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Randall S. Prather - One of the best experts on this subject based on the ideXlab platform.
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removal of hypotaurine from Porcine Embryo culture medium does not impair development of in vitro fertilized or somatic cell nuclear transfer derived Embryos at low oxygen tension
Molecular Reproduction and Development, 2020Co-Authors: Paula R Chen, Lee D. Spate, Eric C Leffeler, Joshua A Benne, Raissa F Cecil, Taylor K Hord, Randall S. PratherAbstract:Hypotaurine (HT) is a routine component of Porcine Embryo culture medium, functioning as an antioxidant, but its requirement may be diminished as most Embryo culture systems now use 5% O2 instead of atmospheric (20%) O2 . Our objective was to determine the effects of removing HT from the culture medium on Porcine preimplantation Embryo development. Embryos cultured in 20% O2 without HT had decreased blastocyst development compared to culture with HT or in 5% O2 with or without HT. Notably, differences in blastocyst development or total cell number were not detected between Embryos cultured in 5% O2 with or without HT. After culture in 5% O2 without HT and Embryo transfer, healthy fetuses were retrieved from two pregnancies on Day 42, confirming in vivo developmental competence. Transcript abundance of proapoptotic markers was decreased in Embryos cultured without HT regardless of oxygen tension; however, assays for apoptosis did not demonstrate differences between groups. Additionally, no differences were observed in the development or apoptosis of somatic cell nuclear transfer-derived Embryos cultured in 5% O2 with or without HT. With decreased utility in 5% O2 , removing HT from Porcine Embryo culture medium would also have economic advantages because it is undoubtedly the most expensive component.
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69 Phosphorylation of mechanistic target of rapamycin in Porcine blastocyst-stage Embryos is dependent on the concentration of glutamine in the medium
Reproduction Fertility and Development, 2020Co-Authors: Paula R Chen, Lee D. Spate, Randall S. PratherAbstract:Supplementation of glutamine to Porcine Embryo culture medium improves blastocyst development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the phosphorylation and activation of mechanistic target of rapamycin (MTOR) to support rapid cellular proliferation. The objective of this study was to determine if phosphorylation of MTOR in Porcine blastocysts was dependent upon the concentration of glutamine in the medium and presence of leucine, another activator of MTOR. Presumptive zygotes (n = 732 per treatment across 6 replicates) were split into four groups and cultured in 0, 1, 3.75, or 10 mM GlutaMAX, an L-glutamine alternative, with or without 0.2 mM leucine. On Day 6, percentages of Embryos developed to the blastocyst stage were recorded. Blastocyst-stage Embryos (n = 100 per treatment) were collected for immunoblotting to detect total MTOR and phosphorylated (Ser2448) MTOR (pMTOR) with 3 replicates per target. To assess MTOR and lysosomal colocalization as another indicator of MTOR activation, blastocyst-stage Embryos (n = 15 per treatment) were fixed, probed with antibodies against MTOR and lysosomal associated membrane protein 1 (LAMP1), and imaged on a confocal microscope. Data were analysed by using the GLM procedure in SAS 9.4 (SAS Institute Inc.), and differences between means were detected by using one-way ANOVA followed by a least significant difference test with P
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71 removal of hypotaurine from Porcine Embryo culture medium decreases message for pro apoptotic genes but does not affect development at low oxygen tension
Reproduction Fertility and Development, 2019Co-Authors: Paula R Chen, Lee D. Spate, Eric C Leffeler, Randall S. PratherAbstract:Hypotaurine (HT) is a routine additive to Embryo culture medium, acting primarily as an oxygen radical scavenger. However, the practice of culturing Embryos at ~5% O2 has been widely adopted because this is more physiologically relevant to the oxygen tensions detected in the oviduct and uterus. Thus, the utility of HT may be diminished as fewer oxygen radicals are produced during culture at 5% O2. The objective here was to determine the effects of removing HT from our Porcine Embryo culture medium (MU2) on the development of Embryos incubated at a lower oxygen tension (5% O2) compared with atmospheric oxygen (~20% O2). Porcine cumulus-oocyte complexes were aspirated, matured, and fertilized with standard procedures from our laboratory. Presumptive zygotes were cultured in 1 of 4 conditions: MU2 with 5 mM HT at 5% O2 (low O2 +HT; control), MU2 without HT at 5% O2 (low O2 −HT), MU2 with 5 mM HT at 20% O2 (high O2 +HT), or MU2 without HT at 20% O2 (high O2 −HT). The percentage of presumptive zygotes that developed to the blastocyst stage on Day 6 and total number of nuclei in the blastocysts were determined. The RNA was extracted from pools of 30 blastocysts, and cDNA was synthesised for quantitative PCR for genes associated with HT synthesis, oxidative stress, and apoptosis. Damage to DNA was assessed by TUNEL staining of Day 6 blastocysts. Data were analysed for normality by using a Shapiro-Wilk test, and differences between means were detected by using 2-way ANOVA followed by Tukey’s honest significant difference test with P < 0.05 considered significant. Embryos cultured with high O2 −HT had significantly decreased blastocyst development compared with all other groups (26.2 ± 2.5% v. 36.9-41.7 ± 3.3-4.3%). Embryos cultured with low O2 −HT had significantly more nuclei than those cultured with high O2 +HT (50.5 ± 1.5 v. 45.5 ± 1.2). Notably, differences in blastocyst development (41.7 ± 3.3% v. 36.9 ± 3.3%) or total number of nuclei (50.0 ± 1.8 v. 50.5 ± 1.5) were not detected between Embryos cultured with low O2 +HT or low O2 −HT. The abundance of messages for genes involved in HT synthesis (cysteine sulfinic acid decarboxylase [CSAD]) and oxidative stress (superoxide dismutase 1 [SOD1] and glutathione peroxidase 6 [GPX6]) did not differ between groups. Contrarily, messages for 2 pro-apoptotic markers (BCL2 associated agonist of cell death [BAD] and caspase 3 [CASP3]) were significantly increased in Embryos cultured with +HT regardless of oxygen tension; however, percentages of DNA-damaged nuclei in blastocysts after TUNEL staining were not different between groups (5.4-6.5 ± 0.5-1.0%). These results indicate that HT is not necessary for Porcine pre-implantation development at 5% O2 but is beneficial at atmospheric oxygen tension. Further investigation is required to confirm if HT promotes apoptosis in pre-implantation Embryos. This research was supported by Food for the 21st Century at the University of Missouri and R01 HD080636.
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impairment of preimplantation Porcine Embryo development by histone demethylase kdm5b knockdown through disturbance of bivalent h3k4me3 h3k27me3 modifications
Biology of Reproduction, 2015Co-Authors: Jiaojiao Huang, Hongyong Zhang, Xianlong Wang, Kyle B Dobbs, Jing Yao, Guosong Qin, Kristin M Whitworth, Eric M Walters, Randall S. PratherAbstract:KDM5B (JARID1B/PLU1) is a H3K4me2/3 histone demethylase that is implicated in cancer development and proliferation and is also indispensable for Embryonic stem cell self-renewal, cell fate, and murine Embryonic development. However, little is known about the role of KDM5B during preimplantation Embryo development. Here we show that KDM5B is critical to Porcine preimplantation development. KDM5B was found to be expressed in a stage-specific manner, consistent with demethylation of H3K4me3, with the highest expression being observed from the 4-cell to the blastocyst stages. Knockdown of KDM5B by morpholino antisense oligonucleotides injection impaired Porcine Embryo development to the blastocyst stage. The impairment of Embryo development might be caused by increased expression of H3K4me3 at the 4-cell and blastocyst stages, which disturbs the balance of bivalent H3K4me3-H3K27me3 modifications at the blastocyst stage. Decreased abundance of H3K27me3 at blastocyst stage activates multiple members of homeobox genes (HOX), which need to be silenced for faithful Embryo development. Additionally, the histone demethylase KDM6A was found to be upregulated by knockdown of KDM5B, which indicated it was responsible for the decreased abundance of H3K27me3 at the blastocyst stage. The transcriptional levels of Ten-Eleven Translocation gene family members (TET1, TET2, and TET3) are found to be increased by knockdown of KDM5B, which indicates cross talk between histone modifications and DNA methylation. The studies above indicate that KDM5B is required for Porcine Embryo development through regulating the balance of bivalent H3K4me3-H3K27me3 modifications.
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oxamflatin treatment enhances cloned Porcine Embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Mingtao Zhao, Lee D. Spate, Kristin M Whitworth, Eric M Walters, Clifton N Murphy, Chad Ogorman, Melissa Samuel, Kevin D Wells, Rocio Melissa Rivera, Randall S. PratherAbstract:Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT Embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on Embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT Embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) Embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the c...
