54 Choline alters the pattern of DNA methylation and lipid content of pre-implantation bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 152
Author(s):  
E. Estrada-Cortés ◽  
P. J. Hansen
Keyword(s):  
Dna Methylation ◽  
Lipid Content ◽  
Cell Membranes ◽  
Culture Medium ◽  
Cytosine Methylation ◽  
Choline Chloride ◽  
Nile Red ◽  
Dna Demethylation ◽  
Methylation Of Dna ◽  
Methyl Cytosine

Choline is a methyl donor for DNA methylation and a precursor for phosphatidylcholine, which is the major phospholipid of cell membranes. Early embryonic development involves processes of DNA demethylation and remethylation as well as synthesis of new cell membranes. Addition of choline chloride (ChCl) to culture medium of embryos produced invitro increased birthweight of Brahman calves after embryo transfer. The objective was to determine whether the addition of ChCl to culture medium alters the pattern of DNA methylation and lipid content of pre-implantation embryos produced invitro. Embryos were incubated after fertilisation in BBH7 culture medium containing 0.0, 0.004, 1.3, or 1.8mM ChCl (adjusted with NaCl to maintain isotonicity). Concentrations were chosen to approximate free choline (0.004mM) and total choline (1.30mM) in plasma of cows at week 1 postpartum and concentration of total choline in plasma of cows fed rumen-protected choline (1.8mM). Cleavage and blastocyst rate (n=8 replicates) were evaluated at Days 3 and 7.5 post-insemination, respectively. Embryos ≥8 cells (range 8 to 24 cells; stages near embryonic genome activation) and expanded blastocysts were harvested at Day 3.75 (n=232) and 7.5 (n=204) to estimate global DNA methylation by immunostaining for 5-methyl-cytosine. Methylation of DNA was estimated by calculating the ratio of fluorescence for 5-methyl-cytosine to that of propidium iodide (DNA). Another group of expanded blastocysts was harvested (n=99) to estimate lipid content using Nile Red. Embryo development was analysed by GLIMMIX procedure and fluorescence data by GLM procedure of SAS (SAS Institute Inc.). The proportion of zygotes that cleaved after fertilisation (77.5±2.3, 78.1±2.3, 74.5±2.4, and 80.1±2.2% for 0.0, 0.004, 1.3, and 1.8mM ChCl; P=0.2736) and cleaved embryos that became blastocysts (37.8±4.4, 41.5±4.6, 42.8±4.6, and 39.6±4.4%; P=0.5764) was similar between treatments. The DNA methylation at both days was affected by treatment (P<0.001). At Day 3.75, 1.3mM choline reduced methylation and there were no effects of other concentrations (1.13a±0.03, 1.04a±0.03, 0.92b±0.03, and 1.13a±0.04; means with different superscripts differ at P<0.05). For blastocysts, in contrast, DNA methylation was increased for embryos treated with 1.3 and 1.8mM choline (0.98a±0.04, 1.04ab±0.03, 1.25c±0.03, and 1.11b±0.03). Lipid content in blastocysts was also affected by treatment (P=0.0139). In particular, lipid content was higher for embryos treated with 1.3 and 1.8mM choline (409.1a±54.3, 542.3ab±62.3, 651.3b±54.3, and 583.9b±55.0). In conclusion, addition of ChCl to culture medium altered DNA methylation in bovine pre-implantation embryos produced invitro in a manner dependent on developmental stage and choline concentration. Likewise, ChCl increased lipid content in the resultant blastocysts. Support was provided by the Red Larson Endowment.

