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RISS 인기검색어
- 검색키워드
- 저자 : Hong‐Thuy Bui (검색결과 25 건)
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Effect of Vanadate on the Chromatin Configuration in Pig GV-oocytes
BUI, Hong-Thuy,HWANG, Kyu-Chan,KIM, Jin-Hoi,VAN THUAN, Nguyen,WAKAYAMA, Teruhiko,MIYANO, Takashi Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.4
<P>Vanadate, an inhibitor of tyrosine phosphatases, has been reported to prevent germinal vesicle breakdown in mammalian oocytes. We examined the effect of vanadate on the chromatin configuration of fully grown pig oocytes. In the presence of human menopausal gonadotropin (hMG), vanadate (0.5-5 mM) resulted in a dose-dependent change in oocyte chromatin in germinal vesicles from the condensed state to a decondensed filamentous or stringy configuration. The effect of vanadate and hMG on chromatin configuration could be replicated with 2 mM dibutyryl cyclic AMP (dbcAMP) in place of hMG. Western blot analysis showed that vanadate caused a massive accumulation in the oocytes of tyrosine-phosphorylated proteins with a range of molecular weights that was enhanced by both hMG and dbcAMP in a similar manner. These results suggest that inhibition of tyrosine phosphatase(s) in the presence of an effective level of cAMP induces a change in chromatin configuration of pig oocytes.</P>
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Bui, Hong-Thuy,Wakayama, Sayaka,Kishigami, Satoshi,Park, Keun-Kyu,Kim, Jin-Hoi,Thuan, Nguyen Van,Wakayama, Teruhiko Society for the Study of Reproduction [etc.] 2010 BIOLOGY OF REPRODUCTION Vol.83 No.3
<P>Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5'-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.</P>
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Bui, Hong-Thuy,Seo, Hyeon-Jeong,Park, Mi-Rung,Park, Jong-Yi,Thuan, Nguyen Van,Wakayama, Teruhiko,Kim, Jin-Hoi Society for the Study of Reproduction [etc.] 2011 BIOLOGY OF REPRODUCTION Vol.85 No.5
<P>Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos. In the present study, using highly sensitive fluorescence in situ hybridization (FISH) with probes complementary to mouse rDNA, the effect of Scriptaid on the onset of rRNA synthesis was examined in cloned embryos. In addition, to determine how Scriptaid affects pre-rRNA processing machinery in SCNT embryos with activated rDNA transcription, functional nucleolar formation was analyzed in detail by combined assessment of rRNA synthesis and nucleolar protein allocation in preimplantation embryos. In this experiment, at least part of the rRNA localization by FISH was substituted by 5-bromouridine 5'-triphosphate staining after alpha-amanitin treatment. The results show that in the late 2-cell stage, a number of SCNT embryos initiated transcriptional activation while having one blastomere showing inactivated rRNA transcription and another blastomere showing activated rRNA transcription and despite both nuclei being in interphase. In addition, in some SCNT embryos, the same nuclei contained a mixture of inactively and actively transcribed rRNA, which was rarely observed in intracytoplasmic sperm injection embryos. This asynchronous transcription induced a delay of one cell cycle in SCNT embryo activation of functional nucleoli. Scriptaid can overcome this failure in the timely onset of embryonic gene transcription by activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of EGA.</P>
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Identification and characterization of putative stem cells in the adult pig ovary
Bui, Hong-Thuy,Van Thuan, Nguyen,Kwon, Deug-Nam,Choi, Yun-Jung,Kang, Min-Hee,Han, Jae-Woong,Kim, Teoan,Kim, Jin-Hoi The Company of Biologists Limited 2014 Development (Cambridge) Vol.141 No.11
<P>Recently, the concept of ‘neo-oogenesis’ has received increasing attention, since it was shown that adult mammals have a renewable source of eggs. The purpose of this study was to elucidate the origin of these eggs and to confirm whether neo-oogenesis continues throughout life in the ovaries of the adult mammal. Adult female pigs were utilized to isolate, identify and characterize, including their proliferation and differentiation capabilities, putative stem cells (PSCs) from the ovary. PSCs were found to comprise a heterogeneous population based on c-kit expression and cell size, and also express stem and germ cell markers. Analysis of PSC molecular progression during establishment showed that these cells undergo cytoplasmic-to-nuclear translocation of Oct4 in a manner reminiscent of gonadal primordial germ cells (PGCs). Hence, cells with the characteristics of early PGCs are present or are generated in the adult pig ovary. Furthermore, the <I>in vitro</I> establishment of porcine PSCs required the presence of ovarian cell-derived extracellular regulatory factors, which are also likely to direct stem cell niche interactions <I>in vivo</I>. In conclusion, the present work supports a crucial role for c-kit and kit ligand/stem cell factor in stimulating the growth, proliferation and nuclear reprogramming of porcine PSCs, and further suggests that porcine PSCs might be the culture equivalent of early PGCs.</P>
