scholarly journals Human embryonic genome activation initiates at the one-cell stage

2021 ◽  
Author(s):  
Maki Asami ◽  
Brian Y.H. Lam ◽  
Marcella K. Ma ◽  
Kara Rainbow ◽  
Stefanie Braun ◽  
...  
Keyword(s):  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
The One ◽  
Genome Activation

69 GLOBAL H3k9ac AND H3k27me3 EXPRESSION IN BLASTOMERES FROM 8- TO 16-CELL STAGE BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 148
Author(s):  
C. S. Oliveira ◽  
N. Z. Saraiva ◽  
L. Z. Oliveira ◽  
R. V. Serapião ◽  
M. R. de Lima ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Histone Modifications ◽  
Pearson Correlation ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Group A ◽  
Genome Activation

Embryonic genome activation is a crucial step in early embryo development, and is accompanied by a dramatic change in the epigenetic profile of blastomeres. Histone modifications related to euchromatin and heterochromatin can be important parameters to infer developmental competence, as they are affected by manipulation and environmental stress conditions. The aim of this study was to characterise permissive (H3k9ac) and repressive (H3k27me3) histone modifications during the embryonic genome activation cell cycle in bovine embryos, regarding correlation between those marks and variance among blastomeres. For that, bovine embryos were produced by IVF and cultured in SOF medium supplemented with 5 mg mL–1 of BSA and 2.5% FCS in 5% O2 in an air atmosphere for 5 days (70 h after IVF). The 8 to 16 cell embryos were fixed in 4% paraformaldehyde and submitted to H3k9ac and H3k27me3 immunofluorescence assay (mouse anti-H3K9ac monoclonal antibody, 1 : 200; Sigma; rabbit anti-H3k27me3 monoclonal antibody, 1 : 200; Upstate, Charlottesville, VA, USA). Nuclei were counterstained with Hoechst 33342. Images of each embryo were captured (AxioCam, Carl Zeiss, São Paulo, Brazil) and measured for nuclear fluorescence intensity in each blastomere using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA). Mean levels were compared using the Mann-Whitney test and variances were compared using F-test (SAS 9.1, SAS Institute Inc., Cary, NC, USA; P = 0.05). We evaluated 2 replicates and 12 embryos during the transition from the 8 to 16 cell stages, totaling 169 blastomeres. Global H3k27me3 levels varied accordingly to H3k9ac levels, as indicated by a high Pearson correlation coefficient (r = 0.913). Levels of each blastomere were normalized to the lowest level obtained within each embryo. Some embryos displayed a high variation between blastomeres, and, for further analysis, we divided the embryos into groups: group A for embryos that presented similar H3k9ac levels between blastomeres (8 embryos, 66%), and group B for embryos that exhibited higher heterogeneity between blastomeres (at least 2 blastomeres presenting a 2-fold increase compared to the lowest blastomere; 4 embryos, 33%). Mean H3k9ac and H3k27me3 normalized levels were lower for group A [H3k9ac: 1.35 ± 0.29 (A), 1.94 ± 1.02* (B); H3k27me3: 1.33 ± 0.24 (A), 1.99 ± 0.77 (B)], and group A displayed lower variance values (H3k9ac: 0.07 (A), 1.05* (B); H3k27me3: 0.06 (A), 0.60 (B)]. Within each embryo, blastomeres were sorted in ascending order for H3k9ac level (1 to 16), and compared between groups A and B. We detected that mean levels differed (P < 0.05) between groups from blastomere 9 to 16 for H3k9ac and 10 to 16 for H3k27me3. Therefore, in 8- to 16-cell stage embryos, the H3k27me3 repressive mark is highly correlated with the H3k9ac permissive mark. Also, our results describe the presence of 2 distinguishable populations of bovine embryos at this stage, considering their epigenetic status. One population presented similar levels of repressive and permissive marks among blastomeres, whereas the second one displayed a remarkable variation among their blastomeres. This observation should be further studied, as it might reflect distinct cleavage pattern embryos and blastomere competence. The authors acknowledge FAPESP, FAPERJ and CNPq for financial support.

