IVMBIX-01294, an inhibitor of the histone methyltransferase EHMT2, disrupts histone H3 lysine 9 (H3K9) dimethylation in the cleavage-stage porcine embryo

2012 ◽  
Vol 24 (6) ◽  
pp. 813 ◽  
Author(s):  
Ki-Eun Park ◽  
Christine M. Johnson ◽  
Ryan A. Cabot
Keyword(s):  
Potent Inhibitor ◽  
Histone H3 ◽  
Specific Inhibitor ◽  
Developmental Potential ◽  
Histone Methyltransferases ◽  
Histone Lysine ◽  
Porcine Embryo ◽  
H3k9 Dimethylation ◽  
Vitro Development

Global patterns of histone methylation are remodelled during cleavage development. Of the five histone methyltransferases known to mediate methylation of the lysine 9 residue of histone H3 (H3K9), euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) has been shown to be a primary mediator of H3K9 dimethylation; BIX-01294 has been shown to be a specific inhibitor of EHMT2. The objective of the present study was to determine the effect of BIX-01294 treatment on global H3K9 dimethylation in porcine embryos. We hypothesised that inhibition of EHMT2 by BIX-01294 would result in reduced levels of H3K9 dimethylation and compromised embryo development. Our results showed that incubation in 5 µM BIX-01294 markedly reduced global levels of H3K9 dimethylation at the pronuclear, 2-cell and 4-cell stages of development and resulted in developmental arrest before blastocyst formation. Although transient exposure of embryos to BIX-01294 did not alter in vitro development, embryos transiently exposed to BIX-01294 did not establish pregnancy. These data demonstrate that BIX-01294 is a potent inhibitor of H3K9 dimethylation and that transient alterations in global histone modifications can have profound effects on embryo developmental potential.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P<0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P<0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P<0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

scholarly journals Melatonin enhances porcine embryo development via the Nrf2/ARE signaling pathway

2019 ◽  
Vol 63 (3) ◽  
pp. 175-185 ◽  
Author(s):  
Eui Hyun Kim ◽  
Geon A Kim ◽  
Anukul Taweechaipaisankul ◽  
Seok Hee Lee ◽  
Muhammad Qasim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Antioxidant Properties ◽  
Specific Inhibitor ◽  
Treated Group ◽  
Related Factor ◽  
Porcine Embryo ◽  
Responsive Element

Oxidative stress (OS) is a major problem during in vitro culture of embryos. Numerous studies have shown that melatonin, which is known to have antioxidant properties, prevents the occurrence of OS in embryos. However, the molecular mechanisms by which melatonin prevents OS in embryos are still unclear. The present study suggests a possible involvement of the nuclear factor erythroid 2-related factor 2/antioxidant-responsive element (Nrf2/ARE) signaling pathway, which is one of the prominent signals for OS prevention through Nrf2 activation, connecting melatonin, OS prevention and porcine embryonic development. The aim of this study was to investigate the effects of melatonin (10−7 M) on porcine embryonic development via the Nrf2/ARE signaling pathway; brusatol (50 nM; Nrf2 specific inhibitor) was used to validate the mechanism. Treatment of porcine embryo with melatonin significantly increased formation rates of blastocysts and their total cell numbers and also upregulated the expression of Nrf2/ARE signaling and apoptosis-related genes (MT2, NRF2, UCHL, HO-1, SOD1 and BCL-2). Furthermore, the expression of proteins (NRF2 and MT2) was also upregulated in the melatonin-treated group. Concomitantly, brusatol significantly inhibited these effects, upregulating the expression of KEAP1 and BAX, including the expression level of KEAP1 protein. These results provide evidences that melatonin prevents OS through Nrf2/ARE signaling pathway in porcine in vitro fertilization -derived embryos.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

scholarly journals Differential Histone H3 Lys-9 and Lys-27 Methylation Profiles on the X Chromosome

2004 ◽  
Vol 24 (12) ◽  
pp. 5475-5484 ◽  
Author(s):  
Claire Rougeulle ◽  
Julie Chaumeil ◽  
Kavitha Sarma ◽  
C. David Allis ◽  
Danny Reinberg ◽  
...  
Keyword(s):  
X Chromosome ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Dynamic Changes ◽  
Histone Methyltransferases ◽  
H3k27 Methylation ◽  
H3k9 Dimethylation ◽  
Inactivation Process ◽  
Kinetics Of ◽  
Relative Kinetics