Ryan A. Cabot - One of the best experts on this subject based on the ideXlab platform.
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KPNA7, an oocyte- and Embryo-specific karyopherin α subtype, is required for Porcine Embryo development
Reproduction Fertility and Development, 2012Co-Authors: Xin Wang, Ki-eun Park, S. L. Koser, Shihong Liu, Luca Magnani, Ryan A. CabotAbstract:Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in Porcine oocytes and Embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage Embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect Porcine cleavage development. To test this hypothesis, in vitro matured and fertilised Porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all Embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P
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kpna7 an oocyte and Embryo specific karyopherin α subtype is required for Porcine Embryo development
Reproduction Fertility and Development, 2012Co-Authors: Xin Wang, Ki-eun Park, S. L. Koser, Shihong Liu, Luca Magnani, Ryan A. CabotAbstract:Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in Porcine oocytes and Embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage Embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect Porcine cleavage development. To test this hypothesis, in vitro matured and fertilised Porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all Embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P < 0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that Embryos have differential requirements for individual karyopherin α subtypes and that these karyopherin α subtypes differentially transport intracellular cargo during cleavage development.
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IVMBIX-01294, an inhibitor of the histone methyltransferase EHMT2, disrupts histone H3 lysine 9 (H3K9) dimethylation in the cleavage-stage Porcine Embryo
Reproduction Fertility and Development, 2012Co-Authors: Ki-eun Park, Christine M. Johnson, Ryan A. CabotAbstract: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.
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differential remodeling of mono and trimethylated h3k27 during Porcine Embryo development
Molecular Reproduction and Development, 2009Co-Authors: Ki-eun Park, Luca Magnani, Ryan A. CabotAbstract:Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage Porcine Embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation). We found that monomethylated H3K27 was detectable in the nuclei of oocytes and pronuclear, 2-cell, 4-cell, 8-cell, and blastocyst stage Embryos. Trimethylated H3K27 was detectable in the nuclei of GV stage oocytes, the chromosome of MII stage oocytes and a single pronucleus of the pronuclear stage Embryos produced by fertilization; the signals were faint or absent in nuclei of two-cell through blastocyst stage Embryos. In addition, EED transcripts were increased from the four-cell stage (P < 0.05) in Embryos obtained by in vitro fertilization, parthenogenetic activation and in vivo fertilization. EZH2 transcript levels were highest in the GV-stage oocyte (P < 0.05). SUZ12 transcripts were transiently increased at the four-cell stage (P < 0.05) in parthenogenetic and in vivo derived Embryos. Our results suggest that H3K27 trimethylation is an epigenetic marker of maternally derived chromatin that is globally remodeled during Porcine Embryogenesis. Mol. Reprod. Dev. 76: 1033–1042, 2009. © 2009 Wiley-Liss, Inc.
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cleavage stage Porcine Embryos may have differing developmental requirements for karyopherins α2 and α3
Molecular Reproduction and Development, 2003Co-Authors: Ryan A. Cabot, Randall S. PratherAbstract:Numerous cellular proteins are able to localize to the nucleus due to the fact that they possess a nuclear localization signal (NLS) in their amino acid sequence. Nuclear localization sequences recognized by the importin α/β heterodimer are found in cellular proteins capable of performing many diverse functions, ranging from chromatin remodeling to cell cycle regulation. Evidence has been presented that suggests individual importin α homologues are present at varying levels in different adult tissues. Other data have shown that specific subsets of NLSs found in different cellular proteins are recognized by individual importin α homologues with varying affinities. This evidence led us to hypothesize that due to the specific cargoes they carry, the mammalian Embryo has different developmental requirements for individual importin α homologues. The results of the studies presented here indicate that importin α/β-mediated import occurs throughout early cleavage in the Porcine Embryo, as determined by a reporter protein microinjection assay, and that multiple importin α homologues are present throughout early cleavage, as determined by immunocytochemical analysis. An RNA interference approach was used in an attempt to determine the developmental requirements for specific importin α homologues during early cleavage in the Porcine Embryo. Results from this study showed that fertilized Porcine Embryos injected with double stranded RNA (dsRNA) corresponding to the importin α homologue karyopherin α3 had significantly fewer nuclei following four days of culture than did Embryos injected with dsRNA for another importin α homologue, karyopherin α2, or two control groups. This is the first report indicating that mammalian Embryos may have differential developmental requirements for specific nuclear trafficking pathways. Mol. Reprod. Dev. 64: 292–301, 2003. © 2003 Wiley-Liss, Inc.
