Quantitative morphological analysis of early mouse embryogenesis in vitro

Development ◽  
1977 ◽  
Vol 40 (1) ◽  
pp. 91-100
Author(s):  
Russell L. Deter
Keyword(s):  
Morphological Analysis ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Perfusion Culture ◽  
Blastocyst Stage ◽  
Mouse Development ◽  
Mesh Structure ◽  
Early Mouse ◽  
Quantitative Morphological Analysis

To facilitate a quantitative morphological analysis of early mouse development under controlled conditions, a perfusion culture system capable of supporting embryogenesis to blastocyst stage has been developed. The use of a mesh system allows identification of individual embryos by position, and control of their orientation during culture and preparation for light and electron microscopy. Quantitative evaluation of tissue-processing procedures has permitted selection of conditions which reduce changes in linear dimensions to −1·6 ± 1·8 % in two-cell embryos. Through the definition of a coordinate system based on mesh structure and the development of a special sectioning procedure, sections can be localized within the intact embryo and three-dimensional coordinates given to any element of embryo volume.

scholarly journals H3K27me3 at Pericentromeric Heterochromatin is a Defining Feature of the Early Mouse Blastocyst

2022 ◽  
Author(s):  
Mélanie Pailles ◽  
Mélanie Hirlemann ◽  
Vincent Brochard ◽  
Martine Chebrout ◽  
Jean-François Oudin ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
Pericentromeric Heterochromatin ◽  
Embryonic Cells ◽  
Mouse Development ◽  
Mouse Blastocyst ◽  
Major Satellite ◽  
Post Translational Modifications ◽  
Early Mouse ◽  
Diffuse Distribution

Abstract Early mouse development is characterized by structural and epigenetic changes at the chromatin level while cells progress towards differentiation. At blastocyst stage, the segregation of the three primordial lineages is accompanied by establishment of differential patterns of DNA methylation and post-translational modifications of histones, such as H3K27me3. In this study, we have analysed the dynamics of H3K27me3 at pericentromeric heterochromatin (PCH) during development of the mouse blastocyst, in comparison with cultured embryonic cells. We show that this histone modification is first enriched at PCH in the whole embryo and evolves into a diffuse distribution in epiblast during its specification and maturation. Concomitantly, the level of transcription from major satellite decreases. Stem cells derived from blastocyst (naïve ESCs and TSCs) do not fully maintain the H3K27me3 enrichment at PCH. Moreover, the dynamic of H3K27me3 at PCH during in vitro conversion from naïve to primed pluripotent state and during ESCs derivation suggests that the mechanisms underlying the control of this histone mark at PCH are different in embryo and in vitro. We also conclude that the non-canonical presence of H3K27me3 at PCH is a defining feature of embryonic cells in the young blastocyst before epiblast segregation.

Lethality of a tritiated amino acid in early mouse embryos

Development ◽  
1985 ◽  
Vol 88 (1) ◽  
pp. 209-217
Author(s):  
Janet L. Wiebold ◽  
Gary B. Anderson
Keyword(s):  
Specific Activity ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Radiation Induced ◽  
Specific Activities ◽  
Tritiated Amino Acids ◽  
Early Mouse

2- to 4-cell and morula- to blastocyst-stage mouse embryos were cultured for 1 h in tritiated leucine at two specific activities and their subsequent development followed in vitro and in vivo (after transfer to recipients), respectively. 2- to 4-cell embryos that incorporated an average of 42 d.p.m. per embryo were impaired in their ability to develop to the morula and blastocyst stage. Recipients receiving morulae and blastocysts that had incorporated an average of 384 d.p.m. per embryo failed to produce young. Reduction of the specific activity improved the viability of embryos both in vitro and in vivo but development was still less than that of unlabelled embryos. Protein degradation curves were different for both 2- to 4-cell and morulato blastocyst-stage embryos labelled at the two different specific activities. Most studies using tritiated amino acids have employed higher specific activities than those used here and they may have to be reevaluated due to the possibility of radiation-induced artifacts.