161 LIPID CONTENT, RESISTANCE TO CRYOPRESERVATION, AND SEX RATIO OF IN VITRO BOVINE BLASTOCYSTS PRODUCED IN A SERUM-FREE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 188
Author(s):  
F. George ◽  
C. Daniaux ◽  
G. Genicot ◽  
F. Focant ◽  
B. Verhaeghe ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Lipid Content ◽  
Culture Medium ◽  
Nile Red ◽  
Embryo Cryopreservation ◽  
Hatching Rate ◽  
Free System ◽  
Serum Free

In vitro-produced (IVP) bovine blastocysts are known to be more sensitive to cryopreservation than their in vivo counterparts. Removing serum from the culture medium decreases sanitary risk and could improve embryo resistance to cryopreservation by preventing the accumulation of intracellular lipids. Our objectives were to evaluate the lipid content, resistance to cryopreservation, and sex ratio of IVP embryos cultured in a serum-free system. Oocytes from slaughterhouse ovaries were matured in a serum-free enriched medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) and cultured in 5% O2 in modified SOF supplemented with 5% FCS (FCS) or with insulin-transferrin-selenium (ITS) and 0.1 mg/mL polyvinylpyrrolidone (PVP) (ITS-PVP) or 4 mg/mL BSA (ITS-BSA) (Daniaux et al. 2005 Reprod. Fertil. Dev. 17, 217). Day 5 morulae were stained with the fluorescent dye Nile Red in order to evaluate their lipid content (Genicot et al. 2005 Theriogenology 63, 1181). Day 7 blastocysts (diameter ≥160 µm) were selected, classified according to their size, and frozen in HEPES-SOF containing 1.5 M ethylene glycol, 0.1 M sucrose, and 1.8 mg/mL wheat peptones (George et al. 2002 Reproduction 29, 51). The lipid content was significantly lower in morulae cultured in ITS-BSA compared with the two other media (320 ± 10 arbitrary fluorescence units vs. 383 ± 12 in FCS and 406 ± 10 in ITS-PVP; n = 271; ANOVA2: P < 0.01). After cryopreservation, a higher total hatching rate was found 24 h post-thawing in blastocysts cultured in ITS-BSA and for both serum-free conditions at 48 h (Table 1). In particular, embryos ≤180 µm cultured in FCS were less resistant to cryopreservation than embryos of the same size produced without serum. Expanded blastocysts cultured in ITS-BSA were sexed by PCR (Grisart et al. 1995 Theriogenology 43, 1097) and a higher proportion of male embryos was found (62.7%; n = 51). In conclusion, a complete serum-free system was set up from oocyte maturation to embryo cryopreservation that gave high quality embryos resistant to cryo-preservation. Embryos produced in ITS-BSA presented a lower lipid content, but a shift of the expanded blastocyst sex ratio toward males was observed. Table 1. Hatching rates post-thawing as a function of the blastocyst size and the culture medium

scholarly journals Current Advances in DNA Methylation Analysis Methods

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Ehsan Khodadadi ◽  
Leila Fahmideh ◽  
Ehsaneh Khodadadi ◽  
Sounkalo Dao ◽  
Mehdi Yousefi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Large Scale ◽  
Primary Methods ◽  
Bisulfite Treatment ◽  
Advantages And Disadvantages ◽  
Methylation Of Dna ◽  
Biological Identification ◽  
Methyl Cytosine ◽  
Sequencing Platforms ◽  
New Generation

DNA methylation is one of the epigenetic changes, which plays a major role in regulating gene expression and, thus, many biological processes and diseases. There are several methods for determining the methylation of DNA samples. However, selecting the most appropriate method for answering biological questions appears to be a challenging task. The primary methods in DNA methylation focused on identifying the state of methylation of the examined genes and determining the total amount of 5-methyl cytosine. The study of DNA methylation at a large scale of genomic levels became possible following the use of microarray hybridization technology. The new generation of sequencing platforms now allows the preparation of genomic maps of DNA methylation at the single-open level. This review includes the majority of methods available to date, introducing the most widely used methods, the bisulfite treatment, biological identification, and chemical cutting along with their advantages and disadvantages. The techniques are then scrutinized according to their robustness, high throughput capabilities, and cost.

scholarly journals DNA demethylation is initiated in the central cells of Arabidopsis and rice

2016 ◽  
Vol 113 (52) ◽  
pp. 15138-15143 ◽  
Author(s):  
Kyunghyuk Park ◽  
M. Yvonne Kim ◽  
Martin Vickers ◽  
Jin-Sup Park ◽  
Youbong Hyun ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
Dna Demethylation ◽  
Central Cell ◽  
Dna Modification ◽  
Active Dna Demethylation ◽  
Genome Wide ◽  
Regulatory Functions ◽  
Global Reduction