Lee D. Spate - One of the best experts on this subject based on the ideXlab platform.
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removal of hypotaurine from Porcine Embryo culture medium does not impair development of in vitro fertilized or somatic cell nuclear transfer derived Embryos at low oxygen tension
Molecular Reproduction and Development, 2020Co-Authors: Paula R Chen, Lee D. Spate, Eric C Leffeler, Joshua A Benne, Raissa F Cecil, Taylor K Hord, Randall S. PratherAbstract:Hypotaurine (HT) is a routine component of Porcine Embryo culture medium, functioning as an antioxidant, but its requirement may be diminished as most Embryo culture systems now use 5% O2 instead of atmospheric (20%) O2 . Our objective was to determine the effects of removing HT from the culture medium on Porcine preimplantation Embryo development. Embryos cultured in 20% O2 without HT had decreased blastocyst development compared to culture with HT or in 5% O2 with or without HT. Notably, differences in blastocyst development or total cell number were not detected between Embryos cultured in 5% O2 with or without HT. After culture in 5% O2 without HT and Embryo transfer, healthy fetuses were retrieved from two pregnancies on Day 42, confirming in vivo developmental competence. Transcript abundance of proapoptotic markers was decreased in Embryos cultured without HT regardless of oxygen tension; however, assays for apoptosis did not demonstrate differences between groups. Additionally, no differences were observed in the development or apoptosis of somatic cell nuclear transfer-derived Embryos cultured in 5% O2 with or without HT. With decreased utility in 5% O2 , removing HT from Porcine Embryo culture medium would also have economic advantages because it is undoubtedly the most expensive component.
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69 Phosphorylation of mechanistic target of rapamycin in Porcine blastocyst-stage Embryos is dependent on the concentration of glutamine in the medium
Reproduction Fertility and Development, 2020Co-Authors: Paula R Chen, Lee D. Spate, Randall S. PratherAbstract:Supplementation of glutamine to Porcine Embryo culture medium improves blastocyst development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the phosphorylation and activation of mechanistic target of rapamycin (MTOR) to support rapid cellular proliferation. The objective of this study was to determine if phosphorylation of MTOR in Porcine blastocysts was dependent upon the concentration of glutamine in the medium and presence of leucine, another activator of MTOR. Presumptive zygotes (n = 732 per treatment across 6 replicates) were split into four groups and cultured in 0, 1, 3.75, or 10 mM GlutaMAX, an L-glutamine alternative, with or without 0.2 mM leucine. On Day 6, percentages of Embryos developed to the blastocyst stage were recorded. Blastocyst-stage Embryos (n = 100 per treatment) were collected for immunoblotting to detect total MTOR and phosphorylated (Ser2448) MTOR (pMTOR) with 3 replicates per target. To assess MTOR and lysosomal colocalization as another indicator of MTOR activation, blastocyst-stage Embryos (n = 15 per treatment) were fixed, probed with antibodies against MTOR and lysosomal associated membrane protein 1 (LAMP1), and imaged on a confocal microscope. Data were analysed by using the GLM procedure in SAS 9.4 (SAS Institute Inc.), and differences between means were detected by using one-way ANOVA followed by a least significant difference test with P
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71 removal of hypotaurine from Porcine Embryo culture medium decreases message for pro apoptotic genes but does not affect development at low oxygen tension
Reproduction Fertility and Development, 2019Co-Authors: Paula R Chen, Lee D. Spate, Eric C Leffeler, Randall S. PratherAbstract:Hypotaurine (HT) is a routine additive to Embryo culture medium, acting primarily as an oxygen radical scavenger. However, the practice of culturing Embryos at ~5% O2 has been widely adopted because this is more physiologically relevant to the oxygen tensions detected in the oviduct and uterus. Thus, the utility of HT may be diminished as fewer oxygen radicals are produced during culture at 5% O2. The objective here was to determine the effects of removing HT from our Porcine Embryo culture medium (MU2) on the development of Embryos incubated at a lower oxygen tension (5% O2) compared with atmospheric oxygen (~20% O2). Porcine cumulus-oocyte complexes were aspirated, matured, and fertilized with standard procedures from our laboratory. Presumptive zygotes were cultured in 1 of 4 conditions: MU2 with 5 mM HT at 5% O2 (low O2 +HT; control), MU2 without HT at 5% O2 (low O2 −HT), MU2 with 5 mM HT at 20% O2 (high O2 +HT), or MU2 without HT at 20% O2 (high O2 −HT). The percentage of presumptive zygotes that developed to the blastocyst stage on Day 6 and total number of nuclei in the blastocysts were determined. The RNA was extracted from pools of 30 blastocysts, and cDNA was synthesised for quantitative PCR for genes associated with HT synthesis, oxidative stress, and