scholarly journals The absence of a Ca2+ signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 45-57 ◽  
Author(s):  
N T Rogers ◽  
G Halet ◽  
Y Piao ◽  
J Carroll ◽  
M S H Ko ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Expression Patterns ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Distinct Pattern ◽  
Egg Activation ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genome Activation

A series of Ca2+ oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca2+ signal. We have triggered parthenogenetic egg activation with cycloheximide that causes no Ca2+ increase, with ethanol that causes a single large Ca2+ increase, or with Sr2+ that causes Ca2+ oscillations. Eggs were co-treated with cytochalasin D to make them diploid and they formed pronuclei and two-cell embryos at high rates with each activation treatment. However, far fewer of the embryos that were activated by cycloheximide reached the blastocyst stagecompared tothose activated by Sr2+ orethanol. Any cycloheximide-activated embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr2+-activated embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca2+ increase because they developed to blastocyst stages at high rates when co-treated with Sr2+ or ethanol. Embryos activated by either Sr2+ or cycloheximide showed similar signs of initial embryonic genome activation (EGA) when measured using a reporter gene. However, microarray analysis of gene expression at the eight-cell stage showed that activation by Sr2+ leads to a distinct pattern of gene expression from that seen with embryos activated by cycloheximide. These data suggest that activation of mouse eggs in the absence of a Ca2+ signal does not affect initial parthenogenetic events, but can influence later gene expression and 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.

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.

Effect of the oxidative phosphorylation uncoupler 2,4-dinitrophenol on hypoxia-inducible factor-regulated gene expression in bovine blastocysts

2004 ◽  
Vol 16 (7) ◽  
pp. 665 ◽  
Author(s):  
A. J. Harvey ◽  
K. L. Kind ◽  
J. G. Thompson
Keyword(s):  
Gene Expression ◽  
Oxidative Phosphorylation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Hypoxia Inducible Factor ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Vegf Mrna ◽  
Embryonic Genome ◽  
Regulated Gene Expression

In cattle embryos, development to the blastocyst stage is improved in the presence of 10 μm 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, coincident with an increase in glycolytic activity following embryonic genome activation. The present study examined redox-sensitive gene expression and embryo development in response to the addition of DNP post-compaction. 2,4-Dinitrophenol increased the expression of hypoxia-inducible factor 1α and 2α (HIF1α, HIF2α) mRNA. Although HIF1α protein remained undetectable in bovine blastocysts, HIF2α protein was localised within the nucleus of trophectoderm and inner cell mass (ICM) cells of blastocysts cultured in the presence or absence of DNP, with a slight increase in staining evident within the ICM in blastocysts cultured in the presence of DNP. However, the expression of GLUT1 and VEGF mRNA, genes known to be regulated by HIFs, was unaffected by the addition of DNP to the culture. Although the development of Grade 1 and 2 blastocysts was unaltered by the addition of DNP post compaction in the present study, a significant increase in the proportion of ICM cells was observed. Results indicate that 10 μm DNP improves the quality of bovine embryos, coincident with increased HIF2α protein localisation within ICM cells and increased HIFα mRNA levels. Therefore, the results demonstrate redox-regulated expression of HIF2.

Tracing the origin of the placental trophoblast cells in mouse embryo development†

2019 ◽  
Vol 102 (3) ◽  
pp. 598-606
Author(s):  
Shanshan Guo ◽  
Xiuhong Cui ◽  
Xiangxiang Jiang ◽  
Shuguang Duo ◽  
Shiwen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Inner Cell Mass ◽  
Expression Patterns ◽  
Cell Mass ◽  
Cell Fates ◽  
Cell Stage ◽  
Mouse Fetus ◽  
Cell Fate Decisions ◽  
Distinct Cell

Abstract The placenta, which originates from the trophectoderm (TE), is the first organ to form during mammalian embryogenesis. Recent studies based on bioinformatics analysis have revealed that heterogeneous gene expression initiates cell-fate decisions and directs two distinct cell fates by modulating the balance of pluripotency and differentiation as early as the four-cell stage. However, direct developmental evidence to support this is still lacking. To address at which stage the cell fate of the TE and inner cell mass (ICM) is determined, in this study, we administered a microinjection of Cre mRNA into a single blastomere of the mTmG mouse at different cleavage stages before implantation to examine the distributions of the descendants of the single-labeled cell in the mouse fetus and the placenta at E12.5. We found that the descendants of the labeled cells at the two-cell stage contributed to both the placenta and the fetus. Notably, the derivatives of the labeled cells at the four-cell stage fell into three categories: (1) distributed in both embryonic and extraembryonic lineages, (2) distributed only in mouse placental trophoblast layers, or (3) distributed only in the lineage derived from the ICM. In addition, these results fell in line with single-cell studies focusing on gene expression patterns that characterize particular lineages within the blastocyst. In conclusion, this study shows that the four-cell blastomeres differ in their individual developmental properties insofar as they contribute to either or both the ICM and trophoblast fate.

scholarly journals SMCHD1 terminates the first embryonic genome activation event in mouse two-cell embryos and contributes to a transcriptionally repressive state

2019 ◽  
Vol 317 (4) ◽  
pp. C655-C664 ◽  
Author(s):  
Meghan L. Ruebel ◽  
Kailey A. Vincent ◽  
Peter Z. Schall ◽  
Kai Wang ◽  
Keith E. Latham
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Marker Genes ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Protein Levels ◽  
Embryonic Genome ◽  
Maternal Effect Gene ◽  
Integral Role ◽  
Genome Activation