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GV Extract Highly Induce Pig Stem Like Cells from Differentiated Somatic Cells
Hong-Thuy Bui,Deug-Nam Kwon,Mi-Ryung Park,Min-Hui Kang,Mihye Oh,Nguyen Van Thuan,Jin-Hoi Kim 한국동물번식학회 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s
Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre-exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. In this study, a pig GV oocyte extract (pGV extract) was developed. Treatment of pig fibroblasts with the pGV extract promoted colony formation after 2-3 weeks in culture, concomitant with the expression of stem cell markers (Oct-4, Rex1, Nanog, Sox2) and repression of differentiated cell markers (CKAP2, NPR3 ). Using fibroblasts transfected with human Oct-4 promoter-driven enhanced green fluorescent protein (Oct4-EGFP), pGV extract treatment induced the reactivation of the Oct-4 promoter in Oct4 - EGFP cells by 10 days post-treatment. These transgenic donor cells were injected into 8-cell embryos. Oct-4 promoter activity was subsequently detected in most ICM cells of the host blastocyst. Interestingly, reconstructed embryos with pGV extract-treated Oct4- EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Additionally, the pGV extract promoted somatic cell reprogramming and cloned embryo development when assessed by measuring histone H3-K9 hypomethylation, the expression of Oct4 and Nanog in blastocysts, and the production of increased numbers of high- quality blastocysts. Under specific culture conditions, pGV extract-treated fibroblast cells differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig.
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Establishment and Characterization of Pig iPS and Female Germ Line Stem Cells
Hong-Thuy Bui,Jin-Hoi Kim 한국동물번식학회 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s
Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre-exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. To overcome difficulties in preparing mice oocyte extract, a pig GV oocyte extract (pGV extract) was developed to investigate the epigenetic reprogramming events in treated somatic cell nuclei. The pGV extract promoted colony formation concomitant with the expression of stem cell markers and repression of differentiated cell markers in treated cells. Using fibroblasts transfected with human Oct-4 promoter- driven enhanced green fluorescent protein (Oct4-EGFP), pGV extract treatment induced the reactivation of the Oct-4 promoter in Oct4-EGFP cells by 10 days post-treatment. Interestingly, reconstructed embryos with pGV extract-treated Oct4-EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Using donor nuclei treated with pGV extract, increase the number of high-quality blastocysts that expressed Me-H3-K9, Oct4 and Nanog at levels comparable to in vitro fertilized embryos. The pGV extracttreated fibroblast cells can differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig. Next, we identified germ line stem cells that supported oogenesis. female germ line stem cells (FGSC) from neonatal pig was established and cultured for more than 6 months. After long-term culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. To further study developmental potential of oocyte-like cells generated from FGSCs, these cells were aggregated with granulosa cells collected from neonatal pig ovaries. Interestingly after overnight culture in hanging drops, oocyte-like cells aggregated with granulosa cells and formed structures very similar to primordial follicles containing the oocyte-like cell in the middle and a layer of granulosa cells around it. Our results demonstrate the presence of a population of germ line stem cells in postnatal pig ovary with the ability to self-renew and differentiate to oocyte-like cells that might be useful for follicle engineering and assisted reproductive technologies. However, the functionality of FGSC-derived oocytes us-ing in vitro maturation, fertilization and embryo development as well as ovarian transplantation is currently under investigation. In conclusion, gene manipulation of FGSCs or iPS cells is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.
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