61 REVERSIBLE INHIBITION OF BOVINE MINOR EMBRYONIC GENOME ACTIVATION IMPAIRS PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 138
Author(s):  
R. P. Nociti ◽  
R. V. Sampaio ◽  
V. F. M. H. de Lima ◽  
R. M. Schultz ◽  
P. J. Ross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Reversible Inhibitor ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Embryonic Genome Activation ◽  
Transcriptional Inhibition ◽  
Embryonic Genome ◽  
Genome Activation

Bovine pre-implantation embryos can develop in the absence of gene expression up to the 8/16-cell stage, the time when the major embryonic genome activation (EGA) occurs. Some embryonic genes, however, are transcribed before EGA (minor EGA). This study used a reversible inhibitor of RNA Polymerase II (5,6 dichlorobenzimidazole 1-β-D-ribofuranoside; DRB) to assess the importance of minor EGA for development to the blastocyst stage. Oocytes were matured and inseminated in vitro, and the fertilized eggs were cultured in supplemented KSOMaa and allocated to different treatments 16 h post-insemination (hpi). Development was recorded at 44 and 72 hpi, and the incidence of blastocyst formation on Day 7 (IVF = Day 0) was recorded. Data were analysed by ANOVA followed by Duncan test. First, we tested different DRB concentrations [50 μM (D50), 75 μM (D75), 100 μM (D100), and dimethyl sulfoxide vehicle control (CTRL)] to block development to blastocyst when continuously present. Only embryos in CTRL produced blastocysts (45.0 ± 5.8%; 4 replicates with a total of 391 oocytes examined). No difference in development was observed at 44 h (57.9 ± 16.5, 53.3 ± 10.5, 60.5 ± 19.0, and 52.3 ± 5.8% for D50, D75, D100, and CTRL, respectively) and 72 h (78.9 ± 8.8, 66.1 ± 11.7, 71.5 ± 16.5, and 70.8 ± 5.6% for D50, D75, D100, and CTRL, respectively). Next, in 7 replicates (751 oocytes) we determined the effect of blocking transcription (50 μM DRB) spanning 2 embryo stages (periods of 28 h), initiated at 16 hpi (1&2C), 30 hpi (2&4C), and 44 hpi (4&8C). Controls included DRB treatment from 16 to 72 hpi (1–8C) and CTRL. There was no difference in development at 44 and 72 h. The incidence of blastocyst formation, however, was significantly decreased in all treatment groups compared with CTRL (27.7 ± 4.7; 15.1 ± 3.5; 23.3 ± 3.1; 20.5 ± 1.9; and 42.1 ± 3.2% for 1&2C, 2&4C, 4&8C, 1–8C, and CTRL, respectively). Finally, in 12 replicates (1499 oocytes), the effect of blocking transcription for 14-h periods, spanning mostly a unique cleavage stage, was evaluated. The DRB treatment (50 μM) started at 16 hpi (1C), 30 hpi (2C), 44 hpi (4C), and 58 hpi (8C). Furthermore, 1–16C and CTRL treatments were included. No difference in development at 44 and 72 h were observed. Development to the blastocyst was significantly lower from CTRL (46.0 ± 3.2%) in 2C, 4C, 8C, and 1–16C (28.9 ± 3.9, 26.1 ± 4.2, 30.1 ± 4.8, and 18.9 ± 3.2%, respectively) but not in 1C (34.7 ± 4.4%). In summary, continuous transcriptional inhibition using DRB resulted in a developmental block at the time of major EGA, similar to α-amanitin treatment (an irreversible RNA Polymerase II inhibitor). Transcriptional inhibition during single cleavage stages was sufficient to decrease the developmental potential of the embryo. We conclude that minor EGA has an important role for bovine development. This work was funded by NIH-NICHD R01HD070044 to P. J. Ross. R. P. Nociti was sponsored by CNPQ; R. V. Sampaio was sponsored by FAPESP.

