scholarly journals Embryonic fate after somatic cell nuclear transfer in non-enucleated goldfish oocytes is determined by first cleavages and DNA methylation patterns

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexandra Depincé ◽  
Pierre-Yves Le Bail ◽  
Charlène Rouillon ◽  
Catherine Labbé
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
High Variability ◽  
Marker Genes ◽  
Cycle Duration ◽  
Genetic Origin ◽  
Critical Determinant ◽  
Cell Cycles ◽  
Cell Cycle Duration ◽  
Methylation Patterns

AbstractReducing the variability in nuclear transfer outcome requires a better understanding of its cellular and epigenetic determinants, in order to ensure safer fish regeneration from cryobanked somatic material. In this work, clones from goldfish were obtained using cryopreserved fin cells as donor and non-enucleated oocytes as recipients. We showed that the high variability of clones survival was not correlated to spawn quality. Clones were then characterized for their first cleavages pattern in relation to their developmental fate up to hatching. The first cell cycle duration was increased in clones with abnormal first cleavage, and symmetric first two cleavages increased clone probability to reach later on 24 h- and hatching-stages. At 24 h-stage, 24% of the clones were diploids and from donor genetic origin only. However, ploidy and genetic origin did not determine clones morphological quality. DNA methylation reprogramming in the promoter region of pou2, nanog, and notail marker genes was highly variable, but clones with the nicest morphologies displayed the best DNA methylation reprogramming. To conclude, non-enucleated oocytes did allow authentic clones production. The first two cell cycles were a critical determinant of the clone ability to reach hatching-stage, and DNA methylation reprogramming significantly influenced clones morphological quality.

Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1807-1817 ◽  
Author(s):  
Jiyoung Lee ◽  
Kimiko Inoue ◽  
Ryuichi Ono ◽  
Narumi Ogonuki ◽  
Takashi Kohda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genomic Imprinting ◽  
Nuclear Transfer ◽  
Monoallelic Expression ◽  
Parental Origin ◽  
Imprinted Genes ◽  
Specific Expression ◽  
Male And Female ◽  
Imprinted Gene ◽  
Methylation Patterns

Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.

scholarly journals DNA-methylation patterns imply a common cellular origin of virus- and UV-associated Merkel cell carcinoma

Oncogene ◽  
2021 ◽  
Author(s):  
Jan Gravemeyer ◽  
Ivelina Spassova ◽  
Monique E. Verhaegen ◽  
Andrzej A. Dlugosz ◽  
Daniel Hoffmann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Merkel Cell Carcinoma ◽  
Marker Genes ◽  
Methylation Data ◽  
Merkel Cell ◽  
Scoring Model ◽  
Methylation Patterns ◽  
Neuroendocrine Cancers

AbstractMerkel cell carcinoma (MCC) is a neuroendocrine tumor either induced by integration of the Merkel cell polyomavirus into the cell genome or by accumulation of UV-light-associated mutations (VP-MCC and UV-MCC). Whether VP- and UV-MCC have the same or different cellular origins is unclear; with mesenchymal or epidermal origins discussed. DNA-methylation patterns have a proven utility in determining cellular origins of cancers. Therefore, we used this approach to uncover evidence regarding the cell of origin of classical VP- and UV-MCC cell lines, i.e., cell lines with a neuroendocrine growth pattern (n = 9 and n = 4, respectively). Surprisingly, we observed high global similarities in the DNA-methylation of UV- and VP-MCC cell lines. CpGs of lower methylation in VP-MCC cell lines were associated with neuroendocrine marker genes such as SOX2 and INSM1, or linked to binding sites of EZH2 and SUZ12 of the polycomb repressive complex 2, i.e., genes with an impact on carcinogenesis and differentiation of neuroendocrine cancers. Thus, the observed differences appear to be rooted in viral compared to mutation-driven carcinogenesis rather than distinct cells of origin. To test this hypothesis, we used principal component analysis, to compare DNA-methylation data from different epithelial and non-epithelial neuroendocrine cancers and established a scoring model for epithelial and neuroendocrine characteristics. Subsequently, we applied this scoring model to the DNA-methylation data of the VP- and UV-MCC cell lines, revealing that both clearly scored as epithelial cancers. In summary, our comprehensive analysis of DNA-methylation suggests a common epithelial origin of UV- and VP-MCC cell lines.

