1 ABERRANT DNA METHYLATION IN PORCINE IN VITRO-, PARTHENOGENETIC-, AND NUCLEAR TRANSFER-PRODUCED BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 109 ◽  
Author(s):  
A. Bonk ◽  
M. Samuel ◽  
L. Lai ◽  
Y. Hao ◽  
R. Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
The Other ◽  
Specific Gene ◽  
Aberrant Dna Methylation

Aberrant DNA methylation of in vitro-, parthenogenetic-, and nuclear transfer-derived embryos has been implicated in the low developmental competence of early embryos. Demethylation of the genome occurs immediately after fertilization and continues through the blastocyst stage. Remethylation or reprogramming of the genome occurs around the time of implantation and is maintained in somatic tissues. The aim of this study was to analyze DNA methylation in porcine gametes and blastocysts. Differential DNA methylation hybridization was conducted to analyze the methylation status of the Bstu I site (CGCG) in the gamete and blastocyst epigenomes. Germinal vesicle oocytes were aspirated from ovaries collected at an abattoir, sperm was isolated from a fresh ejaculate, and blastocysts were derived and collected from in vivo, in vitro, nuclear transfer, and parthenogenetic sources. Genomic clones were selected from a porcine CpG Island library based on the presence of a Bstu I site. The inserts from these clones were PCR amplified and spotted on glass slides. DNA was digested with Mse I, ligated to linkers, and digested with Bstu I. Fragments with methylated Bstu I sites remained intact whereas fragments with unmethylated Bstu I sites were cut. Intact fragments were amplified by PCR and labeled with amino allyl-dUTP. Liver DNA served as the reference and was labeled with Cy5; the other samples were labeled with Cy3. An Axon Genepix 4000B scanner (Axon Instruments, Inc., Union City, CA, USA) was used to scan the slides. Initial analysis of the microarray image was performed with GenePix Pro 4.0 software. Additional analysis, performed by using Genespring 7.0 ANOVA (P < 0.05), identified 221 clones as being significantly different in at least one of the biological conditions of the gametes or the blastocysts. Forty-six clones were sequenced and BLAST analysis identified 18 clones that were unique, 16 clones that had no similarity, and 12 clones that had similarity to multiple genes. Ribosomal (RPS20, RPL18) and protoporphyrinogen oxidase (PPOX) genes were identified in several clones. Components of the immune system (CCRs, TLRs), a transcription factor (ATF2), and an embryo-specific gene (WNT8B) were also identified. A condition tree was created according to the standard correlation similarity measure for the spots identified as significantly different. The condition tree shows that the methylation profiles are most similar in the germinal vesicle oocyte, parthenogenetic blastocyst, nuclear transfer blastocyst, in vitro-produced blastocyst, and sperm. In vivo-produced blastocysts grouped separately from the other samples. These results are consistent with previous studies that have shown that gametes undergo demethylation after fertilization on through the blastocyst stage when the genome is remethylated. Additionally, these results suggest that the reprogramming events that occur during the development of the in vivo-produced blastocysts are less likely to occur in in vitro-, nuclear transfer-, and parthenogenetic-produced blastocysts. This work was funded by a grant from the NIH (RR13438) and Food for the 21st Century.

Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 81-87 ◽  
Author(s):  
P.N. Moreira ◽  
R. Fernández-Gonzalez ◽  
M.A. Ramirez ◽  
M. Pérez-Crespo ◽  
D. Rizos ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Differential Effects ◽  
Glut 1 ◽  
Mouse Somatic Cell

It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMα, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMα, as well as on in vivo cultured and MEMα cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMα cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

scholarly journals Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts

2007 ◽  
Vol 75 (2) ◽  
pp. 250-264 ◽  
Author(s):  
Aaron J. Bonk ◽  
Rongfeng Li ◽  
Liangxue Lai ◽  
Yanhong Hao ◽  
Zhonghua Liu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Aberrant Dna Methylation

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

scholarly journals Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells

2004 ◽  
Vol 24 (3) ◽  
pp. 1270-1278 ◽  
Author(s):  
Jonathan C. Cheng ◽  
Daniel J. Weisenberger ◽  
Felicidad A. Gonzales ◽  
Gangning Liang ◽  
Guo-Liang Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Promoter Regions ◽  
Bladder Cancer Cells ◽  
Continuous Application ◽  
Aberrant Dna Methylation ◽  
Human Bladder Cancer Cells ◽  
Partial Depletion

ABSTRACT During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(β-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5′ region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2′-deoxycytidine followed by zebularine hindered the remethylation of the p16 5′ region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.

Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses

1999 ◽  
Vol 11 (8) ◽  
pp. 457 ◽  
Author(s):  
Christopher G. Grupen ◽  
Paul J. Verma ◽  
Zhong Tao Du ◽  
Stephen M. McIlfatrick ◽  
Rodney J. Ashman ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Single Treatment ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
Electrical Pulses ◽  
Number Of Cells

The current protocols used to activate pig nuclear transfer embryos are less efficient than those used for other species. To address this problem, the effect of multiple sets of electrical pulses on the parthenogenetic development of in vivo- and in vitro-derived porcine oocytes was examined. Each set of pulses consisted of two 1.5 kV cm–1 DC pulses of 60 s duration each, administered 1 s apart. For in vivo-derived oocytes, application of a second set of pulses 30 min after the first set increased the proportion of oocytes that developed to the blastocyst stage compared with a single treatment (51 v. 34%). Application of a third set of pulses 30 min after the second set reduced the rate of blastocyst formation compared with two sets of pulses. In contrast, the rate of blastocyst formation was greater with one set of pulses compared with two sets for in vitro matured oocytes (31 v. 16%). Additional sets of electrical pulses did not affect the number of cells in blastocysts obtained from either group of oocytes compared with a single treatment. In summary, the study demonstrates that the application of a second set of activating pulses 30 min after the first set is beneficial to in vivo-derived oocytes, but detrimental to in vitro matured oocytes, in terms of their ability to develop parthenogenetically to the blastocyst stage.

5 TRANSCRIPTIONAL PROFILING OF PIG IN IN VIVO, IN VITRO-FERTILIZED, AND NUCLEAR TRANSFER-DERIVED BLASTOCYSTS AND THE DONOR SOMATIC CELL LINE

2008 ◽  
Vol 20 (1) ◽  
pp. 83
Author(s):  
K. M. Whitworth ◽  
L. D. Spate ◽  
R. Li ◽  
A. Rieke ◽  
D. M. Wax ◽  
...  
Keyword(s):  
Cell Line ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Reference Sample ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Electrical Activation ◽  
Shock Proteins

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix� 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

264 ANALYSIS OF DNA METHYLATION PATTERN IN PRE-IMPLANTATION-STAGE EMBRYOS DERIVED FROM NUCLEAR TRANSFER USING PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 248
Author(s):  
D.-H. Choi ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
H.-S. Kim ◽  
H.-Y. Son ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Perinatal Death ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Paternal Allele ◽  
High Birth Weight ◽  
Aberrant Methylation

Somatic cell nuclear transfer (SCNT) has been successfully used to produce live cloned offspring in various mammals. However, some studies had reported that cloned embryos by SCNT had many problems in reprogramming or epigenetic modification, such as DNA methylation. DNA methylation is an essential process in epigenetic modification for development, and aberrant methylation in cloned embryos gives rise to abortion, high birth weight, and perinatal death. In this study, embryonic germ (EG) cells were used as donor cells for nuclear transfer. EG cells may have less reprogramming or demethylation than SCNT because these are already in erased status. However, little is known about methylation state or developmental capacity of the EG cell as a donor. The objective of this study was to analyze the methylation pattern of pre-implantation embryos cloned from porcine EG cells. Two regions, PRE-1 and microsatellite (MS), were analyzed for methylation patterns of cloned embryos from porcine EG cells and compared with the pattern of mature oocytes and in vitro-fertilized (IVF) embryos as a control. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro for 44 h, followed by use for IVF and NT with porcine EG cells. The porcine EG cells were prepared from 28-day-old fetuses after mating; genital ridges were isolated from fetuses, and then transferred into a culture medium on a feeder layer. The number of embryos for analysis was 300 for matured oocytes, 50–80 for 4–8 cell embryos, 30–40 for morulae, and 20–30 for blastocysts. The genomic DNA was isolated from the embryos and treated with bisulfite solution. PCR was performed for the amplification of PRE-1 and MS regions. The PCR products were sequenced by using an automatic DNA sequencer. The methylation rates of the PRE-1 and MS regions in IVF embryos showed that the demethylation process had occurred during the pre-implantation stage, which is a typical phenomenon of in vivo counterparts (Kang et al. 2001 J. Biol. Chem. 276, 39 980). However, compared to IVF embryos, embryos derived from NT using EG cells showed differences at the morula (PRE-1) and blastocyst (MS) stage. These results indicate that porcine EG cells also have problems in reprogramming during NT. For detailed and reliable results, the methylation pattern analysis of the imprinting region, for example, H19 in maternal allele and Igf2 in paternal allele, must be examined. Table 1.Methylation of PRE-1 and MS regions in embryos derived from IVF and NT using porcine EG cells

