Cell relationships during aggregation between preimplantation embryos and teratocarcinoma-derived cells

Development ◽  
1984 ◽  
Vol 81 (1) ◽  
pp. 17-35
Author(s):  
E. Lehtonen ◽  
J. Wartiovaara ◽  
I. Reima
Keyword(s):  
Gap Junctions ◽  
Tight Junctions ◽  
Preimplantation Embryos ◽  
Aggregate Formation ◽  
Cell Stage ◽  
F9 Cells ◽  
Cell Processes ◽  
Ec Cells ◽  
Early Phases ◽  
Close Contacts

Cleavage-stage mouse embryos aggregate and form chimaeric blastocysts with embryonal carcinoma (EC) cells. We used scanning and transmission electron microscopy to study cell relationships during aggregate formation between 8-cell-stage embryos and F9 EC cells. Relations between heterotypic cells were similarly studied in aggregation experiments with embryos and teratocarcinoma-derived visceral (PSA5-E) and parietal (PYS-2) endoderm cells and in experiments with EC cells and endoderm cells. The embryos and F9 cells always adhered to each other and rapidly formed compacted aggregates. Numerous microvilli and cell processes, originating from both embryo and EC cells, extended between the two cell types during adhesion and early phases of aggregation. The aggregation process involved spreading of the blastomeres on the EC cells. Frequent adherent junctions and close contacts, including possible focal gap or tight junctions were observed between the embryo and F9 cells after 3 h of culture. Apparent gap or tight junctions were infrequent during the early phases of aggregation but during further culture, extensive typical gap junctions were also seen between embryo and EC cells. The embryos adhered only irregularly and loosely to PSA5-E and PYS-2 cells; this interaction never led to aggregate formation comparable to that seen in the experiments with embryos and EC cells. Close contacts but no gap or tight junctions could be observed between the embryo and endoderm cells. On the other hand, both PSA5-E and PYS-2 cells readily adhered to and aggregated with EC cells. The present results suggest that microvilli and cell processes mediate membrane interactions during adhesion and early phases of aggregation between embryos and EC cells. During aggregation, blastomeres spread over the EC cells, and rapid formation of adherent junctions and close contacts, including possible focal gap or tight junctions is involved during the early phases of this process. After this initial phase, typical gap junctions are also seen between the embryo and EC cells. Interestingly, adhesive properties of embryo and EC cells differ: the former aggregate only with EC cells, whereas the latter do so also with teratocarcinoma-derived visceral and parietal endoderm cells. Mechanisms operating in the morphogenetic movement of cells in this experimental setup may be involved also in the development of the blastocyst in vivo.

5-AZA-2′-deoxycytidine (5-AZA-CdR) leads to down-regulation of Dnmt1o and gene expression in preimplantation mouse embryos

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 137-145 ◽  
Author(s):  
Jian-Ning Yu ◽  
Chun-Yang Xue ◽  
Xu-Guang Wang ◽  
Fei Lin ◽  
Chun-Yi Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gap Junctions ◽  
Tight Junctions ◽  
Preimplantation Embryos ◽  
Cell Nuclei ◽  
Developmental Gene ◽  
Down Regulation ◽  
Cell Stage ◽  
Developmental Gene Expression ◽  
Developmental Arrest

Summary5-AZA-2′-deoxycytidine (5-AZA-CdR) is a demethylating, teratogenic agent and a mutagen, which causes defects in the developing mouse and rat after implantation. Our previous data indicated that 5-AZA-CdR (0.2 and 1.0 μM) inhibited the development of mouse preimplantation embryos. Pronuclear embryos exposed to 5-AZA-CdR at the pronuclear stage were unable to form 8-cell embryos, while 2-cell-stage embryos exposed to 5-AZA-CdR only developed into uncompacted 8-cell-stage embryos. And there was no formation of blastocysts when 4-cell embryos cultured in 5-AZA-CdR. In our present study, we detected Dnmt1o protein and some developmental gene expression in order to find the reasons for the developmental arrest. Dnmt1o could not traffic to 8-cell nuclei as control when embryos were exposed to 5-AZA-CdR. Dnmt1o was in cytoplasm at 2-cell and 4-cell stages before and after treated with 5-AZA-CdR. Gene expression changes were also detected in this research. Our data indicated that connexin 31 (Cx31), connexin 43 (Cx43), connexin 45 (Cx45), E-cadherin (Cdh1) and β-catenin (Ctnnb1) were all downregulated by 5-AZA-CdR. Cx31, Cx43 and Cx45 are members of connexins family, which have a central role in gap junctions. Cdh1 and Ctnnb1 are necessary for the foundation of tight junctions. Therefore, developmental arrest induced by 5-AZA-CdR may be caused by the failure of Dnmt1o cytoplasmic–nuclear traffic and the down-regulation of developmental gene expression. Normal compaction and blastocoel cavitation need Dnmt1o traffic to 8-cell nuclei and the right gene expression, especially the correlative genes in gap junctions and tight junctions.

