The development potential of parthenogenetically derived cells in chimeric mouse embryos: implications for action of imprinted genes

Development ◽  
1988 ◽  
Vol 104 (1) ◽  
pp. 175-182 ◽  
Author(s):  
H.J. Clarke ◽  
S. Varmuza ◽  
V.R. Prideaux ◽  
J. Rossant
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Abnormal Development ◽  
Imprinted Genes ◽  
Chimeric Mouse ◽  
Paternal Genome ◽  
Transgenic Mouse Line ◽  
Parthenogenetic Embryos

Parthenogenetic embryos of mice die shortly after implantation and characteristically contain poorly developed extraembryonic tissue. To investigate the basis of the abnormal development of parthenotes, we combined them with normal embryos to produce chimeras and examined the distribution of the parthenogenetically derived cells during preimplantation and early postimplantation development. The parthenogenetic embryos were derived from a transgenic mouse line bearing a large insert, which allowed these cells to be identified in histological sections using in situ hybridization. At the blastocyst stage, the parthenogenetic embryos contributed cells to the trophectoderm (TE) and inner cell mass (ICM) of chimeras. By 6.5 days, however, in almost every embryo, parthenogenetically derived cells were not detected in the extraembryonic trophoblast tissue descended from the TE. In contrast, parthenogenetically derived cells could contribute to all descendants of the ICM of 6.5-and 7.5-day chimeras, including the extraembryonic visceral and parietal endoderm. Quantitative analysis of the degree of chimerism in the embryonic ectoderm at 6.5-7.5 days indicated that parthenogenetically derived cells could contribute as extensively as normal cells. These results indicate that normal trophoblast development requires gene expression from the paternally inherited genome before 6.5 days of embryogenesis. Tissues of the ICM lineage, however, apparently can develop independently of the paternal genome at least to 7.5 days of embryogenesis. Comparison of these results with those of others suggests that the influence of imprinted genes is manifested at different times and in a variety of tissues during development.

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.

Defects in the allocation of cells to the inner cell mass and trophectoderm of parthenogenetic mouse blastocysts

1996 ◽  
Vol 8 (8) ◽  
pp. 1193 ◽  
Author(s):  
B Mognetti ◽  
D Sakkas
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Substantial Reduction ◽  
Cell Lineage ◽  
Blastocyst Stage ◽  
Trophectoderm Cells ◽  
Parthenogenetic Development ◽  
Preimplantation Stage ◽  
Parthenogenetic Mouse Embryos ◽  
Parthenogenetic Embryos

Diploid parthenogenetic mouse embryos (which possess two maternally-derived genomes) can develop only as far as the 25-somite stage when transferred in utero and exhibit a substantial reduction in trophoblast tissue. The loss of cultured parthenogenetic embryos during postimplantation indicates that a defect in cell lineage may be evident as early as the blastocyst stage. The possibility that a defect may already be reflected at the preimplantation stage was investigated by examining the allocation of cells to the trophectoderm (trophoblast progenitor cells) and the inner cell mass of haploid and diploid parthenogenetic mouse blastocysts. Utilizing a differential labelling technique for counting cells, diploid parthenogenetic blastocysts were found to have fewer inner cell mass cells and trophectoderm cells than their haploid counterparts and normal blastocysts. In addition, both haploid and diploid parthenogenetic blastocysts had a lower inner cell mass: trophectoderm ratio than normal blastocysts. Thus, the relatively poor development of the trophectoderm lineage at the postimplantation stage is not reflected by a reduction in its allotment of cells at its first appearance. Nevertheless, the findings indicate that parthenogenetic development is already compromised at the blastocyst stage, and provide evidence that the expression of imprinted genes has significance for the development of the embryo at the preimplantation stage.