Embryonic genome activation (EGA) in mammals begins with transient expression of a large group of genes (EGA1). Importantly, entry into and exit from the 2C/EGA state is essential for viability. Dux family member genes play an integral role in EGA1 by activating other EGA marker genes such as Zscan4 family members. We previously reported that structural maintenance of chromosomes flexible hinge domain-containing protein 1 ( Smchd1) is expressed at the mRNA and protein levels in mouse oocytes and early embryos and that elimination of Smchd1 expression inhibits inner cell mass formation, blastocyst formation and hatching, and term development. We extend these observations here by showing that siRNA knockdown of Smchd1 in zygotes results in overexpression of Dux and Zscan4 in two-cell embryos, with continued overexpression of Dux at least through the eight-cell stage as well as prolonged expression of Zscan4. These results are consistent with a role for SMCHD1 in promoting exit from the EGA1 state and establishing SMCHD1 as a maternal effect gene and the first chromatin regulatory factor identified with this role. Additionally, bioinformatics analysis reveals that SMCHD1 also contributes to the creation of a transcriptionally repressive state to allow correct gene regulation.

scholarly journals 246 EXPRESSION PATTERN OF NANOG AND Par3 GENES IN IN VITRO-DERIVED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 231 ◽  
Author(s):  
F. Gandolfi ◽  
F. Cillo ◽  
S. Antonini ◽  
S. Colleoni ◽  
I. Lagutina ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cdna Sequence ◽  
Inner Cell Mass ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Genome Activation

Homeobox genes have been demonstrated to be important in patterning and lineage specification during early embryogenesis. Nanog belongs to the family of DNA-binding transcription factors and has been shown to maintain pluripotency of embryonic stem cells, both in murine and human. Par3 plays an essential role in determining cell fate of the early mouse embryo, leading to the generation of the inner cell mass and the trophectoderm. No information is available on these genes in the bovine; therefore, the aim of the present study was to identify and characterize Nanog and Par3 expression in bovine embryos. Oocytes recovered from slaughterhouse ovaries were matured for 22 h, fertilized in vitro and then cultured in mSOFaa medium. RNA was extracted from pools of five oocytes and embryos at different stages of development (2-, 4-, 8-, 16-cell, morula and blastocyst). It was then reverse transcribed, and PCR runs were carried out with primers specifically designed for Nanog and Par3, based on the sequence data bank available. The amplified products were separated on a 2% TAE agarose gel, purified, sequenced and aligned using Clustal W. Comparison of the bovine Nanog cDNA sequence (EMBL AM039957) with databases revealed a 84% degree of homology with the human, 97% with the mouse, and 82% with the goat genes. IVF bovine embryos express Nanog only upon genome activation, becoming detectable from the 8-cell stage onward indicating that Nanog is zygotically expressed in the bovine similar to what happens in mouse, pig and goat. Bovine Par3 cDNA sequence (EMBL AM039956) shows a high degree of homology with human (83%), mouse (81%), and rat (79%). Also Par3 is expressed only upon the maternal to embryonic transition (MET) at the 8-cell stage. As opposed to the expression patterns of other early embryo genes, like Oct-4 and Zar-1, Nanog and Par3 expression patterns in bovine embryos closely resemble those described in the mouse. Since both are absent in the ooplasm and before MET, they represent useful markers for genome activation. This work was supported by FIRB RBNE01HPMX, FIRST 2004 and ESF-EuroStells.

scholarly journals Revealing the dynamics of gene expression during embryonic genome activation and first differentiation in the rabbit embryo with a dedicated array screening

2009 ◽  
Vol 36 (2) ◽  
pp. 98-113 ◽  
Author(s):  
R. D. Léandri ◽  
C. Archilla ◽  
L. C. Bui ◽  
N. Peynot ◽  
Z. Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Cdna Array ◽  
Epigenetic Modifications ◽  
Embryonic Cells ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Genome Activation

Early mammalian development is characterized by extensive changes in nuclear functions that result from epigenetic modifications of the newly formed embryonic genome. While the first embryonic cells are totipotent, this status spans only a few cell cycles. At the blastocyst stage, the embryo already contains differentiated trophectoderm cells and pluripotent inner cell mass cells. Concomitantly, the embryonic genome becomes progressively transcriptionally active. During this unique period of development, the gene expression pattern has been mainly characterized in the mouse, in which embryonic genome activation (EGA) spans a single cell cycle after abrupt epigenetic modifications. To further characterize this period, we chose to analyze it in the rabbit, in which, as in most mammals, EGA is more progressive and occurs closer to the first cell differentiation events. In this species, for which no transcriptomic arrays were available, we focused on genes expressed at EGA and first differentiation and established a 2,000-gene dedicated cDNA array. Screening this with pre-EGA, early post-EGA, and blastocyst embryos divided genes into seven clusters of expression according to their regulation during this period and revealed their dynamics of expression during EGA and first differentiation. Our results point to transient properties of embryo transcriptome at EGA, due not only to the transition between maternal and embryonic transcripts but also to the transient expression of a subset of embryonic genes whose functions remained largely uncharacterized. They also provide a first view of the functional consequences of the changes in gene expression program.

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.

scholarly journals Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq

2020 ◽  
Vol 21 (18) ◽  
pp. 6488
Author(s):  
Arkadiusz Kajdasz ◽  
Ewelina Warzych ◽  
Natalia Derebecka ◽  
Zofia E. Madeja ◽  
Dorota Lechniak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Rna Seq ◽  
Expression Of Genes ◽  
Bovine Blastocyst

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

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).

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