Actin nucleator Arp2/3 complex is essential for mouse preimplantation embryo development

2013 ◽  
Vol 25 (4) ◽  
pp. 617 ◽  
Author(s):  
Shao-Chen Sun ◽  
Qing-Ling Wang ◽  
Wei-Wei Gao ◽  
Yong-Nan Xu ◽  
Hong-Lin Liu ◽  
...  
Keyword(s):  
Cell Division ◽  
Embryo Development ◽  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Specific Inhibitor ◽  
Cell Stage ◽  
Diverse Range ◽  
Mouse Embryo Development ◽  
Actin Nucleator

The Arp2/3 complex is a critical actin nucleator, which promotes actin assembly and is widely involved in a diverse range of actin-related processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Previous studies showed that the Arp2/3 complex regulates spindle migration and asymmetric division during mouse oocyte maturation; however, the role of the Arp2/3 complex in early mouse embryo development is still unknown. The results of the present study show that the Arp2/3 complex is critical for cytokinesis during mouse embryo development. The Arp2/3 complex was concentrated at the cortex of each cell at the 2- to 8-cell stage and the peripheral areas of the morula and blastocyst. Inhibition of the Arp2/3 complex by the specific inhibitor CK666 at the zygote stage caused a failure in cell division; mouse embryos failed to undergo compaction and lost apical–basal polarity. The actin level decreased in the CK666-treated group, and two or more nuclei were observed within a single cell, indicating a failure of cell division. Addition of CK666 at the 8-cell stage caused a failure of blastocyst formation, and CDX2 staining confirmed the loss of embryo polarity and the failure of trophectoderm and inner cell mass formation. Taken together, these data suggest that the Arp2/3 complex may regulate mouse embryo development via its effect on cell division.

When and how does cell division order influence cell allocation to the inner cell mass of the mouse blastocyst?

Development ◽  
1987 ◽  
Vol 100 (2) ◽  
pp. 325-332
Author(s):  
C.L. Garbutt ◽  
M.H. Johnson ◽  
M.A. George
Keyword(s):  
Cell Division ◽  
Cell Population ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
The Other ◽  
Mouse Blastocyst ◽  
Short Term ◽  
Cell Stage ◽  
Cell Embryo

Aggregate 8-cell embryos were constructed from four 2/8 pairs of blastomeres, one of which was marked with a short-term cell lineage marker and was also either 4 h older (derived from an early-dividing 4-cell) or 4 h younger (derived from a late-dividing 4-cell) than the other three pairs. The aggregate embryos were cultured to the 16-cell stage, at which time a second marker was used to label the outside cell population. The embryos were then disaggregated and each cell was examined to determine its labelling pattern. From this analysis, we calculated the relative contributions to the inside cell population of the 16-cell embryo of older and younger cells. Older cells were found to contribute preferentially. However, if the construction of the aggregate 8-cell embryo was delayed until each of the contributing 2/8 cell pairs had undergone intercellular flattening and then had been exposed to medium low in calcium to reverse this flattening immediately prior to aggregation, the advantage possessed by the older cells was lost. These results support the suggestion that older cells derived from early-dividing 4-cell blastomeres contribute preferentially to the inner cell mass as a result of being early-flattening cells.

scholarly journals PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1272 ◽  
Author(s):  
Muhammad Idrees ◽  
Lianguang Xu ◽  
Seok-Hwan Song ◽  
Myeong-Don Joo ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Specific Inhibitor ◽  
Mrna Translation ◽  
Bovine Oocyte ◽  
Blastocyst Development

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

213 HOXB9 PROTEIN IS PRESENT THROUGHOUT EARLY EMBRYO DEVELOPMENT IN THE BOVINE

2013 ◽  
Vol 25 (1) ◽  
pp. 255
Author(s):  
C. Sauvegarde ◽  
D. Paul ◽  
R. Rezsohazy ◽  
I. Donnay
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Immature Oocyte ◽  
Cell Stage ◽  
Morula Stage ◽  
Oocyte Stage

Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).

scholarly journals Initiation of X Chromosome Inactivation during Bovine Embryo Development