Cytosine methylation is a DNA modification with important regulatory functions in eukaryotes. In flowering plants, sexual reproduction is accompanied by extensive DNA demethylation, which is required for proper gene expression in the endosperm, a nutritive extraembryonic seed tissue. Endosperm arises from a fusion of a sperm cell carried in the pollen and a female central cell. Endosperm DNA demethylation is observed specifically on the chromosomes inherited from the central cell in Arabidopsis thaliana, rice, and maize, and requires the DEMETER DNA demethylase in Arabidopsis. DEMETER is expressed in the central cell before fertilization, suggesting that endosperm demethylation patterns are inherited from the central cell. Down-regulation of the MET1 DNA methyltransferase has also been proposed to contribute to central cell demethylation. However, with the exception of three maize genes, central cell DNA methylation has not been directly measured, leaving the origin and mechanism of endosperm demethylation uncertain. Here, we report genome-wide analysis of DNA methylation in the central cells of Arabidopsis and rice—species that diverged 150 million years ago—as well as in rice egg cells. We find that DNA demethylation in both species is initiated in central cells, which requires DEMETER in Arabidopsis. However, we do not observe a global reduction of CG methylation that would be indicative of lowered MET1 activity; on the contrary, CG methylation efficiency is elevated in female gametes compared with nonsexual tissues. Our results demonstrate that locus-specific, active DNA demethylation in the central cell is the origin of maternal chromosome hypomethylation in the endosperm.

scholarly journals DNA Methylation in Cancer and Development: An Evolving Paradigm

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-50-SCI-50
Author(s):  
Maria E. Figueroa
Keyword(s):  
Dna Methylation ◽  
Gene Regulation ◽  
Cytosine Methylation ◽  
Conflicts Of Interest ◽  
Cpg Islands ◽  
Dna Methyltransferases ◽  
Dna Demethylation ◽  
Epigenetic Mark ◽  
Cpg Dinucleotides ◽  
Promoter Dna

DNA methylation is an epigenetic mark which, in mammals, occurs primarily on position 5 of cytosines, especially at those found in the context of CpG dinucleotides. This CpG methylation is known to play a major role in gene regulation. Cytosine methylation is regulated by the DNA methyltransferases, responsible for adding the methyl group to unmethylated CpGs, and the TET dioxygenases, involved in the DNA demethylation pathway. Initially, DNA methylation was believed to be important mainly for gene silencing through promoter DNA methylation, especially at CpG-rich promoters containing CpG islands. However, our understanding of the role that DNA methylation plays in gene regulation during normal development and how this process becomes deregulated in cancer, has evolved in recent years. Moreover, the discovery of frequent mutations in DNMT3A and TET2 both in clonal hematopoiesis of indeterminate significance as well as in many hematological malignancies has brought new interest into understanding what role DNA methylation plays in normal HSC function as well as how it contributes to malignant transformation. In this session, we will review the current understanding in the field of DNA methylation and gene regulation, and present data on DNA methylation in normal HSCs as well as the role that this epigenetic mark plays during leukemic transformation in acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

73 LIPID CONTENT AND CRYOTOLERANCE OF PORCINE EMBRYOS CULTURED WITH PHENAZINE ETHOSULFATE

2011 ◽  
Vol 23 (1) ◽  
pp. 142 ◽  
Author(s):  
B. Gajda ◽  
M. Romek ◽  
I. Grad ◽  
E. Krzysztofowicz ◽  
M. Bryla ◽  
...  
Keyword(s):  
Lipid Content ◽  
Culture Medium ◽  
Nile Red ◽  
Cell Number ◽  
Fluorescent Dye ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Phenazine Ethosulfate ◽  
Experimental Group