Localization of cytoskeletal proteins in preimplantation mouse embryos

Development ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 211-225
Author(s):  
E. Lehtonen ◽  
R. A. Badley
Keyword(s):  
Blastocyst Stage ◽  
Cytoskeletal Proteins ◽  
Mouse Embryos ◽  
Trophectoderm Cells ◽  
Apparent Concentration ◽  
Preimplantation Mouse ◽  
Early Mouse ◽  
Filament Protein

The immunofluorescence technique was used to detect the presence and distribution of actin, alpha-actinin, tubulin and 10 nm filament protein in early mouse embryos. Actin and alpha-actinin stainings showed a distinct concentration to a peripheral layer in the cleavage-stage blastomeres and in trophectoderm cells. Dots of fluorescence appeared in this cortical staining pattern. The distribution of tubulin staining in the blastomere cytoplasm was relatively even with apparent concentration at the perinuclear region and frequently at wide intercellular contact areas. 10 nm filament protein was distributed evenly in the blastomere cytoplasm without cortical concentration of the label. At the blastocyst stage, the trophectoderm cells in blastocyst outgrowths in vitro developed well organized cytoskeletons including both microfilament, microtubule and 10 nm filament elements. Comparable structures were not observed in blastocysts in vivo, or in late hatched blastocysts cultured in suspension. The morphogenetic significance of the observations is discussed.

scholarly journals G9a regulates temporal preimplantation developmental program and lineage segregation in blastocyst

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jan J Zylicz ◽  
Maud Borensztein ◽  
Frederick CK Wong ◽  
Yun Huang ◽  
Caroline Lee ◽  
...  
Keyword(s):  
Cell Fate ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Mouse Development ◽  
Cell Stage ◽  
Developmental Progression ◽  
Early Mouse ◽  
The Impact

Early mouse development is regulated and accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2). Previously, we provided insights into its role in post-implantation development (Zylicz et al., 2015). Here we explore the impact of depleting the maternally inherited G9a in oocytes on development shortly after fertilisation. We show that G9a accumulates typically at 4 to 8 cell stage to promote timely repression of a subset of 4 cell stage-specific genes. Loss of maternal inheritance of G9a disrupts the gene regulatory network resulting in developmental delay and destabilisation of inner cell mass lineages by the late blastocyst stage. Our results indicate a vital role of this maternally inherited epigenetic regulator in creating conducive conditions for developmental progression and on cell fate choices.

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.

The paternal allele of the H19 gene is progressively silenced during early mouse development: the acetylation status of histones may be involved in the generation of variegated expression patterns

Development ◽  
1998 ◽  
Vol 125 (1) ◽  
pp. 61-69 ◽  
Author(s):  
K. Svensson ◽  
R. Mattsson ◽  
T.C. James ◽  
P. Wentzel ◽  
M. Pilartz ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Trichostatin A ◽  
Expression Patterns ◽  
Position Effect ◽  
Life Time ◽  
Paternal Allele ◽  
Position Effect Variegation ◽  
Mouse Development ◽  
Early Mouse

Transcriptional silencing can reflect heritable, epigenetic inactivation of genes, either singly or in groups, during the life-time of an organism. This phenomenon is exemplified by parent-of-origin-specific inactivation events (genomic imprinting) for a subset of mammalian autosomal genes, such as H19. Very little is known, however, about the timing and mechanism(s) of silencing of the paternal H19 allele during mouse development. Using a novel in situ approach, we present evidence that the silencing of the paternal H19 allele is progressive in the trophectodermal lineage during early mouse development and generates variegated expression patterns. The silencing process apparently involves recruitment of histone deacetylases since the mosaic paternal-specific H19 expression reappears in trichostatin A-treated mouse conceptuses, undergoing in vitro organogenesis. Moreover, the paternal H19 alleles of PatDup.d7 placentas, in which a region encompassing the H19 locus of chromosome 7 is bipaternally derived, partially escape the silencing process and are expressed in a variegated manner. We suggest that allele-specific silencing of H19 share some common features with chromatin-mediated silencing in position-effect variegation.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