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1016 ◽  
Author(s):  
Bo Yu ◽  
Helena T. A. van Tol ◽  
Tom A.E. Stout ◽  
Bernard A. J. Roelen
Keyword(s):  
Embryo Development ◽  
X Chromosome ◽  
Developmental Process ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Morula Stage

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

142 DYNAMICS OF Tet FAMILY DURING PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 183 ◽  
Author(s):  
J. Teson ◽  
K. Lee ◽  
L. Spate ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Profile ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Dna Demethylation ◽  
Dimensional Structure ◽  
Cell Stage ◽  
Donor Cells

One of the key regulators of gene expression in mammals is DNA methylation. The Tet family (Tet1–3) is suggested to be involved in regulating the level of methylation by hydroxylating a methyl group from 5-methylcytosine to form 5-hydroxymethylcystosine. This hydroxylation alters the 3-dimensional structure of the DNA and results in altered gene expression. Previous studies conducted in the mouse have shown that Tet1 is important for inner cell mass specification by regulating the apparent level of methylation on a specific promoter region in blastocysts and Tet3 is related to the apparent paternal DNA demethylation after fertilization by hydroxylating the paternal genome. The objective of this study was to investigate the expression profile of the Tet family in porcine oocytes and pre-implantation-stage embryos derived from IVF and somatic cell nuclear transfer (SCNT). The RNA was isolated from donor cells, germinal vesicle (GV), MII and 2-cell and blastocyst stage embryos (20 oocytes or embryos per group). Levels of mRNA for each Tet gene were measured by quantitative real-time RT-PCR. The levels of each mRNA transcript were compared to YWHAG, a housekeeping gene that shows a constant level of expression throughout pre-implantation embryo development and normalized to the GV stage. The analysis was repeated with 3 biological replications and 2 experimental replications. Differences in gene expression were compared by ANOVA and P < 0.05 was considered significant. No difference was found in the levels of the Tet family members between GV and MII stage oocytes. Compared with GV stage oocytes, up-regulation of Tet3 at the 2-cell stage was detected in both IVF and SCNT embryos, 4.7 and 6.2 fold, respectively. A dramatic increase in Tet1 was also observed at the blastocyst stage in IVF and SCNT embryos when compared with the GV stage, 65.7 and 79.7 fold increases, respectively. Interestingly, the level of Tet3 was down-regulated in blastocyst embryos at a 25 or more fold decrease compared with GV. The level of Tet2 remained constant throughout embryo development. Embryos (2-cell and blastocyst) compared from IVF and SCNT showed no difference in Tet expression levels. Donor cells had significantly lower levels of Tet2 and Tet3 when compared with GV. Our results indicate that the Tet family shows a dynamic expression profile during porcine pre-implantation embryo development. High expression of Tet3 in 2-cell stage embryos suggests its importance during the post-activation demethylation process. The increase of Tet1 transcript in blastocysts suggests that Tet1 is involved in regulating the type of methylation at the blastocyst stage. These results are consistent with results from previous mouse studies. There was no misregulated expression of the Tet family in SCNT embryos compared with IVF embryos, thus indicating successful reprogramming of the Tet family after SCNT. Lower levels of Tet2 and Tet3 would indicate that Tet1 is important for maintaining type of methylation in donor cells. This is the first report on the profile of the Tet family during porcine pre-implantation embryo development and further studies are needed to clarify their role during this stage.

Gp330 is specifically expressed in outer cells during epithelial differentiation in the preimplantation mouse embryo

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3289-3299 ◽  
Author(s):  
C. Gueth-Hallonet ◽  
A. Santa-Maria ◽  
P. Verroust ◽  
B. Maro
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Transcriptional Level ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Extensive Cell ◽  
High Level ◽  
Endocytic Activity

During preimplantation development of the mouse embryo, a layer of outer cells differentiates into a perfect epithelium, the trophectoderm. The divergence between the trophectoderm and the inner cell mass takes place from the 8-cell stage to the 64-cell stage and precedes their commitment at the blastocyst stage. In this work, we have investigated the expression of gp330, a 330 × 10(3) M(r) glycoprotein found in clathrin-coated areas of the plasma membrane of some epithelial cells characterized by a high level of endocytic activity. Our results show that gp330 is first synthesized in 16-cell stage embryos and that its appearance is restricted to outer cells until the blastocyst stage. Furthermore, its expression is repressed in inner cells at a post-transcriptional level, probably through the development of extensive cell-cell contacts.