scholarly journals Unusual structural features and assembly of gap and pleated septate junctions in embryonic cockroach CNS

1985 ◽  
Vol 76 (1) ◽  
pp. 269-281
Author(s):  
L.S. Swales ◽  
N.J. Lane
Keyword(s):  
Gap Junctions ◽  
Tight Junctions ◽  
Glial Cell ◽  
Structural Features ◽  
Septate Junctions ◽  
Intramembrane Particles ◽  
Cell Processes ◽  
Gap Junctional ◽  
Perineurial Cells

Junctional assembly in the developing CNS in cockroach embryos has been studied during the last half of neurogenesis. Atypical linear tracts of gap junctions are found to develop between attenuated cytoplasmic glial cell processes and their overlying perineurial cells during the last third of development. During both perineurial and glial gap-junctional formation, 13 nm E face (EF) intramembrane particles (IMPs), such as are characteristic of arthropod gap junctions, are seen initially as free IMPs; these then become arranged in loose irregular clusters or alignments and finally are aggregated in plaques. P face ridges (or EF grooves), typical of tight junctions, are found on the same perineurial membrane face as assembling gap-junctional PF pits (or EF particles). Pleated separate junctions also develop between adjacent perineurial processes during the last third of embryogenesis; these form by the apparent migration of individual 8 nm PF IMPs into meandering rows, which then become aligned in numerous orderly parallel stacks. Although all these junctions occur on the same perineurial membrane face, the IMPs that form the different junctional types never appear to be confused during junctional assembly. The cues to signal the advent of these precise patterns, however, are unknown.

scholarly journals A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Laura Bernhardt ◽  
Marcus Dittrich ◽  
Rabih El-Merahbi ◽  
Antoine-Emmanuel Saliba ◽  
Tobias Müller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Negative Impact ◽  
Preimplantation Embryos ◽  
High Fat ◽  
Cell Stage ◽  
Genetic Transmission ◽  
Diet Induced Obesity ◽  
Genome Wide ◽  
A Genome ◽  
Paternal Obesity

AbstractPaternal obesity is known to have a negative impact on the male’s reproductive health as well as the health of his offspring. Although epigenetic mechanisms have been implicated in the non-genetic transmission of acquired traits, the effect of paternal obesity on gene expression in the preimplantation embryo has not been fully studied. To this end, we investigated whether paternal obesity is associated with gene expression changes in eight-cell stage embryos fathered by males on a high-fat diet. We used single embryo RNA-seq to compare the gene expression profile of embryos generated by males on a high fat (HFD) versus control (CD) diet. This analysis revealed significant upregulation of the Samd4b and Gata6 gene in embryos in response to a paternal HFD. Furthermore, we could show a significant increase in expression of both Gata6 and Samd4b during differentiation of stromal vascular cells into mature adipocytes. These findings suggest that paternal obesity may induce changes in the male germ cells which are associated with the gene expression changes in the resulting preimplantation embryos.

scholarly journals Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 301-311 ◽  
Author(s):  
Paolo Rinaudo ◽  
Richard M Schultz
Keyword(s):  
Gene Expression ◽  
Preimplantation Embryos ◽  
Expression Analysis Systematic Explorer ◽  
Cell Stage ◽  
Global Pattern ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Global Patterns ◽  
The One

Culture of preimplantation embryos affects gene expression. The magnitude of the effect on the global pattern of gene expression, however, is not known. We compared global patterns of gene expression in blastocysts cultured from the one-cell stage in either Whitten’s medium or KSOM + amino acids (KSOM/AA) with that of blastocysts that developed in vivo, using the Affymetrix MOE430A chip. The analysis revealed that expression of 114 genes was affected after culture in Whitten’s medium, whereas only 29 genes were mis-expressed after culture in KSOM/AA. Expression Analysis Systematic Explorer was used to identify biological and molecular processes that are perturbed after culture and indicated that genes involved in protein synthesis, cell proliferation and transporter function were down-regulated after culture in Whitten’s medium. A common set of genes involved in transporter function was also down-regulated after culture in KSOM/AA. These results provide insights as to why embryos develop better in KSOM/AA than in Whitten’s medium, and highlight the power of microarray analysis to assess global patterns of gene expression.

scholarly journals Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

Reproduction ◽  
2016 ◽  
Vol 152 (5) ◽  
pp. 417-430 ◽  
Author(s):  
Atsushi Fukuda ◽  
Atsushi Mitani ◽  
Toshiyuki Miyashita ◽  
Hisato Kobayashi ◽  
Akihiro Umezawa ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Splice Variants ◽  
Protein Localization ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Sequencing Analysis ◽  
Dependent Manner ◽  
Cell Stage ◽  
Protein Levels ◽  
Oct4 Protein

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8–16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3–5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.