62 Investigating differences in gene expression between invitro-produced bovine embryos and parthenotes

2021 ◽  
Vol 33 (2) ◽  
pp. 138
Author(s):  
K. Stoecklein ◽  
K. Clark ◽  
K. Pohler ◽  
M. S. Ortega
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Male Gamete ◽  
Rna Isolation ◽  
Blastocyst Stage ◽  
Suitable Model ◽  
Cleavage Rate ◽  
Paternal Genome ◽  
Cellular Division

Parthenogenic activation allows for the development of an oocyte without male gamete contribution and may serve as a suitable model for understanding maternal and paternal contributions during development. In the bovine, parthenotes lack the ability to survive to term once transferred into females. The goal of this study was to investigate the gene expression profile at the blastocyst stage in parthenotes and embryos by characterising expression of developmentally important genes, such as markers for pluripotency (OCT4, NANOG), hypoblast (GATA6), epiblast (SOX2), trophectoderm (CDX2), and maternal-embryo communication (IFNT2). To test this, IVM oocytes were either fertilized to a bull with known fertility invitro or activated. To activate, oocytes were denuded, and placed in ionomycin calcium salt for 5min. They were incubated for 3h in 6-(dimethylamino)purine (6-DMAP) and placed in synthetic oviductal fluid (SOF). Putative zygotes and activated oocytes were cultured in SOF for 8 days. Cleavage (at least one cellular division) was recorded on Day 3 and development to the blastocyst stage was recorded on Day 8 after insemination or activation. Cleavage rate was 85.9% and 83.2% for parthenotes and embryos, respectively. Both groups produced a similar blastocyst rate, 32.3% for parthenotes and 33.7% for embryos. On Day 8, blastocyst stage parthenotes and embryos were collected. Data were analysed by ANOVA (Tukey HSD post hoc test) using the GLM procedure of SAS version 9.4. Pools of 5 embryos or parthenotes (3 replicates) were flash frozen and stored until RNA isolation (PicoPure™ RNA Isolation Kit). Real-time PCR was used for quantification of gene expression. Genes were analysed relative to a housekeeping gene, GAPDH. There was no difference in gene expression detected for OCT4 (P=0.25), NANOG (P=0.11), GATA6 (P=0.32), SOX2 (P=0.25), or IFNT2 (P=0.52). Expression of CDX2 was lower in the parthenotes than the embryos (P=0.05). In a second experiment, the proteins GATA6, NANOG, and CDX2 were immunolocalized in 17 parthenotes and 15 embryos. Fixed embryos were permeabilized, blocked, and placed in primary antibodies overnight. After, they were placed into the secondary antibody for 1h, followed by nuclear stain. There was a decreased mean intensity of CDX2 in the parthenotes compared to embryos (P=0.005). No difference (P>0.05) in GATA6 or NANOG was observed between the 2 groups. The ratio of inner cell mass to trophectoderm was higher (P=0.04) in the parthenotes (2.5±0.23) than in the embryos (1.7±0.25). Here we analysed and confirmed the expression of developmentally important genes at the blastocyst stage in embryos and parthenotes. CDX2, a marker of the trophectoderm that will later give rise to the placenta, was downregulated in parthenotes. This highlights the importance of the contribution of the paternal genome to development. Further research is necessary to elucidate the ability of the parthenotes to establish and maintain pregnancy. This research was supported by the National Needs Fellowship funded by USDA NIFA Grant 2019-38420-28972.

scholarly journals Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eszter Posfai ◽  
Sophie Petropoulos ◽  
Flavia Regina Oliveira de Barros ◽  
John Paul Schell ◽  
Igor Jurisica ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Global Gene Expression ◽  
Blastocyst Stage ◽  
Egfp Expression ◽  
Hippo Signaling ◽  
Ability To Regenerate ◽  
Early Mouse ◽  
Cell Commitment

The segregation of the trophectoderm (TE) from the inner cell mass (ICM) in the mouse blastocyst is determined by position-dependent Hippo signaling. However, the window of responsiveness to Hippo signaling, the exact timing of lineage commitment and the overall relationship between cell commitment and global gene expression changes are still unclear. Single-cell RNA sequencing during lineage segregation revealed that the TE transcriptional profile stabilizes earlier than the ICM and prior to blastocyst formation. Using quantitative Cdx2-eGFP expression as a readout of Hippo signaling activity, we assessed the experimental potential of individual blastomeres based on their level of Cdx2-eGFP expression and correlated potential with gene expression dynamics. We find that TE specification and commitment coincide and occur at the time of transcriptional stabilization, whereas ICM cells still retain the ability to regenerate TE up to the early blastocyst stage. Plasticity of both lineages is coincident with their window of sensitivity to Hippo signaling.

Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocyst

Development ◽  
1984 ◽  
Vol 84 (1) ◽  
pp. 63-90
Author(s):  
Tom P. Fleming ◽  
Paul D. Warren ◽  
Julia C. Chisholm ◽  
Martin H. Johnson
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Central Area ◽  
Ultrastructural Changes ◽  
Trophectoderm Cells ◽  
Junctional Complexes ◽  
Secondary Lysosomes ◽  
Cytoplasmic Processes ◽  
Cellular Cover

Mouse blastocysts, aged 0, 2, 6 and 12 h from the onset of cavitation, were examined by transmission (TEM) and scanning (SEM) electron microscopy. In TEM sections, trophectoderm cells (TE) differed morphologically from those of the inner cell mass (ICM) by their flattened shape, paler cytosol staining and polarized disposition of both junctional complexes (apicolateral) and intracellular secondary lysosomes (SL; basal). Throughout this period of development, cytoplasmic processes, characterized by abundant SLs, cover approximately 80 % of the juxtacoelic face of the ICM. These processes are shown to be derived from the basal surface of TE cells intermediately placed between polar and mural regions. In SEM preparations of the juxtacoelic ICM surface, revealed by ‘cracking open’ blastocysts, the processes appear as tongue-shaped, centripetally oriented structures which terminate collectively at a central area on the ICM surface. The potential of cultured ICMs to generate TE was demonstrated following their immunosurgical isolation from blastocysts aged up to 12 h post cavitation and by examining the sequence of ultrastructural changes associated with TE generation by ICMs from 2 h blastocysts. In contrast, the juxtacoelic cells of similarly aged ICMs observed in situ in ultrasections of intact embryos showed little or no evidence of totipotency expression as judged by the absence of TE characteristics. Since TE expression within presumptive ICM cells is thought to be generated by an asymmetry of cell contacts (Johnson & Ziomek, 1983), we propose that the juxtacoelic TE processes, by providing a cellular cover to the ICM, function in suppressing the expression in situ of ICM totipotency.

Stem cell defects in parthenogenetic peri-implantation embryos

Development ◽  
1995 ◽  
Vol 121 (7) ◽  
pp. 2069-2077
Author(s):  
E.D. Newman-Smith ◽  
Z. Werb
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Insulin Like Growth Factor ◽  
Embryonic Antigen ◽  
Parietal Endoderm

Mouse embryos containing only maternal chromosomes (parthenotes) develop abnormally in vivo, usually failing at the peri-implantation stage. We have analyzed the development of parthenote embryos by using an inner cell mass (ICM) outgrowth assay that mimics peri-implantation development. ICMs from normal embryos maintained undifferentiated stem cells positive for stage-specific embryonic antigen-1 and Rex-1 while differentiating into a variety of cell types, including visceral endoderm-like cells and parietal endoderm cells. In contrast, ICMs from parthenotes failed to maintain undifferentiated stem cells and differentiated almost exclusively into parietal endoderm. This suggests that parthenote ICMs have a defect that leads to differentiation, rather than maintenance, of the stem cells, and a defect that leads to a parietal endoderm fate for the stem cells. To test the hypothesis that the ICM population is not maintained owing to a lack of proliferation of the stem cells, we investigated whether mitogenic agents were able to maintain the ICM population in parthenotes. When parthenote blastocysts were supplied with the insulin-like growth factor-1 receptor (Igf-1r) and insulin-like growth factor-2 (Igf-2), two genes not detectable in parthenote blastocysts by in situ hybridization, the ICM population was maintained. Similarly, culture of parthenote blastocysts in medium conditioned by embryonic fibroblasts and supplemented with the maternal factor leukemia inhibitory factor maintained the ICM population. However, once this growth factor-rich medium was removed, the parthenote ICM cells still differentiated predominantly into parietal endoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of the murine cyclin B2 cDNA and characterization of the lineage and temporal specificity of expression of the B1 and B2 cyclins during oogenesis, spermatogenesis and early embryogenesis

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 229-240 ◽  
Author(s):  
D.L. Chapman ◽  
D.J. Wolgemuth
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Northern Blot ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Development ◽  
Early Embryogenesis ◽  
Northern Blot Hybridization ◽  
Germ Cell Development