Cytokeratin filament assembly in the preimplantation mouse embryo

Development ◽  
1987 ◽  
Vol 101 (3) ◽  
pp. 565-582 ◽  
Author(s):  
J.C. Chisholm ◽  
E. Houliston
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Lineage Tracing ◽  
Cell Stage ◽  
Filament Assembly ◽  
Filament Networks ◽  
Tracing Techniques ◽  
Cytokeratin Filaments

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.

Membrane organization in the preimplantation mouse embryo

Development ◽  
1985 ◽  
Vol 90 (1) ◽  
pp. 101-121
Author(s):  
Hester P. M. Pratt
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Freeze Fracture ◽  
Molecular Organization ◽  
Surface Polarity ◽  
Cell Stage ◽  
Preimplantation Mouse Embryo ◽  
Preimplantation Mouse ◽  
Polarized Epithelium

The preimplantation mouse blastocyst consists of two differentiated tissues, the trophectoderm (a structurally and functionally polarized epithelium) and the inner cell mass. The divergence of these two cell types can be traced back to a contact dependent polarization of the surface and cytoplasm at the 8-cell stage. Membrane/cytocortical organization during this preimplantation period has been studied using freeze fracture in conjunction with the sterol-binding antibiotic filipin in an attempt to discern the molecular basis and origin of these surface asymmetries. The distribution of filipin reactivity within the different membrane domains showed that the surface polarity exhibited by trophectoderm and by blastomeres of the 8-cell stage is underlain by a heterogeneity in molecular organization of the membrane/cytocortex which may originate prior to the appearance of any overt surface polarity. The results are discussed in terms of the likely basis of this membrane/cytocortical asymmetry, its probable origins and the use of the preimplantation mouse embryo as a model system for studying the assembly of a polarized epithelium.

Size regulation in the mouse embryo

Development ◽  
1986 ◽  
Vol 98 (1) ◽  
pp. 209-217
Author(s):  
G. F. Rands
Keyword(s):  
Growth Rate ◽  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Normal Growth ◽  
Mouse Embryos ◽  
Down Regulation ◽  
Cell Stage ◽  
Size Regulation ◽  
Size Dependent

The study describes an analysis of the development of mouse embryos halved at the 2-cell stage by the destruction of one blastomere, in comparison with control embryos of parallel derivation, at 2·5–13·5 days post coitum. The results showed that: (1) half embryos achieve size regulation some time between 7·5 and 10·5 days; (2) there is an indication that by 13·5 days half embryos may have again dropped back significantly in size relative to controls; (3) preregulation half embryos are slightly retarded developmental, but this does not wholly account for their smaller size: morphogenesis is not size-dependent; (4) early postimplantation half embryos contain a significantly decreased proportion of inner cell mass derivatives and increased proportion of trophectoderm derivatives when compared with controls. A comparison is also made between the up-regulation of half embryos and the down-regulation of aggregate embryos, and it is suggested that size regulation may occur by delaying a change in the normal growth rate.

The establishment of the embryonic-abembryonic axis in the mouse embryo

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 125-134 ◽  
Author(s):  
C.L. Garbutt ◽  
J.C. Chisholm ◽  
M.H. Johnson
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Dividing Cells ◽  
Intercellular Cooperation

The influence of cell division order on the establishment of the embryonic-abembryonic axis (EA axis) of the mouse embryo was investigated. Aggregate embryos were constructed in which a labelled cell (or pair of cells) was combined with a group of unlabelled cells all of which were up to one cell cycle earlier or later in their progress through development to the blastocyst stage. The aggregates were cultured first to the nascent blastocyst stage and then to the expanded blastocyst stage. The positions of the progeny of the labelled cells in relation to the nascent blastocoel and to the orientation of the embryonic-abembryonic axis were recorded. It was concluded that cell division order does influence the establishment of the EA axis, early dividing cells tending to be associated with the nascent blastocoel and the site of the nascent blastocoel tending to mark the site of the abembryonic pole. However, the influence of division order was diminished by a requirement for intercellular cooperation during blastocoel formation and by a counteracting influence of division order arising from its effects on the allocation of cells to the inner cell mass.

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