apoptosis. Damage to DNA was assessed by TUNEL staining of Day 6 blastocysts. Data were analysed for normality by using a Shapiro-Wilk test, and differences between means were detected by using 2-way ANOVA followed by Tukey’s honest significant difference test with P < 0.05 considered significant. Embryos cultured with high O2 −HT had significantly decreased blastocyst development compared with all other groups (26.2 ± 2.5% v. 36.9-41.7 ± 3.3-4.3%). Embryos cultured with low O2 −HT had significantly more nuclei than those cultured with high O2 +HT (50.5 ± 1.5 v. 45.5 ± 1.2). Notably, differences in blastocyst development (41.7 ± 3.3% v. 36.9 ± 3.3%) or total number of nuclei (50.0 ± 1.8 v. 50.5 ± 1.5) were not detected between Embryos cultured with low O2 +HT or low O2 −HT. The abundance of messages for genes involved in HT synthesis (cysteine sulfinic acid decarboxylase [CSAD]) and oxidative stress (superoxide dismutase 1 [SOD1] and glutathione peroxidase 6 [GPX6]) did not differ between groups. Contrarily, messages for 2 pro-apoptotic markers (BCL2 associated agonist of cell death [BAD] and caspase 3 [CASP3]) were significantly increased in Embryos cultured with +HT regardless of oxygen tension; however, percentages of DNA-damaged nuclei in blastocysts after TUNEL staining were not different between groups (5.4-6.5 ± 0.5-1.0%). These results indicate that HT is not necessary for Porcine pre-implantation development at 5% O2 but is beneficial at atmospheric oxygen tension. Further investigation is required to confirm if HT promotes apoptosis in pre-implantation Embryos. This research was supported by Food for the 21st Century at the University of Missouri and R01 HD080636.
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oxamflatin treatment enhances cloned Porcine Embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Mingtao Zhao, Lee D. Spate, Kristin M Whitworth, Eric M Walters, Clifton N Murphy, Chad Ogorman, Melissa Samuel, Kevin D Wells, Rocio Melissa Rivera, Randall S. PratherAbstract:Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT Embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on Embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT Embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) Embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the c...
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oxamflatin treatment enhances cloned Porcine Embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Jiude Mao, Lee D. Spate, Kristin M Whitworth, Eric M Walters, Clifton N Murphy, Mingtao Zhao, Chad Ogorman, Melissa Samuel, Kiho Lee, Kevin D WellsAbstract:Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT Embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on Embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT Embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) Embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned Embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF Embryos, a higher methylation level in day-7 cloned Embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned Embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo Embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned Embryos was delayed again when compared to in vivo Embryos. However, methylation of XIST in Oxamflatin-treated Embryos was comparable with in vivo Embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency.
Ki-eun Park - One of the best experts on this subject based on the ideXlab platform.
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KPNA7, an oocyte- and Embryo-specific karyopherin α subtype, is required for Porcine Embryo development
Reproduction Fertility and Development, 2012Co-Authors: Xin Wang, Ki-eun Park, S. L. Koser, Shihong Liu, Luca Magnani, Ryan A. CabotAbstract:Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in Porcine oocytes and Embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage Embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect Porcine cleavage development. To test this hypothesis, in vitro matured and fertilised Porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all Embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P
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kpna7 an oocyte and Embryo specific karyopherin α subtype is required for Porcine Embryo development
Reproduction Fertility and Development, 2012Co-Authors: Xin Wang, Ki-eun Park, S. L. Koser, Shihong Liu, Luca Magnani, Ryan A. CabotAbstract:Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in Porcine oocytes and Embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage Embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect Porcine cleavage development. To test this hypothesis, in vitro matured and fertilised Porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all Embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P < 0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that Embryos have differential requirements for individual karyopherin α subtypes and that these karyopherin α subtypes differentially transport intracellular cargo during cleavage development.