ABSTRACT Histone H3 tail modifications are among the earliest chromatin changes in the X-chromosome inactivation process. In this study we investigated the relative profiles of two important repressive marks on the X chromosome: methylation of H3 lysine 9 (K9) and 27 (K27). We found that both H3K9 dimethylation and K27 trimethylation characterize the inactive X in somatic cells and that their relative kinetics of enrichment on the X chromosome as it undergoes inactivation are similar. However, dynamic changes of H3K9 and H3K27 methylation on the inactivating X chromosome compared to the rest of the genome are distinct, suggesting that these two modifications play complementary and perhaps nonredundant roles in the establishment and/or maintenance of X inactivation. Furthermore, we show that a hotspot of H3K9 dimethylation 5′ to Xist also displays high levels of H3 tri-meK27. However, analysis of this region in G9a mutant embryonic stem cells shows that these two methyl marks are dependent on different histone methyltransferases.

Development of porcine oocytes from preovulatory follicles of different sizes after maturation in media supplemented with follicular fluids

2000 ◽  
Vol 12 (4) ◽  
pp. 133 ◽  
Author(s):  
Ki-Won Yoon ◽  
Tae-Young Shin ◽  
Jong-Im Park ◽  
Sangho Roh ◽  
Jeong M. Lim ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
Developmental Potential ◽  
Large Follicle ◽  
In Vitro Development ◽  
Maturation Medium ◽  
Preovulatory Follicles ◽  
Vitro Development ◽  
Small Follicle ◽  
Follicular Fluids

The development of porcine oocytes from large (3.1–8.0 mm in diameter) or small (<3.1 mm) follicles was examined after maturation culture in medium containing porcine follicular fluid (pFF). Large follicles yielded larger (256 m v. 221 m; P<0.05) cumulus–oocyte complexes and more (22 v. 14%) morphologically normal oocytes than small follicles (Experiment 1). In Experiments 2–4, maturation media supplemented with mixed pFF (10%) from small and large follicles was used. More oocytes from large follicles matured (58% v. 91%), formed pronuclei (81% v. 90%) and developed to the blastocyst stage (2% v. 10%) than oocytes from small follicles. In Experiments 5–7, the effects of pFF collected from either small or large follicles on oocyte development were examined. Regardless of the source of oocytes, large-follicle-derived pFF more significantly enhanced preimplantation development than did small-follicle-derived pFF. The highest rate of blastocyst formation (16%) was found when oocytes from large follicles were cultured in maturation medium containing large-follicle-derived pFF. These results suggest that oocytes from large follicles have greater developmental potential than oocytes from small follicles, and that the origin of pFF, which is added to the maturation media, might be an important factor for improving in vitro development of porcine oocytes.

scholarly journals Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 230
Author(s):  
Hyo-Gu Kang ◽  
Sanghoon Lee ◽  
Pil-Soo Jeong ◽  
Min Ju Kim ◽  
Soo-Hyun Park ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caspase 3 ◽  
Developmental Competence ◽  
Oxygen Species ◽  
Cell Numbers ◽  
Porcine Embryo ◽  
Apoptosis Pathways ◽  
Vitro Development

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

scholarly journals Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination

2007 ◽  
Vol 178 (6) ◽  
pp. 925-936 ◽  
Author(s):  
Roberta Benetti ◽  
Susana Gonzalo ◽  
Isabel Jaco ◽  
Gunnar Schotta ◽  
Peter Klatt ◽  
...  
Keyword(s):  
Telomere Length ◽  
Histone H3 ◽  
The Novel ◽  
Epigenetic Alterations ◽  
Lysine Methylation ◽  
Histone H4 ◽  
Histone Methyltransferases ◽  
Telomere Elongation ◽  
Histone Lysine Methylation ◽  
Histone Lysine

Mammalian telomeres have heterochromatic features, including trimethylated histone H3 at lysine 9 (H3K9me3) and trimethylated histone H4 at lysine 20 (H4K20me3). In addition, subtelomeric DNA is hypermethylated. The enzymatic activities responsible for these modifications at telomeres are beginning to be characterized. In particular, H4K20me3 at telomeres could be catalyzed by the novel Suv4-20h1 and Suv4-20h2 histone methyltransferases (HMTases). In this study, we demonstrate that the Suv4-20h enzymes are responsible for this histone modification at telomeres. Cells deficient for Suv4-20h2 or for both Suv4-20h1 and Suv4-20h2 show decreased levels of H4K20me3 at telomeres and subtelomeres in the absence of changes in H3K9me3. These epigenetic alterations are accompanied by telomere elongation, indicating a role for Suv4-20h HMTases in telomere length control. Finally, cells lacking either the Suv4-20h or Suv39h HMTases show increased frequencies of telomere recombination in the absence of changes in subtelomeric DNA methylation. These results demonstrate the importance of chromatin architecture in the maintenance of telomere length homeostasis and reveal a novel role for histone lysine methylation in controlling telomere recombination.