77 OVIDUCT - EMBRYO INTERACTIONS: TWO-WAY TRAFFIC OR A ONE-WAY STREET? TRANSCRIPTOMIC RESPONSE OF THE BOVINE OVIDUCT TO THE PRESENCE OF AN EMBRYO

2014 ◽  
Vol 26 (1) ◽  
pp. 152 ◽  
Author(s):  
V. Maillo ◽  
P. O'Gaora ◽  
J. P. Mehta ◽  
C. De Frutos ◽  
N. Forde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Corpus Luteum ◽  
Differential Expression ◽  
Microarray Analysis ◽  
Differentially Expressed ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Oviduct Epithelium ◽  
Genome Activation

Despite clear evidence of a two-way interaction between the developing conceptus and the uterine endometrium in early pregnancy, the evidence for reciprocal cross-talk during the transit of the embryo through the oviduct is less clear. The aims were (1) to characterise the transcriptome of the bovine oviduct at the initiation of embryonic genome activation (EGA), (2) to examine the effect, if any, of the presence of an embryo on the oviduct transcriptome, and (3) to compare gene expression in the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum. The oestrous cycles of cross-bred beef heifers were synchronized and those recorded in standing oestrus were randomly allocated to control group, nonbred (n = 7), or AI group (n = 11). All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut in half (ampulla and isthmus). Each portion was flushed with 500 μL of PBS to confirm the presence of an oocyte/embryo and was then opened and scraped longitudinally to obtain epithelial cells. Cells were snap-frozen in liquid nitrogen for microarray analysis. All recovered oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum. The recovery rate was 72.7% (8/11) and 83.3% (5/6) for pregnant and cyclic animals, respectively. The stage of the recovered embryos was as follows: 4-cell stage (n = 1), 8-cell stage (n = 5), and 8–16 cell stage (n = 2), whereas in the cyclic group all recovered structures were unfertilized oocytes. The cells of the isthmus from ipsilateral and contralateral oviducts from 5 cyclic and 5 pregnant animals (8-cell embryos) and the ipsilateral ampulla cells from the pregnant animals were used for microarray analysis (Affymetrix Bovine ST array, Affymetrix, Santa Clara, CA, USA). Array data were analysed using BioConductor packages in R and custom CDF files downloaded from MBNI. Preprocessing of raw data was performed with RMA, and differential expression was assessed by linear modelling implemented in the limma package. Genes displaying P < 0.05 after adjustment for multiple testing were considered differentially expressed. A total of 18 809 probe sets were assessed for differential expression. Comparison of pregnant and cyclic oviduct epithelium revealed no significantly altered genes. However, comparison of the isthmus and ampulla of the ipsilateral oviduct in pregnant animals revealed 4011 (P < 0.05) and 2327 (P < 0.01) differentially expressed genes. Some of the gene ontologies involved in biological processes included fatty acid metabolism, cell adhesion, cell morphogenesis, cellular developmental process, and reproduction. In conclusion, we have characterised the transcriptome of the bovine oviduct epithelium at the initiation of embryonic genome activation on Day 3 post-oestrus in pregnant and cyclic heifers. Although large differences in gene expression were observed between the isthmus and ampulla, data suggest that the presence of an 8-cell embryo had no effect on the transcriptome of the cells of the isthmus, although a local effect at the precise position of the embryo cannot be ruled out.

scholarly journals Improving the Quality of Oocytes with the Help of Nucleolotransfer Therapy

2021 ◽  
Vol 14 (4) ◽  
pp. 328
Author(s):  
Michal Benc ◽  
Frantisek Strejcek ◽  
Martin Morovic ◽  
Alexandra Bartkova ◽  
Matej Murin ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rrna Synthesis ◽  
Comprehensive Overview ◽  
Compact Structure ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Early Embryos ◽  
The One ◽  
Almost All ◽  
Genome Activation

