A. Adams ◽  
M. L. Norris ◽  
M. A. H. Surani
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Paternal Genome ◽  
Developmental Potential ◽  
Somite Stage ◽  
Tissue Degeneration ◽  
Equivalent Control ◽  
Complete Failure ◽  
Almost All ◽  
Insight Into

The developmental potential of inner cell mass (ICM) and trophectoderm (TE) derived from parthenogenetic or biparental gynogenetic embryos was examined in reconstituted blastocysts with normal TE or ICM, respectively. The results demonstrate that when a normal ICM was introduced inside a trophectoderm vesicle derived from parthenogenetic or gynogenetic blastocysts, postimplantation development was characterized by the almost complete failure of trophoblast proliferation and without compensating cellular contribution from the normal ICM to the outer trophoblast lineage. Consequently, the normal ICMs also failed to develop adequately and only a few retarded embryos were detected on day 11–12 of pregnancy. In most respects, development of these reconstituted blastocysts resembled that obtained with unoperated gynogenetic and a parthenogenetic blastocyst. By contrast, an ICM from a parthenogenetic or gynogenetic embryo introduced inside a normal trophectoderm vesicle induced substantial proliferation of the trophoblast but again without a detectable cellular contribution from the ICM to the outer trophoblast lineage. However, with the improved development of the trophoblast, both the parthenogenetic and gynogenetic ICMs developed substantially better and without a detectable cellular contribution from the TE to the embryo. Almost all the embryos developed at least up to the 25-somite stage and many of them reached the 30- to 40-somite stage. Some of the most advanced day-11 and -12 gynogenones and parthenogenones yet seen have now been obtained in this way. Nevertheless, all the embryos were still smaller than the equivalent control embryos and showed signs of some tissue degeneration. The yolk sac was also suboptimal with poor blood supply and may need to be improved to obtain further improvement in the development of the embryos. The combined results demonstrate that the trophoblast proliferates very poorly even in the presence of a normal ICM, if the TE tissue lacks a paternal genome. However, ICM tissues which lack a paternal genome can develop to an advanced embryonic stage if they are introduced inside a normal trophectoderm vesicle. The results give further insight into the differential roles of maternal and paternal genomes during development of the embryo and extraembryonic tissues in the mouse.

Embryonic stem cells alone are able to support fetal development in the mouse

Development ◽  
1990 ◽  
Vol 110 (3) ◽  
pp. 815-821 ◽  
Author(s):  
A. Nagy ◽  
E. Gocza ◽  
E.M. Diaz ◽  
V.R. Prideaux ◽  
E. Ivanyi ◽  
...  
Keyword(s):  
Fetal Development ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Developmental Potential ◽  
The Moment ◽  
Genetically Manipulated ◽  
Almost All

The developmental potential of embryonic stem (ES) cells versus 3.5 day inner cell mass (ICM) was compared after aggregation with normal diploid embryos and with developmentally compromised tetraploid embryos. ES cells were capable of colonizing somatic tissues in diploid aggregation chimeras but less efficiently than ICMs of the same genotype. When ICM in equilibrium with tetraploid and ES in equilibrium with tetraploid chimeras were made, the newborns were almost all completely ICM- or ES-derived, as judged by GPI isozyme analysis, but tetraploid cells were found in the yolk sac endoderm and trophectoderm lineage. Investigation of ES contribution in 13.5 day ES in equilibrium with tetraploid chimeras by DNA in situ hybridization confirmed the complete tetraploid origin of the placenta (except the fetal blood and blood vessels) and the yolk sac endoderm. However, the yolk sac mesoderm, amnion and fetus contained only ES-derived cells. ES-derived newborns failed to survive after birth, although they had normal birthweight and anatomically they appeared normal. This phenomenon remains unexplained at the moment. The present results prove that ES cells are able to support complete fetal development, resulting in ES-derived newborns, and suggest a useful route for studying the development of genetically manipulated ES cells in all fetal lineages.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

P–516 Evidence for self-correction in preimplantation embryos by comparative molecular karyotyping of blastocoele fluid, trophectoderm and inner cell mass

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
D Zhigalina ◽  
N Skryabin ◽  
O Kanbekova ◽  
V Artyukhova ◽  
A Svetlakov ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Morphological Characteristics ◽  
Preimplantation Embryos ◽  
Molecular Karyotyping ◽  
Embryonic Cells ◽  
Developmental Potential ◽  
Positive Correlation ◽  
Registration Number ◽  
Molecular Karyotype