In this study, the addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on cytoplasmic lipid content in cultured pig embryo and survival after open pulled straw (OPS) vitrification (Vajta et al. 1997 Acta Vet. Scand. 38, 363–366). In addition, in cultured blastocysts, the total cell number per blastocyst and the degree of apoptosis were assessed. Porcine zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 0 (control, n = 146) or 0.05 μM PES (n = 150). To evaluate the lipid content in embryos, we employed Nile Red (NR), a fluorescent dye specific for intracellular lipids (Genicot et al. 2005 Theriogenology 63, 1181–1194). We measured the amount of fluorescence originating from NR using LSM 510 Meta Zeiss confocal microscope and ImageJ version 1.38x software (National Institutes of Health, Bethesda, MD, USA) and an Integrated Density (ID) parameter. The total amount of fluorescence per embryo (TF), proportional to the amount of lipids, was calculated as the sum of ID measured for all optical slices in each individual z-stack. Blastocysts that were cultured with (n = 48) or without PES (n = 34) were vitrified using OPS technology. Results were analysed using chi-square, Fisher, and Student’s t-tests. The total number of cells and the percentage of TUNEL-positive nuclei of PES-treated blastocysts were significantly different than for the control group (43.6 v. 37.6; P < 0.05 and 1.6 v. 2.9; P < 0.01, respectively). Blastocysts stained with Nile Red fluorescent dye showed intracellular lipid mainly localised to the lipid droplets. They were present both in the embryoblast and trophoblast cells. Mean values of TF estimated for the experimental group was lower by ∼23% than those of the control group. Thereby, blastocysts of the control group possess a higher content of lipids then those found in the experimental group cultured in medium with 0.05 μM PES (P < 0.001). The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (44.8 and 37.1%, respectively). These results showed that culturing porcine embryos in medium with phenazine ethosulfate supplementation increased the total cell number per blastocyst and reduced the index of DNA fragmentation of cultured blastocysts. Use of PES in porcine culture medium reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. However, the use of PES during in vitro culture had a limited effect on porcine blastocyst survival after vitrification. This study was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.

scholarly journals 49 EFFECTS OF DIETARY CONJUGATED LINOLEIC ACID SUPPLEMENTATION ON BOVINE OOCYTE LIPID METABOLISM, LIPID COMPOSITION, AND EMBRYO CRYOTOLERANCE

2015 ◽  
Vol 27 (1) ◽  
pp. 117
Author(s):  
C. L. Bailey ◽  
J. A. Sarmiento-Guzmán ◽  
S. E. Farmer ◽  
G. T. Gentry ◽  
J. W. Lynn ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Lipid Content ◽  
Milk Fat ◽  
Culture Medium ◽  
Nile Red ◽  
Dietary Supplementation ◽  
Milk Composition ◽  
Lipid Supplement ◽  
Relative Gene

Reduced tolerance to chilling and cryotolerance of oocytes and embryos has been associated with greater cytoplasmic lipids. Previous studies in the cow have demonstrated nutrition-induced modification of follicular components. trans-10, cis-12 conjugated linoleic acid (CLA) was identified as a potent inhibitor of milk fat synthesis in lactating cows, and inclusion of CLA in bovine embryo culture medium reduced embryo lipid content and improved post-thaw embryo survival. Dietary supplementation of cows with CLA could alter oocyte fatty acid metabolism, oocyte lipid composition, and embryo cryotolerance, and responses may be different between Bos indicus and Bos taurus cattle. Therefore, a series of experiments were conducted to evaluate effects of dietary CLA on bovine oocyte lipid content and lipid metabolism and cryosurvival of in vitro-produced embryos from CLA-supplemented oocyte donor cows. Lactating Holstein cows (n = 39) were supplemented 100 g per head/d CLA or Ca salts of palm oil to determine effects of dietary CLA on milk production and milk composition. Nonlactating Holstein (n = 8) and Brahman (n = 17) cows were individually supplemented with 150 g per head/d CLA or no lipid supplement. Cumulus-oocyte complexes (COC) were collected after 41 ± 1 day of CLA supplementation, and mRNA was isolated for qPCR. Relative gene expression in COC from CLA- and control-fed cows was evaluated for genes involved in lipid metabolism (CPT1, FADS2, and PPARα). Crossbred (Angus × Red Angus × Brangus) cows (n = 28) were randomly allotted to a 2 × 2 factorial experiment and fed 150 g per head/d CLA or no lipid supplement. Oocytes were collected (Day 129 and 143 of CLA supplementation), matured, fertilized, and cultured in vitro for 7 days in serum-free culture medium. Embryos were cryopreserved in individual 0.25-mL plastic straws containing 1.5 M ethylene glycol using a slow-cooled method. Post-thaw survival and hatching were evaluated using a 24-h in vitro culture (mSOF with 5% FBS) assay. Milk yield, milk composition, and Nile Red intensity were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). Follicle and oocyte responses were analysed with the Mixed procedure of SAS. Relative gene expression of COC was evaluated using the REST 2009 Software. In vitro embryo production, post-thaw survival, and hatching rates were analysed using Chi Square. Milk fat was depressed (P < 0.001) by 10.1% in lactating Holstein cows fed CLA. Dietary supplementation of Holstien and Brahman cows with CLA did not alter expression of genes (CPT1, FADS2, and PPARα) in COC. Dietary supplementation of crossbred cows with CLA before oocyte collection did not influence cryotolerance of in vitro-produced embryos. Lipid content of oocytes (measured by Nile Red florescence) and follicle, oocyte, and embryo production was not influenced by CLA supplementation of Holstein and Brahman cows. The highly regulated mechanisms involved in fatty acid uptake by ovarian components may help explain the lack of ovarian response to dietary CLA in the current study.