The nucleolus is an important nucleus sub-organelle found in almost all eukaryotic cells. On the one hand, it is known as a differentiated active site of ribosome biogenesis in somatic cells, but on the other hand, in fully grown oocytes, zygotes, and early embryos (up to the major embryonic genome activation), it is in the form of a particular homogenous and compact structure called a fibrillar sphere. Nowadays, thanks to recent studies, we know many important functions of this, no doubt, interesting membraneless nucleus sub-organelle involved in oocyte maturation, embryonic genome activation, rRNA synthesis, etc. However, many questions are still unexplained and remain a mystery. Our aim is to create a comprehensive overview of the recent knowledge on the fibrillar sphere and envision how this knowledge could be utilized in further research in the field of biotechnology and nucleolotransfer therapy.

68 EPIGENETIC REMODELING OF HISTONE 3 MARKS DURING BOVINE PRE-IMPLANTATION DEVELOPMENT

2014 ◽  
Vol 26 (1) ◽  
pp. 148
Author(s):  
Y. S. Bogliotti ◽  
L. B. Ferré ◽  
D. J. Humpal ◽  
P. J. Ross
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Mixed Model ◽  
Statistical Significance ◽  
Germinal Vesicle ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Histone 3 ◽  
Embryonic Genome ◽  
Genome Activation

During pre-implantation development, substantial epigenetic changes occur that are thought to play key roles in achieving embryonic genome activation and totipotency. Embryonic genome activation occurs at the 8- to 16-cell stage in cattle and, although it is a crucial step of development, the specific mechanisms involved are still poorly understood. The aim of this study was to determine whether 4 histone 3 marks associated with active genes are remodelled during oocyte and early embryo development in cattle. The dynamics of acetylation of lysine 27 (H3K27ac), di-methylation of lysine 79 (H3K79me2), and mono- and tri-methylation of lysine 4 (H3K4me1, H3K4me3) were assessed by immunofluorescence and confocal microscopy. Ovaries were obtained from an abattoir. Immature germinal vesicle stage oocytes were aspirated from small antral follicles and matured for 24 h to the metaphase II stage (MII). Embryos were produced by in vitro fertilization and collected at different stages of development: pronuclear [PN; 18 h post-fertilization (hpf)], 2-cell (30 hpf), 4-cell (44 hpf), 8-cell (56 hpf), 16-cell (72 hpf), morula (120 hpf), and blastocyst (180 hpf). Three to 4 biological replicates were done per antibody and a total of 197 oocytes per embryo were imaged (8 to 16 per stage/antibody). The images were analysed using Fiji (Schindelin et al. 2012 Nat. Methods 9, 676–682). The average nuclear intensity per oocyte per embryo was adjusted by the average of 2 cytoplasmic areas (background). An ANOVA mixed model was used for statistical analysis using SAS (SAS Institute Inc., Cary, NC, USA). The least squares means of the different stages were compared (within each antibody group) using a Tukey-Kramer adjustment and were considered to be significantly different at P < 0.05. The H3K79me2 marks showed a significant increase from germinal vesicle to MII, a change opposite that of H3K27ac, which experienced a significant decrease between these two stages. The H3K4me1/me3 marks showed no significant changes during oocyte maturation. All 3 methylation marks presented a significant reduction in nuclear intensity from MII to PN, indicating that these marks are actively removed right after fertilization. The opposite effect was observed for the acetylation mark, in which the levels increased significantly from MII to PN. The H3K4me1/me3 marks showed a gradual decrease in intensity levels from the 2-cell stage onward, reaching a minimum at the 16-cell per morula stages. The H3K79me2 levels were low from PN to 16-cell stage, at which point its intensity levels began to increase, reaching statistical significance at the blastocyst stage. The H3K27ac marks showed a slow decrease in intensity levels from the PN stage, achieving statistical significance as it dropped to a minimum at the 16-cell stage. These results show that the global levels of the assayed epigenetic marks undergo dynamic changes during oocyte maturation and embryo development, suggesting that their remodelling may be important for early development. The authors thank Alta Genetics for providing the semen.