Methylation characteristics and developmental potential of Guangxi Bama minipig (Sus scrofa domestica) cloned embryos from donor cells treated with trichostatin A and 5-aza-2′-deoxycytidine

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 178-186 ◽  
Author(s):  
Shu-Fang Ning ◽  
Qing-Yang Li ◽  
Ming-Ming Liang ◽  
Xiao-Gan Yang ◽  
Hui-Yan Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Formation Rate ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Donor Cells ◽  
Aberrant Dna Methylation ◽  
Methylation Patterns ◽  
Sus Scrofa Domestica

SummaryReprogramming of DNA methylation in somatic cell nuclear transfer (SCNT) embryos is incomplete, and aberrant DNA methylation patterns are related to the inefficiency of SCNT. To facilitate nuclear reprogramming, this study investigated the effect of treating Guangxi Bama minipig donor cells with trichostatin A (TSA), 5-aza-2′-deoxycytine (5-aza-dC), or combination of TSA and 5-aza-dC prior to nuclear transfer. Analyses showed that there were no major changes in cell-cycle status among all groups. We monitored the transcription of DNMT1, DNMT3a, HDAC1 and IGF2 genes in donor cells. Transcription levels of HDAC1 were decreased significantly after treatment with a combination of TSA and 5-aza-dC, along with a significantly increased level of IGF2 (P < 0.05). Although treatment of donor cells with either TSA or 5-aza-dC alone resulted in non-significant effects in blastocyst formation rate and DNA methylation levels, a combination of TSA and 5-aza-dC significantly improved the development rates of minipig SCNT embryos to blastocyst (25.6% vs. 16.0%, P < 0.05). This change was accompanied by decreased levels of DNA methylation in somatic cells and blastocyst (P < 0.05). Thus in combination with TSA, lower concentrations of 5-aza-dC may produce a potent demethylating activity, and lead to the significantly enhanced blastocyst development percentage of Bama minipig SCNT embryos.

scholarly journals Cell Cycle Kinetics and Development of Hydra Attenuata

1974 ◽  
Vol 16 (2) ◽  
pp. 349-358 ◽  
Author(s):  
R. D. CAMPBELL ◽  
C. N. DAVID
Keyword(s):  
Cell Cycle ◽  
Nuclear Dna ◽  
Cell Function ◽  
Control Point ◽  
Interstitial Cells ◽  
Total Cell ◽  
Cycle Duration ◽  
Cell Cycles ◽  
Cell Cycle Duration ◽  
Hydra Attenuata

The cell cycle parameters of interstitial cells in Hydra attenuata have been determined. Interstitial cells were classified according to cluster size in which they occur (1, 2, 4, 8 or 16 cells) and morphology using maceration preparations and histological sections. The lengths of G1, S, G2 and M were determined by standard methods of cell cycle analysis using pulse-chase and continuous labelling with [3H]- and [14C]thymidine. Nuclear DNA contents were measured microfluorimetrically. All classes of interstitial cells proliferate but the cell cycle of large interstitial cells occurring singly or in pairs is longer than that of interstitial cells occurring in clusters of 4, 8 and 16 cells. The S-phase is 11-12 h long and G1 is less than 1 h for all classes of interstitial cells. G2 is 3-4 h long for interstitial cells in clusters of 4, 8 and 16 cells giving these cells a total cell cycle duration of 16-17 h. In contrast, large interstitial cells occurring as singles and in clusters of 2 have G2 durations ranging from 4 to 22 h. Two subpopulations can be discerned among these cells, one having a G1 of about 6 h and a total cell cycle of about 19 h, the other having an average G2 of 14 h and a total cell cycle of about 27 h. The differences in cell cycle duration appear to be associated with interstitial cell function. Cells having a short cell cycle are probably committed to nematocyte differentiation, while large interstitial cells having long and variable cell cycles appear to be undetermined stem cells responsible for proliferating further interstitial cells. The variable length of G2 in these cells suggest it as a possible control point.