2021 ◽  
Vol 41 ◽  
Author(s):  
Natália P.P. Freitas ◽  
Maria Márcia M.S. Maior ◽  
Beatriz A.P. Silva ◽  
Marcus R.L. Bezerra ◽  
José F. Nunes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Platelet Rich Plasma ◽  
Three Dimensional ◽  
In Vitro Production ◽  
Mesh Structure ◽  
Prp Gel

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

scholarly journals In vivo–mimicking microfluidic perfusion culture of pancreatic islet spheroids

2019 ◽  
Vol 5 (11) ◽  
pp. eaax4520 ◽  
Author(s):  
Yesl Jun ◽  
JaeSeo Lee ◽  
Seongkyun Choi ◽  
Ji Hun Yang ◽  
Maike Sander ◽  
...  
Keyword(s):  
Drug Testing ◽  
Cell Damage ◽  
Three Dimensional ◽  
Perfusion Culture ◽  
Interstitial Flow ◽  
Static Culture ◽  
Culture Models ◽  
Main Component

Native pancreatic islets interact with neighboring cells by establishing three-dimensional (3D) structures, and are surrounded by perfusion at an interstitial flow level. However, flow effects are generally ignored in islet culture models, although cell perfusion is known to improve the cell microenvironment and to mimic in vivo physiology better than static culture systems. Here, we have developed functional islet spheroids using a microfluidic chip that mimics interstitial flow conditions with reduced shear cell damage. Dynamic culture, compared to static culture, enhanced islet health and maintenance of islet endothelial cells, reconstituting the main component of islet extracellular matrix within spheroids. Optimized flow condition allowed localization of secreted soluble factors near spheroids, facilitating diffusion-mediated paracrine interactions within islets, and enabled long-term maintenance of islet morphology and function for a month. The proposed model can aid islet preconditioning before transplantation and has potential applications as an in vitro model for diabetic drug testing.

Early embryology af the marsupials Isoodon macrourus and Perameles nasuta

1976 ◽  
Vol 24 (3) ◽  
pp. 361 ◽  
Author(s):  
AG Lyne ◽  
DE Hollis
Keyword(s):  
Electron Microscopy ◽  
Outer Surface ◽  
Zona Pellucida ◽  
Light And Electron Microscopy ◽  
Blastocyst Stage ◽  
Embryonic Cells ◽  
Crystalloid Inclusions ◽  
Early Mouse ◽  
Number Of Cells ◽  
Rabbit Embryos

Twelve embryos, ranging from a four-celled stage to late unilaminar blastocysts, were obtained from the bandicoots I. macrourus and P. nasuta and examined by light and electron microscopy. These early stages covered at least one-quarter of the 12.5-day gestation period. The three non-cellular egg membranes characteristic of marsupials (zona pellucida, mucoid coat and shell membrane) were present, although the zona was sometimes absent or discontinuous in the intermediate and late unilaminar blastocysts examined. At the four-celled stage the blastomeres were close to the zona, but they had lost contact with each other, probably due to the extrusion of yolk, a phenomenon which has been described in other marsupials. The embryo did not increase in diameter until it was composed of at least 75 contiguous cells, which were in contact with the zona. In several of the larger blastocysts the protoderm cells had lost contact with the zona. Subsequently, the number of cells increased considerably and they were flattened against the egg membranes to form the late unilaminar blastocyst stage. Electron microscopy of the protoderm cells revealed the presence of numerous microvilli, particularly on the outer surface, and a range of other structures as great as those found in eutherian mammals. Remnants of spindle bridges were common in one 75-celled embryo. The yolk material in the blastocoele was also composed of a variety of structures, including small crystalloid inclusions composed of hexagonal units about 8-10 nm in diameter. Similar crystalloids have been described in the cells of early mouse and rabbit embryos and in egg and embryonic cells of various amphibians.

Sign in / Sign up

Export Citation Format

Share Document