198 Germline-Specific Expression of the Murine Oct4-EGFP Transgene in the Pig

2018 ◽  
Vol 30 (1) ◽  
pp. 239
Author(s):  
M. Nowak-Imialek ◽  
D. Herrmann ◽  
A. Frenzel ◽  
H. Niemann
Keyword(s):  
Expression Pattern ◽  
Expression Profile ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Ovarian Cells ◽  
Embryonic Genome ◽  
Oct4 Protein ◽  
Genome Activation ◽  
Egfp Signal ◽  
Egfp Fluorescence

The Oct4 gene is crucial for undisturbed early embryonic development and maintenance of pluripotency in the mouse. It is found in mouse pre-implantation embryos after embryonic genome activation. After gastrulation, expression is restricted to germ cells. Limited research has been performed on OCT4 expression in the domestic pig, which is a valuable large animal model in biomedicine. Previously, we generated Oct4-EGFP reporter pigs carrying the genomic sequence of the murine Oct4 gene fused to the EGFP cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev. 20, 1563-1575, 10.1089/scd.2010.0399). In the present study, we used this animal model to analyse the expression profile of the murine Oct4-EGFP transgene in porcine oocytes, in vivo-derived embryos (4-cell embryos, 8- to 16-cell embryos, morulae, and blastocysts) and ovaries. We studied whether the murine Oct4-EGFP transgene mimics the expression pattern of the endogenous OCT4 protein in transgenic pigs. Immature oocytes were isolated from ovaries of Oct4-EGFP transgenic sows (n = 5) using slicing methods. For collection of porcine embryos, wild-type sows were inseminated with sperm from an Oct4-EGFP transgenic boar. Sows were sacrificed 3, 4, and 5 days after insemination, and embryos were recovered by flushing oviducts and uterus and analysed by confocal microscopy. Ovaries obtained from female animals (5–12 months) were enzymatically dissociated and analysed using flow cytometry. Immature oocytes (n = 19) showed a very low, diffuse EGFP signal in cytoplasm. Embryos up to the 4-cell stage (n = 45) did not show Oct4-EGFP transgene expression. For the first time, EGFP fluorescence was detected at the 8-cell stage (n = 29) and a strong EGFP signal was observed in 16-cell stages and morulae (n = 53). In blastocysts from Day 5 (n = 40) EGFP fluorescence was not restricted to the inner cell mass (ICM), but was also seen in the trophectoderm (TE). Expression of EGFP was not detected in ovarian cells (n = 12). Thereafter, we analysed the expression pattern of endogenous OCT4 protein by immunostaining in nontransgenic porcine oocytes and pre-implantation embryos. As in Oct4-EGFP transgenic embryos, no expression of OCT4 was observed in 4-cell embryos (n = 12). Nuclear staining first became visible at the 8-cell stage (n = 12), with a strong signal observed in 16-cell stages and morulae (n = 18). In blastocysts from Day 5 (n = 26), both ICM and TE cell nuclei showed expression of OCT4 protein. These results demonstrate that the Oct4-EGFP transgene expression pattern reproduces the endogenous OCT4 protein expression profile in porcine oocytes and pre-implantation embryos. The Oct4-EGFP transgene was first detected at the 8-cell stage, consistent with embryonic genome activation, which is initiated at the 4-cell stage. However, Oct4-EGFP expression was not detected in ovarian cells. This might be related to the very low expression pattern of the Oct4-EGFP transgene in primary oocytes. In summary, the Oct4-EGFP transgene in the pig provides a useful marker for monitoring pluripotency in pre-implantation embryos after embryonic genome activation. In ongoing experiments, we are analysing the expression profile of the Oct4-EGFP transgene and endogenous OCT4 protein in porcine pre-implantation embryos from Days 8 and 11.