45 The role of TRIM28 in porcine somatic cell nuclear transfer embryo development

2019 ◽  
Vol 31 (1) ◽  
pp. 148
Author(s):  
Y. H. Zhai ◽  
X. L. An ◽  
Z. R. Zhang ◽  
S. Zhang ◽  
Z. Y. Li
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chromatin Modification ◽  
Blastocyst Stage ◽  
Imprinted Genes ◽  
Cell Stage ◽  
Genomic Methylation

During fertilization, the parental genome undergoes extensive demethylation. Global DNA demethylation is a hallmark of epigenetic reprogramming. Embryos engage non-canonical DNA methylation maintenance mechanisms to ensure inheritance of exceptional germline features. However, the mechanisms ensuring demethylation resistance in light of global reprogramming remain poorly understood. TRIM28 is a maternal-effect factor that controls genomic imprinting during early embryonic reprogramming. In this study, cytoplasmic injections of siRNA were performed into oocytes matured in vitro for 26h to interfere with the expression of TRIM28 in oocytes. The injected oocytes were continually matured in vitro until 42h and used to construct somatic cell nuclear transfer (SCNT) embryos. During 2-cell to blastocyst stages, the expression of development-related genes (NANOG, POU5F1, CDX2, BAX, and BCL2), maternal imprinting genes (IGF2, DIO3, PLAGL1, and DLK1), paternal imprinting genes (H19 and PEG3), TRIM28-recruitment complex-associated genes (ZFP57, PGC7, SETDB1, and DNMT), and epigenetic chromatin modification enzymes were detected by quantitative PCR in the constructed TRIM28-interfered SCNT embryos. The DNA methylation levels in the promoter regions of the imprinted genes (H19 and IGF2) and chromatin repeats (PRE-1 and SATELLITE) were analysed by sodium bisulfite genomic sequencing. The results showed that the TRIM28-interfered SCNT embryos had significantly lower cleavage and blastocyst rates (53.9±3.4% and 12.1±4.3%, respectively) than those in control SCNT embryos (64.8±2.7% and 18.8±1.9%, respectively). The expression levels of development-related genes (NANOG and POU5F1) and TRIM28-recruited transcriptional repression complex-associated genes (PGC7, ZFP57, and DNMT1) in the 4-cell stage were significantly reduced (P&lt;0.05). The imprinted genes were significantly up-regulated (P&lt;0.05) from the 2-cell to blastocyst stage in constructed TRIM28-interfered SCNT embryos, except H19 at the 2-cell and blastocyst stage decreased remarkably (P&lt;0.05). The DNA methylation levels of IGF2 decreased 2-fold from the 2-cell to blastocyst stage in TRIM28-interfered SCNT embryos. The PRE-1 and SATELLITE had a remarkably lower (P&lt;0.05) methylation levels in the TRIM28-interfered 2-cell embryos than in control SCNT embryos. The cluster analysis showed some of the chromatin modification enzymes had abnormal expression in the TRIM28-interfered SCNT embryos, especially in the 8-cell stage, where 48 enzymes were significantly decreased (P&lt;0.05). The down-regulation enzymes were mainly clustered in the histone H3K4 methyl transferase and histone acetylase. These results indicate that down-regulation of maternal TRIM28 breaks the steady-state of genomic methylation at a particular locus of the imprinted gene, disrupts the expression of imprinted gene and epigenetic modifications enzymes, and is detrimental to normal development of SCNT embryos. Maternal TRIM28 is needed in maintaining a stable state of genomic methylation and epigenetic modification state during SCNT embryo development.

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