A cDNA encoding the murine cyclin B2 (cycB2) was isolated from an adult mouse testis cDNA library as part of studies designed to identify cyclins involved in murine germ cell development. This cycB2 cDNA was then used to examine the pattern of cycB2 expression during male and female germ cell development and in early embryogenesis, and to compare this expression with the previously characterized expression of cycB1. A single 1.7 kb cycB2 transcript was detected by northern blot hybridization analysis of total RNA isolated from midgestation embryos and various adult tissues. Northern blot and in situ hybridization analyses revealed that cycB2 expression in the testis was most abundant in the germ cells, specifically in pachytene spermatocytes. This is in contrast to the highest levels of expression of cycB1 being present in early spermatids. In situ analysis of the ovary revealed cycB2 transcripts in both germ cells and somatic cells, specifically in the oocytes and granulosa cells of growing and mature follicles. The pattern of cycB1 and cycB2 expression in ovulated and fertilized eggs was also examined. While the steady state level of cycB1 and cycB2 signal remained constant in oocytes and ovulated eggs, signal of both appeared to decrease following fertilization. In addition, both cycB1 and cycB2 transcripts were detected in the cells of the inner cell mass and the trophectoderm of the blastocyst. These results demonstrate that there are lineage- and developmental-specific differences in the pattern of the B cyclins in mammalian germ cells, in contrast to the co-expression of B cyclins in the early conceptus.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 305-312
Author(s):  
M. Aghayan ◽  
L.V. Rao ◽  
R.M. Smith ◽  
L. Jarett ◽  
M.J. Charron ◽  
...  
Keyword(s):  
Western Blot ◽  
Cellular Localization ◽  
Glucose Transporter ◽  
Developmental Stages ◽  
Molecular Level ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Developmental Expression

Two general mechanisms mediate glucose transport, one is a sodium-coupled glucose transporter found in the apical border of intestinal and kidney epithelia, while the other is a sodium-independent transport system. Of the latter, several facilitated transporters have been identified, including GLUT1 (erythrocyte/brain), GLUT2 (liver) and GLUT4 (adipose/muscle) isoforms. In this study, we used Western-blot analysis and high resolution immunoelectron microscopy (IEM) to investigate the stage-related expression and cellular localization of GLUT1, 2 and 4. The Western blot results demonstrate that GLUT1 is detectable in the oocyte and throughout preimplantation development. GLUT2 isoforms were not detectable until the blastocyst stage, while the GLUT4 isoform was undetectable in the oocyte through blastocyst stages. The present findings confirm previous studies at the molecular level which demonstrated that mRNAs encoding the same GLUT isoforms are detectable at corresponding developmental stages. GLUT1 and GLUT2 display different cellular distributions at the blastocyst stage as shown by IEM studies. GLUT1 has a widespread distribution in both trophectoderm and inner cell mass cells, while GLUT2 is located on trophectoderm membranes facing the blastocyst cavity. This observation suggests a different functional significance for these isoforms during mouse preimplantation development.

Nuclear transplantation of male primordial germ cells in the mouse

Development ◽  
1989 ◽  
Vol 107 (2) ◽  
pp. 407-411 ◽  
Author(s):  
Y. Tsunoda ◽  
T. Tokunaga ◽  
H. Imai ◽  
T. Uchida
Keyword(s):  
Germ Cells ◽  
Inner Cell Mass ◽  
Primordial Germ Cells ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Nuclear Transplantation ◽  
Embryonic Genome ◽  
Donor Nucleus ◽  
Implantation Sites ◽  
Genome Activation

We examined the developmental ability of enucleated eggs receiving embryonic nuclei and male primordial germ cells (PGCs) in the mouse. Reconstituted eggs developed into the blastocyst stage only when an earlier 2-cell nucleus was transplanted (36%) but very rarely if the donor nucleus was derived from a later 2-cell, 8-cell, or inner cell mass of a blastocyst (0–3%). 54–100%, 11–67%, 6–43% and 6–20% of enucleated eggs receiving male PGCs developed to 2-cell, 4-cell, 8-cell and blastocyst stage, respectively, in culture. The overall success rate when taking into account the total number of attempts at introducing germ cells was actually 0–6%. Live fetuses were not obtained after transfer of reconstituted eggs to recipients, although implantation sites were observed. The developmental ability of reconstituted eggs in relation to embryonic genome activation and genomic imprinting is discussed.

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