Developmental capacity is unevenly distributed among single blastomeres of 2-cell and 4-cell stage mouse embryos

AbstractDuring preimplantation development, mammalian embryo cells (blastomeres) cleave, gradually losing their potencies and differentiating into three primary cell lineages: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PE). The exact moment at which cells begin to vary in their potency for multilineage differentiation still remains unknown. We sought to answer the question of whether single cells isolated from 2- and 4-cell embryos differ in their ability to generate the progenitors and cells of blastocyst lineages. We revealed that twins were often able to develop into blastocysts containing inner cell masses (ICMs) with PE and EPI cells. Despite their capacity to create a blastocyst, the twins differed in their ability to produce EPI, PE, and TE cell lineages. In contrast, quadruplets rarely formed normal blastocysts, but instead developed into blastocysts with ICMs composed of only one cell lineage or completely devoid of an ICM altogether. We also showed that quadruplets have unequal capacities to differentiate into TE, PE, and EPI lineages. These findings could explain the difficulty of creating monozygotic twins and quadruplets from 2- and 4-cell stage mouse embryos.

Download Full-text

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

Scientific Reports ◽

10.1038/s41598-021-90653-1 ◽

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.

Download Full-text

Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction

Development ◽

10.1242/dev.62.1.339 ◽

1981 ◽

Vol 62 (1) ◽

pp. 339-350

Author(s):

W. J. D. Reeve ◽

C. A. Ziomek

Keyword(s):

Ligand Binding ◽

Single Cells ◽

Progressive Increase ◽

Mouse Embryos ◽

Cell Stage ◽

Characteristic Distribution ◽

Surface Polarization ◽

Cell Embryo ◽

Fluorescent Ligand ◽

Scanning Electron

Cells of mouse embryos develop a polarization of microvillous distribution at compaction. Cells of the 4-cell embryo show a uniform pattern of fluorescent-ligand binding and an even distribution of microvilli. Each cell of the early 8-cell embryo has a uniform distribution both of microvilli and of fluorescent ligand. During the 8-cell stage, there is a progressive increase in the incidence of cells which show microvilli restricted to a region normally on the exposed surface of the embryo. When late 8-cell embryos were disaggregated to single cells, and these sorted by pattern of fluorescent-ligand binding, each of the four patterns of staining related consistently to a characteristic distribution of microvilli as viewed by scanning electron microscopy. The 16-cell embryo possessed an inside population of uniformly labelled cells with a sparse microvillous distribution, and an outside population of cells, each of which had a microvillous pole.

Download Full-text

Transcript analysis of Heat shock protein 72 and protein 53 of 4-cell mouse embryos following Cryotop vitrification

Czech Journal of Animal Science ◽

10.17221/6750-cjas ◽

2013 ◽

Vol 58 (No. 5) ◽

pp. 217-226

Author(s):

A. Habibi ◽

N. Farrokhi ◽

F. Morreira da Silva ◽

A. Hosseini ◽

B.F. Bettencourt ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Mouse Embryos ◽

Control Group ◽

Heat Shock Protein 72 ◽

Mean Values ◽

Relative Expression ◽

Embryo Cells ◽

Developmental Capacity ◽

Polymerase Chain

The effects of two different concentrations of cryoprotectants on survival and developmental capacity of four-cell mouse embryos were compared by Cryotop vitrification to demonstrate that lower concentrations provide the same results as higher previously reported concentrations with lesser negative molecular impact on embryo cells. For this latest, embryos were compared via transcript analyses of Heat shock protein 72 (Hsp72) and protein 53 (p53). Four-cell embryos were obtained from superovulated female mice and randomly assignedto one of three following groups: (i) control (non-vitrified), (ii) vit1 (15% v/v: 7.5% ethylene glycol(EG) and 7.5% dimethyl sulfoxide (DMSO), and (iii) vit2 (30% v/v: 15% EG + 15% DMSO).The cells vitrified by Cryotop were thawed and side-by-side to the control group divided into two parts: one part was used to analyze the morphological traits, survival rate, and embryo cleavage ability to form blastocysts, and the other part was examined for changes in transcript levels of Hsp72 (Hspa1a + Hspa1b), p53, and Hprt1 (reference gene) by quantitative Real-Time polymerase chain reaction (qPCR). The results were analyzed by One Way Analysis of Variance and the mean values compared with LSD (P < 0.05). The relative expression of p53 in vit2 (30% v/v) was significantly higher than in vit1 (15% v/v) and in vit1 it was higher than in the control. The relative expression of Hsp72 was the same in vit1 and vit2 and significantly higher than in the control.The survival, cleavage, and blastocyst rates were the same for both vitrification treatments and significantly lower than in the control group. The up-regulations of Hsp72 and p53 following vitrification were suggestive of imposed heat shock, cold stress, and DNA damage to the mouse 4-cell embryos.

Download Full-text

Differences in blastomere totipotency in 2-cell mouse embryos are a maternal trait mediated by asymmetric mRNA distribution

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz051 ◽

2019 ◽

Vol 25 (11) ◽

pp. 729-744 ◽

Cited By ~ 2

Author(s):

E Casser ◽

S Wdowik ◽

S Israel ◽

A Witten ◽

S Schlatt ◽

...