81 Effects of Day 5 or 6 HEPES-Buffered Transportation Culture Medium on Developmental Competence of Bovine In Vitro-Produced Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 179
Author(s):  
W. Choi ◽  
C. M. Owen ◽  
M. Barcelo-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Embryo Culture ◽  
Culture Medium ◽  
Nile Red ◽  
Developmental Competence ◽  
Fluorescent Intensity ◽  
Embryo Culture Medium ◽  
Thermo Fisher Scientific ◽  
And Control

Most in vitro-produced (IVP) bovine embryos are transferred fresh. Use of a HEPES/bicarbonate embryo culture medium for transportation would offer flexibility for embryo shipment and transfer. We hypothesized that embryos cultured for 36 (Day 6 embryos) or 60 h (Day 5 embryos) in a novel SCF1T medium (SOF for Conventional Freezing 1 supplemented with HEPES) would maintain developmental competence compared with bicarbonate-buffered medium SCF1 (control). In 5 replicates, IVP embryos were produced by aspirating cumulus–oocyte complexes (COC) from 2-to 8-mm follicles of abattoir ovaries. The COC (n = 1036) were matured for 23 h, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 at 38.5°C, 5% CO2, 5% O2, and 90% N2 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Randomly, on Day 5 and 6 after fertilization, a subset of presumptive embryos were moved into 500-µL polystyrene vials containing 100 µL of SCF1T medium, covered with 300 µL of sterile mineral oil and cultured in a portable incubator (MicroQ iQ2, Scottsdale, AZ, USA) at 38.5°C for 60 and 36 h, respectively. On Day 7.5 post-fertilization, blastocyst rates were evaluated and embryos (n = 8) from each group were stained with 1 µg mL−1 Nile Red for lipid quantification, and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by one-way ANOVA and means separated by Tukey’s HSD. Results (Table 1) indicate similar blastocyst rates and lipid content between embryos cultured for 36 to 60 h in SCF1T and control media (P > 0.05). However, mitochondrial polarity was lower in the Day 5 group (P < 0.05) compared with Day 6 and control groups. Results suggest that culturing embryos in SCF1T medium for 36 h maintains developmental competence compared with bicarbonate-buffered media and offers an alternative for shipment and transfer of IVP embryos. Studies involving evaluation of pregnancy rates of the present study are ongoing. Table 1.Effects of Day 5 or 6 SCF1T embryo culture medium on development, lipid content, and mitochondrial polarity

scholarly journals Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain

2018 ◽  
Vol 115 (9) ◽  
pp. E2125-E2134 ◽  
Author(s):  
Javier Gallego-Bartolomé ◽  
Jason Gardiner ◽  
Wanlu Liu ◽  
Ashot Papikian ◽  
Basudev Ghoshal ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cytosine Methylation ◽  
Catalytic Domain ◽  
Epigenetic Modification ◽  
High Specificity ◽  
Dna Demethylation ◽  
Late Flowering ◽  
Dna Cytosine Methylation ◽  
Activate Gene ◽  
Target Effects

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF–TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF–TET1cd fusions, the SunTag–TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.