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IVMBIX-01294, an inhibitor of the histone methyltransferase EHMT2, disrupts histone H3 lysine 9 (H3K9) dimethylation in the cleavage-stage Porcine Embryo
Reproduction Fertility and Development, 2012Co-Authors: Ki-eun Park, Christine M. Johnson, Ryan A. CabotAbstract: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.
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differential remodeling of mono and trimethylated h3k27 during Porcine Embryo development
Molecular Reproduction and Development, 2009Co-Authors: Ki-eun Park, Luca Magnani, Ryan A. CabotAbstract:Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage Porcine Embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation). We found that monomethylated H3K27 was detectable in the nuclei of oocytes and pronuclear, 2-cell, 4-cell, 8-cell, and blastocyst stage Embryos. Trimethylated H3K27 was detectable in the nuclei of GV stage oocytes, the chromosome of MII stage oocytes and a single pronucleus of the pronuclear stage Embryos produced by fertilization; the signals were faint or absent in nuclei of two-cell through blastocyst stage Embryos. In addition, EED transcripts were increased from the four-cell stage (P < 0.05) in Embryos obtained by in vitro fertilization, parthenogenetic activation and in vivo fertilization. EZH2 transcript levels were highest in the GV-stage oocyte (P < 0.05). SUZ12 transcripts were transiently increased at the four-cell stage (P < 0.05) in parthenogenetic and in vivo derived Embryos. Our results suggest that H3K27 trimethylation is an epigenetic marker of maternally derived chromatin that is globally remodeled during Porcine Embryogenesis. Mol. Reprod. Dev. 76: 1033–1042, 2009. © 2009 Wiley-Liss, Inc.
Luca Magnani - One of the best experts on this subject based on the ideXlab platform.
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KPNA7, an oocyte- and Embryo-specific karyopherin α subtype, is required for Porcine Embryo development
Reproduction Fertility and Development, 2012Co-Authors: Xin Wang, Ki-eun Park, S. L. Koser, Shihong Liu, Luca Magnani, Ryan A. CabotAbstract:Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in Porcine oocytes and Embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage Embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect Porcine cleavage development. To test this hypothesis, in vitro matured and fertilised Porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all Embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P
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kpna7 an oocyte and Embryo specific karyopherin α subtype is required for Porcine Embryo development
Reproduction Fertility and Development, 2012Co-Authors: Xin Wang, Ki-eun Park, S. L. Koser, Shihong Liu, Luca Magnani, Ryan A. CabotAbstract:Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in Porcine oocytes and Embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage Embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect Porcine cleavage development. To test this hypothesis, in vitro matured and fertilised Porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all Embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P < 0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that Embryos have differential requirements for individual karyopherin α subtypes and that these karyopherin α subtypes differentially transport intracellular cargo during cleavage development.
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differential remodeling of mono and trimethylated h3k27 during Porcine Embryo development
Molecular Reproduction and Development, 2009Co-Authors: Ki-eun Park, Luca Magnani, Ryan A. CabotAbstract:Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage Porcine Embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation). We found that monomethylated H3K27 was detectable in the nuclei of oocytes and pronuclear, 2-cell, 4-cell, 8-cell, and blastocyst stage Embryos. Trimethylated H3K27 was detectable in the nuclei of GV stage oocytes, the chromosome of MII stage oocytes and a single pronucleus of the pronuclear stage Embryos produced by fertilization; the signals were faint or absent in nuclei of two-cell through blastocyst stage Embryos. In addition, EED transcripts were increased from the four-cell stage (P < 0.05) in Embryos obtained by in vitro fertilization, parthenogenetic activation and in vivo fertilization. EZH2 transcript levels were highest in the GV-stage oocyte (P < 0.05). SUZ12 transcripts were transiently increased at the four-cell stage (P < 0.05) in parthenogenetic and in vivo derived Embryos. Our results suggest that H3K27 trimethylation is an epigenetic marker of maternally derived chromatin that is globally remodeled during Porcine Embryogenesis. Mol. Reprod. Dev. 76: 1033–1042, 2009. © 2009 Wiley-Liss, Inc.