Abstract Study question Does the molecular karyotype of the cell-free DNA (cfDNA) from the blastocyst fluid (BF) can predict the efficiency of self-correction of karyotype of preimplantation embryo? Summary answer Detection of aneuploidies in the BF potentially can point out on effective self-correction of blastocyst karyotype and consequently on high developmental potential of mosaic embryos. What is known already Correction of aneuploidies in the preimplantation embryos can be provided by several mechanisms, including apoptosis. The predominant death of aneuploid cells was demonstrated in mouse embryos (Bolton, 2016). A positive correlation was also shown between the concentration of cfDNA from the BF of human blastocyst and the morphology of the embryo, as well as between the activity of caspase–3 and the concentration of cfDNA (Rule, 2018). The incidence of failed amplification after WGA being significantly higher among euploid blastocysts (Magli, 2019). The capacity of abnormal cells extruding into the BF would be related to the embryo development potential (Gianaroli, 2019). Study design, size, duration This is a prospective observational study of thirty-one Day 5 human blastocysts. Cryopreserved blastocysts were received after treatment cycles at the IVF Center with informed consent obtained from couples. The average age of 15 women was 32.25±5 years. The morphological characteristics of blastocysts were estimated in accordance with the Gardner classification (Gardner, Schoolcraft, 1999). The procedure of BF aspiration and trophectoderm (TE) and ICM cells separation of the blastocysts was previously described (Tsuiko, 2018). Participants/materials, setting, methods WGA was performed by PicoPLEX kit (Rubicon Genomics, USA) or REPLI-g Mini kit (Qiagen) according to manufacturer’s protocols. The DNA of the BF, ICM and TE were analyzed separately using cCGH, aCGH and NGS. SurePrint G3 Human CGH Microarrays (8x60K, Agilent Technologies) were used according to the manufacturer’s recommendations. Image analysis was done using ISIS (v.5.5) (Metasystems) and Agilent CytoGenomics Software (v.3). VeriSeq™ PGS Kit - MiSeq® System (Illumina) was used for NGS. Main results and the role of chance Molecular karyotypes of all three samples - BF, ICM and TE, were obtained for 23 (74.2%) blastocysts. A correlation between the woman’s age and the number of aneuploidies in cfDNA (p = 0.0009) was found. A positive correlation may indicate that the number of aneuploidies in the embryonic cells increases with the age of a woman, however, the embryonic karyotype undergoes self-correcting through the elimination of aneuploid cells. It was noted that well-developing blastocysts (groups 4–5, according to Gardner’s classification) had fewer aneuploidies in ICM (p = 0.0141) and TE (p = 0.0436). In contrast, there was a tendency to an increase in the number of aneuploidies in the BF during blastocysts transition from stage 3 to 5 (p = 0.3542). We assessed the relationship between the number of aneuploidies in groups of blastocysts with different characteristics of ICM (groups “A” and “B” according to Gardner’s classification). These groups significantly differ in the number of aneuploidies in cfDNA (p = 0.0352), although the statistically significant differences between the number of aneuploidies in ICM (p = 0.5992) and in TE (p = 0.5934) was not detected. Thus, higher-quality embryos in terms of ICM morphology contain more abnormalities in the BF, since in this group the elimination of aneuploid cells is more efficient. Limitations, reasons for caution The number of embryos is limited in this study. More comprehensive studies are required to confirm the observed tendency. Wider implications of the findings: Aneuploid cells elimination can be a cause of increasing cfDNA concentration in the BF, which may be a marker of the viability of mosaic embryos when it is necessary to decide on mosaic embryo transfer. This study was supported by the RFBR (15–04–08265) and by the RSF (20–74–00064). Trial registration number Not applicable

Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo

Development ◽  
1982 ◽  
Vol 68 (1) ◽  
pp. 175-198
Author(s):  
R. L. Gardner
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Early Embryo ◽  
Visceral Endoderm ◽  
Primitive Endoderm ◽  
Developmental Potential ◽  
Extraembryonic Endoderm ◽  
Parietal Endoderm ◽  
Endodermal Cells

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

202 Targeting Galactosyl-α-1,3-Galactose (αGal) Epitopes for Multi-Species Embryo Immunosurgery

2018 ◽  
Vol 30 (1) ◽  
pp. 241
Author(s):  
M. Kurome ◽  
A. Baehr ◽  
K. Simmet ◽  
B. Kessler ◽  
E. Jemiller ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Fluorescein Isothiocyanate ◽  
Transcriptome Profiling ◽  
Hoechst 33342 ◽  
Experimental Variation ◽  
Reliable Source ◽  
German Research ◽  
Almost All ◽  
Pig Serum

Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE cell epitopes, have been used as antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyper-acute rejection of pig-to-primate xenotransplants. Because GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they are expected to produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs can be used as a novel antibody source for embryo immunosurgery. First, the presence of αGal epitopes in mouse (E3.5), rabbit (Day 4), pig (Day 6–7), and bovine (Day 7–8) blastocysts was examined by staining with fluorescein isothiocyanate (FITC)-conjugated BSI-B4 lectin (Sigma, St. Louis, MO, USA) that binds αGal. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all examined species. Next, pig blastocysts were incubated with a medium containing GTKO pig serum. Swollen TE cells were observed in some of the blastocysts already after 2 min and, after 10 min, almost all TE cells of these blastocysts were completely destroyed. No lysis was recorded when the same experiment was done with wild-type pig serum, suggesting the presence of sufficient quantities of anti-αGal antibodies in GTKO serum to coat the TE cells and induce their complement-mediated lysis. Finally, GTKO serum was systematically tested for immunosurgery. Zona-free blastocysts of the species mentioned above were incubated with heat-inactivated GTKO pig serum for 1 h at 38°C. After washing, the blastocysts were labelled with Hoechst 33342 and TE was stained with FITC-conjugated concanavalin A (ConA) to distinguish the ICM from TE cells. Eventually, the blastocysts were individually incubated in complement solution for 30 to 40 min. Complement-mediated lysis of TE cells was efficiently induced in mouse, rabbit, pig, and bovine blastocysts (10/10, 7/7, 10/10, and 5/6, respectively), and intact ICM were successfully recovered from all species (100, 100, 60, and 80%, respectively). Double fluorescent staining with Hoechst 33342 and ConA clearly showed that the majority of isolated ICM was not contaminated with TE cells. Our study demonstrates that GTKO pig serum is a reliable source of antibodies targeting the αGal epitope of TE cells. Major advantages of using GTKO serum for embryo immunosurgery are (1) that it can be produced easily in large batches, thus reducing experimental variation; and (2) that it reacts with a large number of different species, except for humans, apes, and old world monkeys that lack αGal epitopes. Interesting applications include the preparation of TE and ICM for transcriptome profiling or chimeric embryo complementation experiments. This work is supported by the German Research Council (TR-CRC 127).

Putrescine supplementation during in vitro maturation of aged mouse oocytes improves the quality of blastocysts

2017 ◽  
Vol 29 (7) ◽  
pp. 1392 ◽  
Author(s):  
Dandan Liu ◽  
Guolong Mo ◽  
Yong Tao ◽  
Hongmei Wang ◽  
X. Johné Liu
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
In Vitro Fertilisation ◽  
Aged Mouse ◽  
Developmental Potential ◽  
The Impact

Mouse ovaries exhibit a peri-ovulatory rise of ornithine decarboxylase and its product putrescine concurrent with oocyte maturation. Older mice exhibit a deficiency of both the enzyme and putrescine. Peri-ovulatory putrescine supplementation in drinking water increases ovarian putrescine levels, reduces embryo resorption and increases live pups in older mice. However, it is unknown if putrescine acts in the ovaries to improve oocyte maturation. This study examined the impact of putrescine supplementation during oocyte in vitro maturation (IVM) on the developmental potential of aged oocytes. Cumulus–oocyte complexes from 9–12-month-old C57BL/6 mice were subjected to IVM with or without 0.5 mM putrescine, followed by in vitro fertilisation and culture to the blastocyst stage. Putrescine supplementation during IVM did not influence the proportion of oocyte maturation, fertilisation or blastocyst formation, but significantly increased blastocyst cell numbers (44.5 ± 1.9, compared with 36.5 ± 1.9 for control; P = 0.003). The putrescine group also had a significantly higher proportion of blastocysts with top-grade morphology (42.9%, compared with 26.1% for control; P = 0.041) and a greater proportion with octamer-binding transcription factor 4 (OCT4)-positive inner cell mass (38.3%, compared with 19.8% for control; P = 0.005). Therefore, putrescine supplementation during IVM improves egg quality of aged mice, providing proof of principle for possible application in human IVM procedures for older infertile women.

scholarly journals Epigenetic asymmetry in the mammalian zygote and early embryo: relationship to lineage commitment?

2003 ◽  
Vol 358 (1436) ◽  
pp. 1403-1409 ◽  
Author(s):  
Wolf Reik ◽  
Fatima Santos ◽  
Kohzoh Mitsuya ◽  
Hugh Morgan ◽  
Wendy Dean
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Lineage Commitment ◽  
Imprinted Genes ◽  
Mammalian Development ◽  
Paternal Genome ◽  
Developmental Defects ◽  
Set Up

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X–chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.

scholarly journals 127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS

2004 ◽  
Vol 16 (2) ◽  
pp. 186
Author(s):  
J.O. Gjørret ◽  
P. Maddox-Hyttel
Keyword(s):  
Production Systems ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Embryo Cell ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Tunel Reaction

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to<0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P<0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general. Table 1

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.

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