52 PRELIMINARY DATA ON PIG-BOVINE INTERSPECIES NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 134 ◽  
Author(s):  
I. Lagutina ◽  
D. Brunetti ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Double Pulse ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Fetal Fibroblasts ◽  
Interspecies Nuclear Transfer ◽  
Genome Activation

Interspecies nuclear transfer (NT) is a very important tool for study of nuclear–cytoplasm interactions and somatic cell nucleus reprogramming. We constructed, by means of a zona-free method, NT embryos using bovine (Bo) or porcine (Po) oocytes matured in vitro and bovine fetal fibroblasts (BFF), pig adult fibroblasts (PAF), and pig fetal (PFF) green fluorescent protein (GFP)-positive fibroblasts. Constructs were fused by a double pulse of DC 1.2 kV/cm for 30 µs. At 3–4 h post-fusion, embryos with Bo were activated by 5 µM ionomycin for 4 min and incubated in 2 mM 6-DMAP in SOFaa for 4 h, whereas embryos with Po were activated by a double pulse of DC 1.2 kV/cm for 30 µs in the fusion medium with 1 mM Ca++ and incubated in SOFaa containing 5 µg/mL cytochalasin B in for 4 h. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The NT embryo development and GFP expression (D7) were checked. Our results (Table 1) showed that the blastocyst rate of control bovine and pig embryos was 74% and from 20 to 44%, respectively. ‘Pig fibroblasts into Bo’ embryos were arrested at the 8–21-cell stage while ‘BFF into Po’ embryos were arrested at the 4-cell stage. About 84% of ‘PFF GFP+ into Bo’ NT embryos started to express GFP, but only 3.2% (3/95) of the embryos were able to progress through the 16-cell stage suggesting insufficient embryonic genome activation. Overall significantly more ‘Pig fibroblast into Bo’ embryos were able to progress through the 4-cell stage pig developmental block than normal pig NT embryos (57.8 ± 3.5% vs. 47.1 ± 1.3%; t-test, P = 0.02). This study shows that early embryo development is driven by recipient cytoplasm up to the stage when genome activation should occur. The arrest of interspecies NT embryos at the stage of embryonic genome activation suggests that this developmental step is impaired. Table 1. Interspecies NT embryo development This work was funded by grant ISS CS 11 and ESF.

scholarly journals 119 THE LOCALIZATION OF A METHYL BINDING DOMAIN PROTEIN (MBD4) IN MURINE AND BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 210
Author(s):  
N. Ruddock ◽  
J. Xue ◽  
L. Sanchez-Partida ◽  
M. Cooney ◽  
N. Korfiatis ◽  
...  
Keyword(s):  
Binding Domain ◽  
Primary Antibody ◽  
Epifluorescence Microscopy ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Stage Of Development ◽  
Embryonic Genome ◽  
Genome Activation ◽  
Bovine Oocytes