Reduction in cytoplasmic lipid content in bovine embryos cultured in vitro with linoleic acid in semi-defined medium is correlated with increases in cryotolerance

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 485-494 ◽  
Author(s):  
Mônica F. Accorsi ◽  
Beatriz Caetano da Silva Leão ◽  
Nathália Alves de Souza Rocha-Frigoni ◽  
Silvia Helena Venturoli Perri ◽  
Gisele Zoccal Mingoti
Keyword(s):  
Linoleic Acid ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Culture Medium ◽  
Calf Serum ◽  
Nile Red ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Embryo Survival

SummaryWe examined whether culturing embryos with linoleic acid (LA) in semi-defined medium reduces lipid accumulation and improves cryosurvival after vitrification. Embryos were cultured with LA (100 μM) and a semi-defined medium was used during in vitro culture (IVC), in which the fetal calf serum was substituted by bovine serum albumin (BSA). There was a reduction (P < 0.05) in the embryonic development rate (Control: 25.8% versus LA: 18.5%), but the proposed system was effective in promoting the decrease (P = 0.0130) in the intracellular lipid content (Control: 27.3 ± 0.7 versus LA: 24.6 ± 0.7 arbitrary fluorescence units of embryos stained with the fluorescent dye Nile Red), consequently increasing (P = 0.0490) the embryo survival after 24h of culture post-warming (Control: 50.0% versus LA: 71.7%). The results question the criteria used to evaluate the efficiency of an in vitro production system specifically with relation to the maximum number of blastocysts produced and suggest that might be more appropriate to improve the desired characteristics of embryos generated in accordance with the specific purpose of in vitro embryo production, commercial or scientific. In conclusion, supplying LA to serum-free culture medium was found to adversely affect the rates of embryo development to the blastocyst stage, but significantly reduced embryo lipid accumulation and improved cryopreservation survival.

scholarly journals Evidence for divergence of DNA methylation maintenance and a conserved inhibitory mechanism from DNA demethylation in chickens and mammals

2021 ◽  
Vol 43 (3) ◽  
pp. 269-280 ◽  
Author(s):  
Masako Tada ◽  
Ayaka Hayashi ◽  
Yumi Asano ◽  
Musashi Kubiura-Ichimaru ◽  
Takamasa Ito ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cytosine Methylation ◽  
Epigenetic Modification ◽  
B Cell Lymphoma ◽  
Dna Methyltransferases ◽  
Dna Demethylation ◽  
Target Sequence ◽  
Sequence Recognition ◽  
Subtelomeric Repeats ◽  
Dt40 Cells

Abstract Background DNA methylation is a significant epigenetic modification that is evolutionarily conserved in various species and often serves as a repressive mark for transcription. DNA methylation levels and patterns are regulated by a balance of opposing enzyme functions, DNA methyltransferases, DNMT1/3A/3B and methylcytosine dioxygenases, TET1/2/3. In mice, the TET enzyme converts DNA cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) at the beginning of fertilisation and gastrulation and initiates a global loss of 5mC, while the 5mC level is increased on the onset of cell differentiation during early embryonic development. Objective Global loss and gain of DNA methylation may be differently regulated in diverged species. Methods Chicken B-cell lymphoma DT40 cells were used as an avian model to compare differences in the overall regulation of DNA modification with mammals. Results We found that DNA methylation is maintained at high levels in DT40 cells through compact chromatin formation, which inhibits TET-mediated demethylation. Human and mouse chromosomes introduced into DT40 cells by cell fusion lost the majority of 5mC, except for human subtelomeric repeats. Conclusion Our attempt to elucidate the differences in the epigenetic regulatory mechanisms between birds and mammals explored the evidence that they share a common chromatin-based regulation of TET–DNA access, while chicken DNMT1 is involved in different target sequence recognition systems, suggesting that factors inducing DNMT–DNA association have already diverged.

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