158 RELATIONSHIP BETWEEN FOURTH CELL CYCLE DURATION AND POST-TRANSFER VIABILITY IN IN VITRO-FERTILIZED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 191
Author(s):  
S. Sugimura ◽  
Y. Hashiyada ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
H. Matsuda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Embryo Transfer ◽  
T Test ◽  
Cycle Duration ◽  
Bovine Embryos ◽  
Cell Cycles ◽  
Cell Cycle Duration ◽  
Student’S T ◽  
Student’S T Test

In cattle, the prediction of embryonic viability after embryo transfer is an important research target. A previous study has indicated that the duration of the fourth cell cycle at the time of maternal-zygotic transition, which is involved in in vitro embryonic development, may be an indicator of blastocyst formation; this study showed that embryos with a short fourth cell cycle have a better potential of developing into blastocysts than those with a long fourth cell cycle (Lequarre et al. 2003 Biol. Reprod. 69, 1707–1713). However, the relationship between the fourth cell cycle duration and post-transfer viability of embryos is unclear. The aim of the present study was to examine the effect of the fourth cell cycle duration on embryo development after embryo transfer. Twenty-five IVF bovine embryos were cultured in well-of-the-well culture dishes contained 125 μ of CR1aa supplemented with 5% calf serum at 38.5°C in 5% O2 and 5% CO2 for 168 h after insemination. In vitro development of the embryos was monitored using time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). We found that 61% of the blastocysts had a long fourth cell cycle (41.5 ± 5.9 h), which is commonly referred to as the lag phase, whereas the remaining embryos had a short fourth cell cycle (7.4 ± 4.5 h). All the embryos with a short fourth cell cycle exhibited a lag phase in the next cell cycle (32.9 ± 6.6 h). Moreover, embryos with a short fourth cell cycle were found to have a higher blastocyst rate (75.8%) than those with a long fourth cell cycle (48.1%; Student's t-test, P < 0.01). However, embryonic cell number, apoptosis incidence, chromosomal abnormality and O2 consumption were found to be identical between the 2 groups (Student's t-test, P > 0.05). Real-time reverse-transcription PCR results of the individual blastocysts showed that the relative expression of 5 genes related to pregnancy reorganization, placentation and fetal growth—namely, CDX2, IFN-τ, PLAC8, AKR1B1 and IGF2R—did not differ between the 2 groups (Student's t-test, P > 0.05). Furthermore, blastocysts derived from embryos with long (n = 30) and short (n = 19) fourth cell cycles were transferred into 49 recipient cows; we did not observe any difference between the long and short fourth cell cycles on the rates of pregnancy (long vs short fourth cell cycle, 30.0 vs 52.6%) and delivery (long vs short fourth cell cycle, 30.0 vs 47.4%; Yates' corrected chi-square test, P > 0.10). These results show that blastocysts derived from embryos with either long or short fourth cell cycles have identical developmental competence after embryo transfer. Therefore, the fourth cell cycle duration during maternal-zygotic transition appears to be unavailable as the indicator of post-transfer viability of IVF bovine embryos. This work was supported by the Research and Developmental Program for New Bio-Industry Initiatives.

