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

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).

48 EFFECTS OF TRICHOSTATIN A ON DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 142 ◽  
Author(s):  
D. Iwamoto ◽  
K. Saeki ◽  
S. Kishigami ◽  
A. Kasamatsu ◽  
A. Tatemizo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Mammalian Species ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Embryonic Genome ◽  
Blastocyst Rate

Although cloning by somatic cell nuclear transfer (SCNT) has been achieved in various mammalian species, its efficiency has been very low (Han et al. 2003 Theriogenology 59, 33–44). Successful cloning requires conversion from differentiated donor nuclei to embryonic nuclei after transfer of the somatic nuclei into enucleated oocytes. Reprogramming of the transferred somatic nuclei must be completed by the time when normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, both full-term development and pre-implantation development of mouse SCNT embryos were significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The objective of this study was to investigate the effects of TSA on the development of bovine SCNT embryos. Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. The cells were electro-fused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF medium until 168 h post-activation (hpa). The NT embryos were exposed to 0 (control), 5, 50, and 500 nM TSA from the start of activation to 48 hpa. Experiments were repeated 3 times, and the data were analyzed with Fisher's PLSD test following ANOVA. The cleavage rates were the same among the groups (60 to 80&percnt;; P &gt;0.05). However, the blastocyst rate of NT embryos treated with 50 nM TSA was higher than that of control embryos (40&percnt; vs. 19&percnt;, respectively; P &lt; 0.05). On the other hand, the blastocyst rate was lower with 500 nM TSA than with 5 or 50 nM TSA (7&percnt; vs. 33&percnt; or 40&percnt;; P &lt; 0.05). These data suggest that proper TSA treatment after somatic cloning improves the rate of development of bovine cloned embryos to the blastocyst stage. Further research is needed to examine whether NT embryos derived from different cell lines or types have similar susceptibility to TSA.

65 BLASTOCYSTS DERIVED FROM ADULT FIBROBLASTS OF RHESUS MONKEY (MACACA MULATTA) USING INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 191
Author(s):  
D. K. Kwon ◽  
J. T. Kang ◽  
S. J. Park ◽  
M. N. L. Gomez ◽  
S. J. Kim ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Somatic Cell ◽  
Macaca Mulatta ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Cell Stage ◽  
Nuclear Number

Interspecies somatic cell nuclear transfer (iSCNT) has alternatively chosen in primate SCNT because of the difficulty in collecting enough oocytes for research. The purpose of this experiment is to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissueofrhesus monkey (male, 6 years old) was collected by biopsy and fibroblasts were isolated. Immature COCs from cow ovaries were collected and matured in vitro in TCM-199. Squish enucleation was done in the presence of bisbenzimide and cytochalasin B. After enucleation, a single rhesus monkey somatic cell was injected into the perivitelline space of an enucleated oocyte through the slit in the zona pellucida made during enucleation. Subsequently, the rhesus monkey somatic cell and cow oocyte membranes were electrically fused. The nonactivated interspecies cloned couplets were cultured for 2 h to allow reprogramming to occur. Then, couplets were activated using a 2-step protocol consisting of treatment with 5 μM ionomycin for 4 to 5 min and subsequently with 2mM 6-DMAP for 4 h. Activated iSCNT embryos were cultured for 10 days inmodified SOF with various conditions (at 37 to39°C, 5 to 5.5% CO2 and 5 to 20% O2) to examine the effects ofIVC conditions. As a results, most embryos were arrested at the 8- to 16-cell stage and only 3 blastocysts were derived from rhesus monkey iSCNT. The blastocyst developmental rate was 0.26% generated from the total IVC activated interspecies embryos (n = 1153). Among the 3 blastocysts, 2 of them were used for counting nuclear number using bisbenzimide staining. The nuclear number of the 2 iSCNT-derived blastocysts was 51 and 24, respectively. The other iSCNT-derived blastocyst was used for analyzing mitochondrial (mt)DNAto confirm that it contained both cow and rhesus monkey mtDNA. As a result, mtDNA from both rhesus monkey and cow were detected inPCR analysis. The band intensity was more dominant for cow mtDNA than for rhesus monkey mtDNA. Although the blastocyst developmental rate is extremely low, it is confirmed that two phylogenetically distant species including primate could develop in vitro until the blastocyst stage by iSCNT. The in vitro developmental system of this rhesus monkey iSCNT-derived blastocysts provides a platform for further improvement of developmental rate and quality of rhesus monkey iSCNT-derived blastocysts. It also provides an opportunity to establish rhesus monkey iSCNT-derived embryonic stem cell lines for study of rhesus monkey nucleus and cow mitochondria interaction mechanisms during early developmental stages. This study was financially supported by the Korean MEST, through the BK21 program for Veterinary Science, and SNU foundation (Benefactor; RNL Bio).

scholarly journals A comparative approach to somatic cell nuclear transfer in the rhesus monkey

2006 ◽  
Vol 21 (10) ◽  
pp. 2564-2571 ◽  
Author(s):  
Q. Zhou ◽  
S. H. Yang ◽  
C. H. Ding ◽  
X. C. He ◽  
Y. H. Xie ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Comparative Approach

Blastocysts derived from adult fibroblasts of a rhesus monkey (Macaca mulatta) using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 19 (3) ◽  
pp. 199-204 ◽  
Author(s):  
Dae Kee Kwon ◽  
Jung Taek Kang ◽  
Sol Ji Park ◽  
Ma Ninia Limas Gomez ◽  
Su Jin Kim ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Somatic Cell ◽  
Macaca Mulatta ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Development Rate ◽  
Attractive Alternative ◽  
Cell Stage

SummaryIn non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus–oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5–5.5% CO2 under various conditions (37–39 °C and 5–20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.

scholarly journals Functions and Regulation of Endogenous Retrovirus Elements during Zygotic Genome Activation: Implications for Improving Somatic Cell Nuclear Transfer Efficiency

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 829
Author(s):  
Bo Fu ◽  
Hong Ma ◽  
Di Liu
Keyword(s):  
Somatic Cell ◽  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Embryos ◽  
Zygotic Genome Activation ◽  
Gene Regulatory ◽  
Genome Activation ◽  
Zygotic Genome

Endogenous retroviruses (ERVs), previously viewed as deleterious relics of ancestral retrovirus infections, are silenced in the vast majority of cells to minimize the risk of retrotransposition. Counterintuitively, bursts of ERV transcription usually occur during maternal-to-zygotic transition (MZT) in preimplantation embryos; this is regarded as a major landmark event in the zygotic genome activation (ZGA) process, indicating that ERVs play an active part in ZGA. Evolutionarily, the interaction between ERVs and hosts is mutually beneficial. The endogenization of retrovirus sequences rewires the gene regulatory network during ZGA, and ERV repression may lower germline fitness. Unfortunately, owing to various limitations of somatic cell nuclear transfer (SCNT) technology, both developmental arrest and ZGA abnormalities occur in a high percentage of cloned embryos, accompanied by ERV silencing, which may be caused by the activation failure of upstream ERV inducers. In this review, we discuss the functions and regulation of ERVs during the ZGA process and the feasibility of temporal control over ERVs in cloned embryos via exogenous double homeobox (DUX). We hypothesize that further accurate characterization of the ERV-rewired gene regulatory network during ZGA may provide a novel perspective on the development of preimplantation embryos.

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.

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