A novel H+ permeability dominating intracellular pH in the early mouse embryo

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1353-1361
Author(s):  
J.M. Baltz ◽  
J.D. Biggers ◽  
C. Lechene
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Preimplantation Embryos ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
K Concentration ◽  
Early Mouse

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electrochemical gradient: its dependence on external K+ concentration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.

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.

scholarly journals Mitochondrial membrane potential in 2-cell stage embryos correlates with the success of preimplantation development

Reproduction ◽  
2014 ◽  
Vol 147 (5) ◽  
pp. 627-638 ◽  
Author(s):  
Kouji Komatsu ◽  
Akira Iwase ◽  
Miki Mawatari ◽  
Jingwen Wang ◽  
Mamoru Yamashita ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Developmental Process ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Hormonal Stimulation ◽  
Maternal Mrna

Hormonal stimulation in superovulation induces female mice to ovulate more oocytes than spontaneous ovulation. Because the superovulated oocytes contain a number of oocytes that normally regress before spontaneous ovulation or immature oocytes, the development of some embryos that derive from these oocytes by IVF is prevented. Therefore, the quality of superovulated oocytes should differ from that of spontaneously ovulated oocytes. In this study, we evaluated the quality of superovulated oocytes, by examining 1- and 2-cell stage embryos, in which the development mainly depends on the maternal mRNA, proteins, and mitochondria that are contained in the oocytes, and we then measured the mitochondrial membrane potential (ΔΨm) of the 1- and 2-cell stage,in vivo-fertilized, and IVF embryos. The ΔΨmof 1-cell stage IVF embryos was lower than that ofin vivo-fertilized embryos; however, there was no difference between IVF embryos. During the developmental process from 1- to 2-cell stage, the ΔΨmofin vivo-fertilized embryos was highly upregulated, whereas a number of IVF embryos remained unchanged. As a result, 2-cell stage embryos were divided into two groups: high- and low- ΔΨm2-cell stage IVF embryos. The development of low-ΔΨm2-cell stage IVF embryos tended to be arrested after the 2-cell stage. These results indicated that the upregulation of ΔΨmduring the 1- to 2-cell stage was important in the development of early preimplantation embryos; there were some defects in the mitochondria of superovulated oocytes, which prevented their development.

Regulation of desmocollin transcription in mouse preimplantation embryos

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 743-753 ◽  
Author(s):  
J.E. Collins ◽  
J.E. Lorimer ◽  
D.R. Garrod ◽  
S.C. Pidsley ◽  
R.S. Buxton ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Splice Variants ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Cleavage Stages

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527–539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

The conserved amphipatic alpha-helical core motif of RARgamma and RARalpha activating domains is indispensable for RA-induced differentiation of F9 cells

2000 ◽  
Vol 113 (16) ◽  
pp. 2887-2895 ◽  
Author(s):  
J. Plassat ◽  
L. Penna ◽  
P. Chambon ◽  
C. Rochette-Egly
Keyword(s):  
Target Gene ◽  
Activation Function ◽  
Core Motif ◽  
Knock Out ◽  
F9 Cells ◽  
Helix 12 ◽  
Ec Cells ◽  
Induction Of Differentiation ◽  
Parietal Endoderm ◽  
Endodermal Differentiation

In monolayers cultures, retinoic acid (RA) induces the differentiation of F9 embryonal carcinomal (EC) cells into primitive endoderm-like cells, while a combination of RA and dibutyryl cAMP leads to parietal endoderm-like differentiation. Knock out of all RARgamma isoforms (RARgamma(-/-) line) drastically impairs primitive and subsequent parietal endodermal differentiation and affects the induction of many endogenous RA-responsive genes. Using lines that reexpress RARgamma2 or overexpress RARalpha1 lacking their AF-2AD core (RARgammadeltaAF2 and RARalphadeltaAF2, respectively), we show that this conserved amphipatic alpha-helical motif (helix 12) of the ligand binding domain, and therefore the activation function AF-2 of both receptors, is required for the induction of differentiation and target gene expression upon RA treatment of F9 EC cells. We also show that these deletion mutants behave as dominant negatives.

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