Keyword(s):

De Novo ◽

Monozygotic Twins ◽

Super Resolution ◽

Culture Conditions ◽

Mouse Embryos ◽

Gene Products ◽

Cell Stage ◽

Mammalian Embryos ◽

Cytoplasmic Organization ◽

Diverse Culture

Abstract It is widely held that the first two blastomeres of mammalian embryos are equally totipotent and that this totipotency belongs to the group of regulative properties. However, this interpretation neglects an important aspect: evidence only came from successful monozygotic twins which can speak only for those pairs of half-embryos that are able to regulate in the first place. Are the frequently occurring incomplete pairs simply an artefact, or do they represent a real difference, be it in the imperfect blastomere’s ability to regulate growth or in the distribution of any compound X that constrains regulation? Using the model system of mouse embryos bisected at the 2-cell stage after fertilization, we present evidence that the interblastomere differences evade regulation by external factors and are already latent in oocytes. Specifically, an interblastomere imbalance of epiblast production persists under the most diverse culture conditions and applies to the same extent in parthenogenetic counterparts. As a result, cases in which twin blastocysts continued to develop in only one member account for 65 and 57% of zygotic and parthenogenetic pairs, respectively. The interblastomere imbalance is related to the subcellular distribution of gene products, as documented for the epiblast-related gene Cops3, using mRNA FISH in super-resolution mode confocal microscopy. Blastomere patterns of Cops3 mRNA distribution are α-amanitin-resistant. Thus, the imbalance originates not from de novo transcription, but from influences which are effective before fertilisation. These data expose previously unrecognized limits of regulative capacities of 2-cell stage blastomeres and point to aspects of cytoplasmic organization of the mouse oocyte that segregate unequally to blastomeres during cleavage.

Download Full-text

Nuclei of Taxus baccata: Flavanols Linked to Chromatin Remodeling Factors

Journal of Botany ◽

10.1155/2009/842869 ◽

2009 ◽

Vol 2009 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Walter Feucht ◽

Heike Dithmar ◽

Jürgen Polster

Keyword(s):

Wide Spectrum ◽

Single Cells ◽

Cell Lineage ◽

Kinetic Studies ◽

Histochemical Staining ◽

Shoot Tips ◽

Taxus Baccata ◽

Specific Expression ◽

Intimate Contact ◽

Cell Lineages

Microscopic studies of young needles and shoot tips from Taxus baccata showed that flavanols are localized in the nuclei. This observation is based on the histochemical staining of flavanols with the DMACA reagent. The colour that is obtained with this reagent varies from pale to deep blue, depending on the amount of flavanols. This study is focused on nondifferentiated cell lineages and on differentiating cells. The key point to note is that all nuclei of a cell lineage showed a uniform DMACA staining pattern based on the amount and structural appearence of nuclear flavanols. This points to transcriptional and epigenetic programming. However, comparing various cell lineages from different shoot tips and needles revealed a lineage-specific expression of nuclear flavanols. This result implied that both positional and developmental signals from neighbouring cells were involved in the nuclear flavanol binding of lineages. The cells of a developmentally advanced lineage loose their intimate contact and, then, they separate from each other to undergo an autonomous, individual sequence of differentiation. This in turn was accompanied by differences in the nuclear flavanol patterns of the single cells. Investigating different mitotic stages revealed a wide spectrum in flavanol staining intensities of the chromosomes. These observations should be linked to UV-VIS spectroscopical kinetic results indicating that nuclear flavanols bound to histones are involved in epigenetically regulated modification of chromatin. The kinetic studies show that catechin is relatively rapidly degraded by oxygen in the presence of -ions. However, this degradation reaction is strongly inhibited when histone proteins were added. This behaviour is a clear indication that coregulatory interactions exist between catechin and histones.

Download Full-text

Interspecific cell markers and lineage in mammals

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1985.0180 ◽

1985 ◽

Vol 312 (1153) ◽

pp. 91-100 ◽

Cited By ~ 16

Keyword(s):

Dna Hybridization ◽

Cell Lineage ◽

Embryonic Cell ◽

Mammalian Embryo ◽

Marker System ◽

Cell Lineages ◽

Streptavidin Peroxidase ◽

Derivatives Of ◽

Lineage Analysis

Study of cell lineage in the mammalian embryo has relied heavily on the use of chimeras to follow the fate of genetically marked cells in later development. Such studies have often been limited by the types of genetic markers available; there are very few markers that allow analysis of the spatial distribution of individual cells at all stages of development. We have developed a marker system that is based on the identification of cells of Mus musculus origin in M. musculus-M. caroli chimeras by in situ DNA-DNA hybridization using a cloned probe to M. musculus satellite DNA. This provides the first ubiquitous in situ cell marker system for mammalian chimeras. We have recently refined the system by the use of biotin-labelled probes and detection of hybridization by streptavidin-peroxidase binding. This increases both the speed and the resolution of the assay. We have used the marker for cell lineage analysis in both embryonic and adult chimeras and results from analysis of the derivatives of early cell lineages in later development and study of coherent growth versus cell mixing in the postimplantation embryo are presented. The importance of understanding embryonic cell lineages as a prelude to molecular studies is emphasized.

Download Full-text

Sex-Linked Differences in Developmental Potential of Single Blastomeres from in vitro-Fertilized 2-Cell Stage Mouse Embryos

Hormone Research ◽

10.1159/000184653 ◽

1995 ◽

Vol 44 (2) ◽

pp. 4-8 ◽

Cited By ~ 6

Author(s):

Eimei Sato ◽

Meiwei Xian ◽

Ruby P.A. Valdivia ◽

Yutaka Toyoda

Keyword(s):

Mouse Embryos ◽

Cell Stage ◽

Developmental Potential ◽

Single Blastomeres

Download Full-text

Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

Nature Communications ◽

10.1038/s41467-021-21532-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Maya Sen ◽

Dylan Mooijman ◽

Alex Chialastri ◽

Jean-Charles Boisset ◽

Mina Popovic ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Embryonic Stem ◽

Cost Effective ◽

Dna Demethylation ◽

Mouse Embryos ◽

Preimplantation Embryos ◽

Mammalian Development ◽

Cell Stage ◽

Stage Of Development

AbstractDNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells.

Download Full-text