The presence of MBD4, a member of the methyl binding domain family, was investigated in both murine and bovine oocytes and pre-implantation embryos. MBD4 is the only MBD family member that is involved in DNA repair but not active in transcriptional repression or in the formation of complexes with histone deacetylase complexes (HDACs). It contains a mismatch-specific glycosylase domain that acts to repair G:T mismatches within a CpG context. Bovine cumulus oocyte complexes were collected from abattoir-derived ovaries, matured in vitro and used for IVF as described previously (Ruddock et al. 2004 Biol. Reprod. 70, 1131–1135). Samples were analyzed at all steps in this process. Murine oocytes were collected from superovulated mice (C57BL6 × CBA) and subjected to conventional IVF. A polyclonal antibody derived in the rabbit against human peptides from specific regions of MBD4 (Imgenex, San Diego, CA, USA) was used to localize MBD4 protein. This antibody was tested at a variety of concentrations against both human HL60 leukemia cells and bovine embryos. Staining of HL60 cells was optimum at 32–64 μg/mL and embryos at 64 μg/mL. Briefly, the staining protocol consisted of fixing cells and zona-free oocytes or embryos in 4% paraformaldehyde for 15 min, followed by 15 min in 0.1% Triton X-100. Primary antibody incubation was performed overnight at 4°C. Embryos were then washed in blocking buffer for 1 hr prior to incubation at 4°C in mouse anti-rabbit IgG conjugated to FITC in blocking buffer for 30 min in the dark. Lastly, embryos were incubated in 10 μg/L Hoescht 33342 for 15 min, and then washed and mounted with Vectashield (Vector Labs, Burlingame, CA, USA). Negative controls contained no primary antibody. Mounted cells/embryos were viewed by epifluorescence microscopy. MBD4 was found to be expressed in both murine and bovine oocytes and pre-implantation embryos. In the cow, faint nuclear expression was detected at the 2-cell stage, followed by exclusion of the protein from the nucleus until the blastocyst stage of development. At this stage, staining was primarily nuclear and quite intense. In the mouse, staining was cytoplasmic at the 2 pronuclear stage, but was then concentrated in the nucleus from the 2-cell stage onward. It will be interesting to determine if this is due to the different timing of embryonic genome activation between the two species, hence implying a role for MBD4 in this important biological process. Further investigations are underway to compare the subcellular localization of the other MBD proteins in both species during preimplantation development and to identify a role for MBD4 in embryonic genome activation.

Golden hamster embryonic genome activation occurs at the two-cell stage: Correlation with major developmental changes

1992 ◽  
Vol 32 (3) ◽  
pp. 229-235 ◽  
Author(s):  
Polani B. Seshagiri ◽  
Debbie I. McKenzie ◽  
Barry D. Bavister ◽  
Judy L. Williamson ◽  
Judd M. Aiken
Keyword(s):  
Golden Hamster ◽  
Developmental Changes ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Genome Activation

scholarly journals Modelling human zygotic genome activation in 8C-like cells in vitro

2021 ◽  
Author(s):  
Jasmin Taubenschmid-Stowers ◽  
Maria Rostovskaya ◽  
Fatima Santos ◽  
Sebastian Ljung ◽  
Ricard Argelaguet ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Human Embryos ◽  
Marker Genes ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Cell Embryo ◽  
Genome Activation

The remodelling of the epigenome and transcriptome of the fertilised oocyte to establish totipotency in the zygote and developing embryo is one of the most critical processes in mammalian embryogenesis. Zygotic or embryonic genome activation (ZGA, EGA) in the 2-cell embryo in mouse, and the 8-cell embryo in humans, constitutes the first major wave of transcription. Failure to initiate ZGA leads to developmental defects, and contributes to the high attrition rates of human pre-implantation embryos. Due to limitations in cell numbers and experimental tractability, the mechanisms that regulate human embryonic genome activation in the totipotent embryo remain poorly understood. Here we report the discovery of human 8-cell like cells (8CLCs) specifically among naive embryonic stem cells, but not primed pluripotent cells. 8CLCs express ZGA marker genes such as ZSCAN4, LEUTX and DUXA and their transcriptome closely resembles that of the 8-cell human embryo. 8-cell like cells reactivate 8-cell stage specific transposable elements such as HERVL and MLT2A1 and are characterized by upregulation of the DNA methylation regulator DPPA3. 8CLCs show reduced SOX2 protein, and can be identified based on expression of the novel ZGA-associated protein markers TPRX1 and H3.Y in vitro. Overexpression of the transcription factor DUX4 as well as spliceosome inhibition increase ZGA-like transcription and enhance TPRX1+ 8CLCs formation. Excitingly, the in vitro identified 8CLC marker proteins TPRX1 and H3.Y are also expressed in 8-cell human embryos at the time of genome activation and may thus be relevant in vivo. The discovery of 8CLCs provides a unique opportunity to model and manipulate human ZGA-like transcriptional programs in vitro, and might provide critical functional insights into one of the earliest events in human embryogenesis in vivo.

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