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.

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.

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

scholarly journals Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

Reproduction ◽  
2011 ◽  
Vol 141 (4) ◽  
pp. 453-465 ◽  
Author(s):  
Irina Lagutina ◽  
Valeri Zakhartchenko ◽  
Helena Fulka ◽  
Silvia Colleoni ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
De Novo ◽  
Mammalian Species ◽  
Housekeeping Genes ◽  
Cell Stage ◽  
Whole Process ◽  
Embryonic Genome ◽  
Donor Nucleus ◽  
Donor Cells ◽  
Genome Activation

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

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.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

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.

Impairments in Embryonic Genome Activation in Rhesus Monkey Somatic Cell Nuclear Transfer Embryos

2008 ◽  
Vol 10 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Yuyu Niu ◽  
Shihua Yang ◽  
Yang Yu ◽  
Chenhui Ding ◽  
Jifeng Yang ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Genome Activation

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.

35 Interspecies Somatic Cell Nuclear Transfer Embryos that Form Nucleoli do not Always Activate Mitochondrial Functional Differentiation at the Time of Embryonic Genome Activation

2018 ◽  
Vol 30 (1) ◽  
pp. 157 ◽  
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Somatic Cell ◽  
Transcriptional Activation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mitochondrial Genes ◽  
Mitochondrial Mass ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Genome Activation ◽  
Bovine Oocytes

Embryonic genome activation (EGA) is a complex process that needs a good orchestration of all biochemical processes at the time of maternal-to-embryonic transition. Mitochondria are strictly dependent on the nucleus for their correct activity as ~1500 mitochondrial genes have nuclear localisation. The finding of transcriptional activation and accumulation of mRNAs related to mitochondrial biogenesis (Mtango et al. 2008 Reprod. Fertil. Dev. 20, 846-859) around the time of EGA confirmed the role of nucleus in this process. Studying mitochondria behaviour in interspecies somatic cell nuclear transfer (iSCNT) embryos (Lagutina et al. 2010 Reproduction 140, 273-285), we have found that at the time of EGA, mitochondria activation could be demonstrated by JC-1 accumulation. We suggested that comparison of the mean green fluorescence intensity (FI) that corresponds to the fluorescence of the monomeric form of the dye and correlates to relative mitochondrial mass (Mancini et al. 1997) in iSCNT and control nuclear transfer (NT) could serve as a test to assess EGA in iSCNT embryos. The aim of this study was to estimate nuclear-cytoplasmic interaction in iSCNT embryos that formed nucleoli at the time of EGA (Lagutina et al. 2011 Reproduction 141, 453-465) such as embryos derived from bovine oocytes and bovine (control), buffalo or ovine donor nuclei, and from porcine oocytes and porcine (control), horse, or rabbit nuclei. Embryos 72 h after activation were stained with 2 μM JC-1 in SOF-HEPES with 10% FCS at 37°C for 1 h. Images were collected using a fluorescein isothiocyanate (FITC) filter and analysed with Adobe Photoshop Elements 2 (Adobe Systems, San Jose, CA, USA). The data are presented as mean FI of the embryo. To demonstrate the effect of EGA inhibition on mitochondria, bovine and porcine NT embryos were cultured in medium supplemented with 25 μg/mL α-amanitin (AA) from 48 to 72 h after activation. The analyses of mean FI of the embryos showed that ovine and buffalo nuclei were able to support mitochondrial mass accumulation in iSCNT embryos with bovine oocytes equal to control bovine NT embryos (35 ± 11.2; 41.9 ± 14.8; 36.2 ± 7.6, respectively) that was significantly higher than in bovine embryos treated with AA (15.4 ± 4.9; P < 0.05). In the iSCNT embryos composed of porcine oocytes and equine or rabbit nuclei, mean FI values (20 ± 13.4; 18.3 ± 5.5, respectively) were comparable with those in porcine NT embryos treated with AA (16.2 ± 6.2), and were significantly lower than in porcine control (91 ± 47.7; P < 0.05) NT embryos, demonstrating the inability of equine and rabbit nuclei to properly govern the porcine mitochondria mass growth at the time of EGA. In conclusion, nucleolus formation and activation of nuclear encoded mitochondrial genes at the time of EGA cannot serve, per se, as a marker of correct embryonic genome activation in iSCNT embryos, because, in our conditions, no iSCNT embryos developed to blastocyst (Lagutina et al. 2010 Reproduction 140, 273-285). This knowledge about behaviour of different embryo compartments at the time of EGA could extend our understanding of the whole process. This work was funded by Translink (EU FP7 no. 603049) and Xenoislet projects (EU FP7 no. 601827).

Sign in / Sign up

Export Citation Format

Share Document