scholarly journals Histone H3 K4 Demethylation during Activation and Attenuation of GAL1 Transcription in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (22) ◽  
pp. 7856-7864 ◽  
Author(s):  
Kristin Ingvarsdottir ◽  
Chris Edwards ◽  
Min Gyu Lee ◽  
Jung Shin Lee ◽  
David C. Schultz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Gene Activation ◽  
Histone H3 ◽  
Histone Lysine ◽  
Insight Into ◽  
Demethylation Activity

ABSTRACT In mammalian cells, histone lysine demethylation is carried out by two classes of enzymes, the LSD1/BHC110 class and the jumonji class. The enzymes of the jumonji class in the yeast Saccharomyces cerevisiae have recently also been shown to have lysine demethylation activity. Here we report that the protein encoded by YJR119c (termed KDM5), coding for one of five predicted jumonji domain proteins in yeast, specifically demethylates trimethylated histone H3 lysine 4 (H3K4me3), H3K4me2, and H3K4me1 in vitro. We found that loss of KDM5 increased mono-, di-, and trimethylation of lysine 4 during activation of the GAL1 gene. Interestingly, cells deleted of KDM5 also displayed a delayed reduction of K4me3 upon reestablishment of GAL1 repression. These results indicate that K4 demethylation has two roles at GAL1, first to establish appropriate levels of K4 methylation during gene activation and second to remove K4 trimethylation during the attenuation phase of transcription. Thus, analysis of lysine demethylation in yeast provides new insight into the physiological roles of jumonji demethylase enzymes.

scholarly journals Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat KidneyIn Vivoand Rat Mesangial CellsIn Vitrounder Diabetic Conditions

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Xiangjun Li ◽  
Chaoyuan Li ◽  
Xiaoxia Li ◽  
Peihe Cui ◽  
Qifeng Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Mesangial Cells ◽  
Histone H3 ◽  
Diabetic Rats ◽  
Lysine Methylation ◽  
Histone Lysine Methylation ◽  
Histone Lysine

Diabetic nephropathy (DN), a common complication associated with type 1 and type 2 diabetes mellitus (DM), characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM) protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD). Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC) hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs) have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme) mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT) SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG-) treated rat mesangial cells (RMCs). p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP) assays showed decreased histone H3-lysine9-dimethylation (H3K9me2) accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3) and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expressionin vivoandin vitrounder diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN.

scholarly journals 28 CLONED EMBRYOS CAN BE PRODUCED USING DONOR CELLS OBTAINED FROM A 72-HOUR COOLED CARCASS

2005 ◽  
Vol 17 (2) ◽  
pp. 164 ◽  
Author(s):  
S. Arat ◽  
H. Bagis ◽  
H. Odaman Mercan ◽  
A. Dinnyes
Keyword(s):  
Calf Serum ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Penicillin G ◽  
Developmental Potential ◽  
Viable Cells ◽  
Donor Cells ◽  
A Cell ◽  
Vitro Development

There are few reports on the use of cells from a dead mammal for nuclear transfer (NT). So far, most calves have been cloned from live adult cows or fresh fetal samples. The ability to produce cloned animals using postmortem tissue can provide an additional application to the field of NT. This study was conducted to investigate whether viable cells could be obtained from tissues chilled for 72 h and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FBS), 50 μg/mL sodium pyruvate, 1% v:v penicillin-streptomycin (10,000 U/mL penicillin G, 10,000 μg/mL streptomycin), 10 ng/mL EGF, 0.5 μg/mL FSH, and 5 μg/mL LH. A cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 72 h. Tissues from muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 medium supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18 h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500 μm for 25 μs. After fusion, NT units were activated using a combination of calcium ionophore (5 μM), cytochalasin D (2.5 μg/mL) and cycloheximide (10 μg/mL) and cultured for 7 days in BARC or G1.3-G2.3 medium. Differences (developmental potential and cell numbers) among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from muscle of a cow carcass stored at cold temperature for 72 h and that these cells have ability to generate NT blastocysts at rates similar to those obtained with fresh GCs. In addition, G1.3 and G2.3 culture medium supported embryo development better than BARC medium. Table 1. In vitro development of NT embryos This study was supported by a grant from TUBITAK, Turkey (VHAG-1908 and Turkey-Hungary bilateral project VHAG-2022).

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