26 DNA METHYLATION PATTERNS ARE APPROPRIATELY ESTABLISHED IN THE SPERM OF BULLS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER AFTER PASSAGE THROUGH THE GERMLINE

2009 ◽  
Vol 21 (1) ◽  
pp. 113 ◽  
Author(s):  
C. Couldrey ◽  
M. P. Green ◽  
D. N. Wells ◽  
R. S. F. Lee
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cpg Island ◽  
Somatic Cells ◽  
Cpg Sites ◽  
Donor Cells ◽  
Genomic Regions ◽  
Methylation Patterns

Cloning of domestic animals by somatic cell nuclear transfer (SCNT) has permitted the rescue of valuable genetics and has the potential to allow rapid dissemination of desirable traits in production animals through the use of cloned sires. Whilst cloned animals may show developmental deviations and aberrant DNA methylation suggestive of incomplete nuclear reprogramming, it is widely accepted that their offspring are normal, as any aberrant epigenetic marks are believed to be corrected on passage of the genome through the germline. We assessed the extent of reprogramming by comparing DNA methylation patterns in sperm of SCNT bulls (n = 4) with sperm from bulls generated by AI (n = 5) and with the nuclear donor somatic cells (adult skin fibroblasts). The genomic regions examined were 3 repetitive sequences (satellites 1, 2, and alpha) and CpG islands in 5 genes [HAND1, LIT1, MASH2, IGF2, Dickkopf-1(DKK-1)]. Semen was collected from 16-month-old bulls and assessed for volume, sperm number, morphology, and motility. DNA was extracted from washed sperm and somatic donor cells, bisulfite-treated and processed for quantification of CpG methylation using the Sequenom MassArray system. Methylation levels at individual CpG sites/groups of CpGs were compared between sample groups using the t-test with pooled variances. No apparent difference was detected in semen characteristics between SCNT and AI bulls. Sperm DNA methylation levels were very low in single copy genes with the exception of the CpG island in IGF2, which has previously been shown to be completely methylated in sperm. At all genomic regions examined, each CpG site or CpG groups were methylated to different levels, and each region had a distinctive profile, which was almost invariant between individual sperm samples from either the SCNT or AI bulls. In all sites examined, there were no significant differences in methylation profiles between sperm from SCNT and AI bulls. In contrast, DNA methylation profiles were significantly different between SCNT bull sperm and the donor cells. The exception was the CpG island in MASH2, which was essentially unmethylated in both. For the 3 satellite sequences along with LIT1, HAND1, and to a lesser extent, the DKK-1 region, DNA was significantly less methylated in sperm than in the donor cells. Only IGF2 was significantly more methylated in SCNT and AI sperm than in the donor cells at 10/25 CpG sites (P < 0.02). The results indicate that gametes from SCNT bulls had different epigenotypes from the donor somatic cells. This is the first molecular evidence that donor cell genomes have been reprogrammed in these SCNT bulls and that after going through the germline had acquired DNA methylation profiles that were similar to AI-derived bulls. It also suggests that any epigenetic aberrations that SCNT bulls may harbor are unlikely to be passed on to their offspring through their gametes. Supported by FRST contract C10X0311.

scholarly journals Transient depletion of xDnmt1 leads to premature gene activation in Xenopus embryos

2000 ◽  
Vol 14 (3) ◽  
pp. 313-327 ◽  
Author(s):  
Irina Stancheva ◽  
Richard R. Meehan
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Gene Activation ◽  
Marker Genes ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Zygotic Transcription ◽  
Early Expression ◽  
Methyl Cytosine ◽  
Cleavage Stages

In Xenopus laevis zygotic transcription begins at the midblastula transition (MBT). Prior to this the genome is organized into chromatin that facilitates rapid cycles of DNA replication but not transcription. Here we demonstrate that DNA methylation contributes to the overall transcriptional silencing before MBT. Transient depletion of the maternal DNA methyltransferase (xDnmt1) by anti sense RNA during cleavage stages is associated with a decrease in the genomic 5-methyl-cytosine content and leads to the activation of zygotic transcription approximately two cell cycles earlier than normal. Hypomethylation allows the early expression of mesodermal marker genes such as Xbra, Cerberus, and Otx2, which are subsequently down-regulated during gastrulation of thexDnmt1-depleted embryos. The temporal switch in gene expression may account for the appearance of body plan defects that we observe. Loss of xDnmt1 can be rescued by the coinjection of mouse or human Dnmt1 protein. These results demonstrate that DNA methylation has a role in the regulation of immediately early genes in Xenopusat MBT.

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