Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

2004 ◽  
Vol 68 (3) ◽  
pp. 313-318 ◽  
Author(s):  
Tadahiro Shinozawa ◽  
Eiji Mizutani ◽  
Ikuo Tomioka ◽  
Manabu Kawahara ◽  
Hiroshi Sasada ◽  
...  
Keyword(s):  
Differential Effect ◽  
Nuclear Transfer ◽  
Embryonic Cell ◽  
Preimplantation Development ◽  
Microtubule Organization

P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Trout ◽  
P Xie ◽  
A Petrini ◽  
Z Rosenwaks ◽  
G Palermo
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Blastocyst Development ◽  
Time Lapse Imaging

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P < 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P < 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P < 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P < 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P < 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

scholarly journals Preimplantation embryo programming: transcription, epigenetics, and culture environment

Reproduction ◽  
2008 ◽  
Vol 135 (2) ◽  
pp. 141-150 ◽  
Author(s):  
Veronique Duranthon ◽  
Andrew J Watson ◽  
Patrick Lonergan
Keyword(s):  
Gene Expression ◽  
Embryo Culture ◽  
Nuclear Transfer ◽  
Preimplantation Embryo ◽  
Later Life ◽  
Short Review ◽  
Basic Program ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Negative Consequences

Preimplantation development directs the formation of an implantation- or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and fetal development can be sustained. The preimplantation embryo exhibits a form of autonomous development fueled by products provided by the oocyte and also from activation of the embryo's genome. Despite this autonomy, the preimplantation embryo is highly influenced by factors in the external environment and in extreme situations, such as those presented by embryo culture or nuclear transfer, the ability of the embryo to adapt to the changing environmental conditions or chromatin to become reprogrammed can exceed its own adaptive capacity, resulting in aberrant embryonic development. Nuclear transfer or embryo culture-induced influences not only affect implantation and establishment of pregnancy but also can extend to fetal and postnatal development and affect susceptibility to disease in later life. It is therefore critical to define the basic program controlling preimplantation development, and also to utilize nuclear transfer and embryo culture models so that we may design healthier environments for preimplantation embryos to thrive in and also minimize the potential for negative consequences during pregnancy and post-gestational life. In addition, it is necessary to couple gene expression analysis with the investigation of gene function so that effects on gene expression can be fully understood. The purpose of this short review is to highlight our knowledge of the mechanisms controlling preimplantation development and report how those mechanisms may be influenced by nuclear transfer and embryo culture.

scholarly journals Spindle Formation and Microtubule Organization During First Division in Reconstructed Rat Embryos Produced by Somatic Cell Nuclear Transfer

2007 ◽  
Vol 53 (4) ◽  
pp. 835-842 ◽  
Author(s):  
Ikuo TOMIOKA ◽  
Eiji MIZUTANI ◽  
Tomoyuki YOSHIDA ◽  
Atsushi SUGAWARA ◽  
Kentaro INAI ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Microtubule Organization ◽  
Spindle Formation ◽  
Rat Embryos

108. INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER IS DEPENDENT ON COMPATIBLE CYTOPLASMIC FACTORS AND MITOCHONDRIAL DNA

2010 ◽  
Vol 22 (9) ◽  
pp. 26
Author(s):  
Y. Jiang ◽  
R. Kelly ◽  
A. Peters ◽  
H. Fulka ◽  
D. A. Mitchell ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Cell ◽  
Copy Number ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Development ◽  
Nuclear Pore ◽  
Mtdna Copy Number ◽  
Cytoplasmic Factors ◽  
Fetal Fibroblasts

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities to analyze and understand nuclear-cytoplasmic interactions. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility, specifically mitochondrial DNA (mtDNA), on successful development. Transfer of somatic murine fetal fibroblasts into enucleated porcine oocytes resulted in extremely low blastocyst rates (0.4%); increased DNA strand breaks; deficient nuclear pore complex arrangements and increased aberrant karyokinesis than observed in porcine-porcine SCNT embryos. Using allele specific-PCR analysis, murine mtDNA was detected at ever-decreasing levels to the blastocyst stage, with peak levels being 0.14 ± 0.055% in 2-cell embryos. Furthermore, these embryos reduced total mtDNA copy number during preimplantation development in a manner similar to porcine embryos. Injecting mouse embryonic stem cell extract and mitochondria along with the murine donor cell into a mitochondria depleted porcine oocyte, increased blastocyst zona pellucida thinning and blastocyst rates significantly (0.4% vs 3.42%) compared to the non-supplemented iSCNT group. They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout preimplantation development. At later stages of preimplantation development, they possessed 48.00% ± 17.38% murine mtDNA and exhibited a mtDNA copy number profile similar to murine embryos. Overall, these data demonstrate that the addition of species compatible cytoplasmic factors and mitochondrial DNA improve developmental competence of iSCNT embryos.

Functional study of distinct domains of dux in improving mouse SCNT embryonic development

2021 ◽  
Author(s):  
Xingwei Huang ◽  
Xinglin Hu ◽  
Qi Jiang ◽  
Qianzi Cao ◽  
Yanshuang Wu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Critical Factor ◽  
Preimplantation Development ◽  
Functional Study ◽  
Intermediate Region ◽  
Cell Stage ◽  
Terminal Domain ◽  
Reprogramming Efficiency ◽  
And Function

Abstract 2-cell-like (2C-like) embryonic stem cells (ESCs) are a small group of ESCs that spontaneously express zygotic genomic activation (ZGA) genes and repeats, such as Zscan4 and MERVL, and are specifically expressed in 2-cell-stage mouse embryos. Although numerous types of treatment and agents elevate the transition of ESCs to 2C-like ESCs, Dux serves as a critical factor in this transition by increasing the expression of Zscan4 and MERVL directly. However, the loss of Dux did not impair the birth of mice, suggesting that Dux may not be the primary transitioning factor in fertilized embryos. It has been reported that for 2-cell embryos derived from somatic cell nuclear transfer (SCNT) and whose expression of ZGA genes and repeats was aberrant, Dux improved the reprogramming efficiency by correcting aberrant H3K9ac modification via its C-terminal domain. We confirmed that overexpression of full-length Dux mRNA in SCNT embryos improved the efficiency of preimplantation development (62.16% vs. 41.26% with respect to controls) and also increased the expression of Zscan4 and MERVL. Furthermore, we found that the N-terminal double homeodomains of Dux were indispensable for Dux localization and function. The intermediate region was essential for MERVL and Zscan4 activation, and the C-terminal domain was important for elevating level of H3K27ac. Mutant Dux mRNA containing N-terminal double homeodomains with the intermediate region or the C-terminal domain also improved the preimplantation development of SCNT embryos. This is the first report focusing on distinguishing functional domains of Dux in embryos derived from SCNT.

scholarly journals Nuclear Transfer of Embryonic Cell Nuclei to Non-enucleated Eggs in Zebrafish, Danio rerio

2011 ◽  
Vol 7 (4) ◽  
pp. 460-468 ◽  
Author(s):  
Manabu Hattori ◽  
Hisashi Hashimoto ◽  
Ekaterina Bubenshchikova ◽  
Yuko Wakamatsu
Keyword(s):  
Danio Rerio ◽  
Nuclear Transfer ◽  
Embryonic Cell ◽  
Cell Nuclei
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