Publications by authors named "Li-Ying Sung"

47 Publications

CTPS forms the cytoophidium in zebrafish.

Exp Cell Res 2021 Aug 12;405(2):112684. Epub 2021 Jun 12.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom. Electronic address:

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.
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http://dx.doi.org/10.1016/j.yexcr.2021.112684DOI Listing
August 2021

IMPDH forms the cytoophidium in zebrafish.

Dev Biol 2021 May 25;478:89-101. Epub 2021 May 25.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.
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http://dx.doi.org/10.1016/j.ydbio.2021.05.017DOI Listing
May 2021

CTPS and IMPDH form cytoophidia in developmental thymocytes.

Exp Cell Res 2021 Aug 20;405(1):112662. Epub 2021 May 20.

Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan; Animal Resource Center, National Taiwan University, Taipei, 106, Taiwan. Electronic address:

The cytoophidium, a filamentous structure formed by metabolic enzymes, has emerged as a novel regulatory machinery for certain proteins. The rate-limiting enzymes of de novo CTP and GTP synthesis, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH), are the most characterized cytoophidium-forming enzymes in mammalian models. Although the assembly of CTPS cytoophidia has been demonstrated in various organisms including multiple human cancers, a systemic survey for the presence of CTPS cytoophidia in mammalian tissues in normal physiological conditions has not yet been reported. Herein, we examine major organs of adult mouse and observe that CTPS cytoophidia are displayed by a specific thymocyte population ranging between DN3 to early DP stages. Most of these cytoophidium-presenting cells have both CTPS and IMPDH cytoophidia and undergo rapid cell proliferation. In addition, we show that cytoophidium formation is associated with active glycolytic metabolism as the cytoophidium-presenting cells exhibit higher levels of c-Myc, phospho-Akt and PFK. Inhibition of glycolysis with 2DG, however, disrupts most of cytoophidium structures and impairs cell proliferation. Our findings not only indicate that the regulation of CTPS and IMPDH cytoophidia are correlated with the metabolic switch triggered by pre-TCR signaling, but also suggest physiological roles of the cytoophidium in thymocyte development.
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http://dx.doi.org/10.1016/j.yexcr.2021.112662DOI Listing
August 2021

STAT3 Is an Upstream Regulator of in the Maternal-To-Zygotic Transition of Mouse Embryos.

Int J Mol Sci 2021 Jan 5;22(1). Epub 2021 Jan 5.

Department of Life Sciences, and Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length promoter from mouse genomic DNA, FL-p (-1696~+28 nt), was cloned, and four deletion constructs of this promoter, Δ1-p (-1369~+28 nt), Δ2-p (-939~+28 nt), Δ3-p (-711~+28 nt) and Δ4-p (-417~+28 nt), were subsequently generated. Different-sized promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-p promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated by binding cis-elements in the -417~+28-nt promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-p reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.
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http://dx.doi.org/10.3390/ijms22010460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796490PMC
January 2021

Effects of Survival Motor Neuron Protein on Germ Cell Development in Mouse and Human.

Int J Mol Sci 2021 Jan 11;22(2). Epub 2021 Jan 11.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.

Survival motor neuron (SMN) is ubiquitously expressed in many cell types and its encoding gene, survival motor neuron 1 gene (SMN1), is highly conserved in various species. SMN is involved in the assembly of RNA spliceosomes, which are important for pre-mRNA splicing. A severe neurogenic disease, spinal muscular atrophy (SMA), is caused by the loss or mutation of SMN1 that specifically occurred in humans. We previously reported that SMN plays roles in stem cell biology in addition to its roles in neuron development. In this study, we investigated whether SMN can improve the propagation of spermatogonia stem cells (SSCs) and facilitate the spermatogenesis process. In in vitro culture, SSCs obtained from SMA model mice showed decreased growth rate accompanied by significantly reduced expression of spermatogonia marker promyelocytic leukemia zinc finger (PLZF) compared to those from heterozygous and wild-type littermates; whereas SMN overexpressed SSCs showed enhanced cell proliferation and improved potency. In vivo, the superior ability of homing and complete performance in differentiating progeny was shown in SMN overexpressed SSCs in host seminiferous tubule of transplant experiments compared to control groups. To gain insights into the roles of SMN in clinical infertility, we derived human induced pluripotent stem cells (hiPSCs) from azoospermia patients (AZ-hiPSCs) and from healthy control (ct-hiPSCs). Despite the otherwise comparable levels of hallmark iPCS markers, lower expression level of was found in AZ-hiPSCs compared with control hiPSCs during in vitro primordial germ cell like cells (PGCLCs) differentiation. On the other hand, overexpressing in AZ-hiPSCs led to increased level of pluripotent markers such as OCT4 and KLF4 during PGCLC differentiation. Our work reveal novel roles of SMN in mammalian spermatogenesis and suggest new therapeutic targets for azoospermia treatment.
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http://dx.doi.org/10.3390/ijms22020661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827477PMC
January 2021

SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network.

J Cell Sci 2020 05 11;133(9). Epub 2020 May 11.

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.
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http://dx.doi.org/10.1242/jcs.240200DOI Listing
May 2020

Survival Motor Neuron Protein Participates in Mouse Germ Cell Development and Spermatogonium Maintenance.

Int J Mol Sci 2020 Jan 25;21(3). Epub 2020 Jan 25.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.

The defective human survival motor neuron 1 (SMN1) gene leads to spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. We previously reported that loss of SMN results in rapid differentiation of Drosophila germline stem cells and mouse embryonic stem cells (ESCs), indicating that SMN also plays important roles in germ cell development and stem cell biology. Here, we show that in healthy mice, SMN is highly expressed in the gonadal tissues, prepubertal spermatogonia, and adult spermatocytes, whereas low SMN expression is found in differentiated spermatid and sperm. In SMA-like mice, the growth of testis tissues is retarded, accompanied with gamete development abnormalities and loss of the spermatogonia-specific marker. Consistently, knockdown of Smn1 in spermatogonial stem cells (SSCs) leads to a compromised regeneration capacity in vitro and in vivo in transplantation experiments. In SMA-like mice, apoptosis and accumulation of the R-loop structure were significantly elevated, indicating that SMN plays a critical role in the survival of male germ cells. The present work demonstrates that SMN, in addition to its critical roles in neuronal development, participates in mouse germ cell and spermatogonium maintenance.
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http://dx.doi.org/10.3390/ijms21030794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037566PMC
January 2020

Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer.

Int J Mol Sci 2019 Mar 12;20(5). Epub 2019 Mar 12.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.

Mammalian telomere lengths are primarily regulated by telomerase, consisting of a reverse transcriptase protein (TERT) and an RNA subunit (). We previously reported the generation of mouse and embryonic stem cells (ntESCs) by somatic cell nuclear transfer. In the present work, we investigated the germ layer development competence of , and wild-type () ntESCs. The telomere lengths are longest in wild-type but shortest in ntESCs, and correlate reversely with the population doubling time. Interestingly, while in vitro embryoid body (EB) differentiation assay reveals EB size difference among ntESCs of different genotypes, the more stringent in vivo teratoma assay demonstrates that ntESCs are severely defective in differentiating into the mesodermal lineage cartilage. Consistently, in a directed in vitro chondrocyte differentiation assay, the cells failed in forming Collagen II expressing cells. These findings underscore the significance in maintaining proper telomere lengths in stem cells and their derivatives for regenerative medicine.
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http://dx.doi.org/10.3390/ijms20051236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429130PMC
March 2019

Histidine-Dependent Protein Methylation Is Required for Compartmentalization of CTP Synthase.

Cell Rep 2018 09;24(10):2733-2745.e7

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan. Electronic address:

CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. Tetramer conformation-based filament formation restricts CTPS enzymatic activity during nutrient deprivation. CTPS protein levels remain stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth after stress relief. We demonstrate that filament formation is controlled by methylation and that histidine promotes re-methylation of homocysteine by donating one-carbon intermediates to the cytosolic folate cycle. Furthermore, we find that starvation stress and glutamine deficiency activate the GCN2/ATF4/MTHFD2 axis, which coordinates CTPS filament formation. CTPS filament formation induced by histidine-mediated methylation may be a strategy used by cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.
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http://dx.doi.org/10.1016/j.celrep.2018.08.007DOI Listing
September 2018

Interfilament interaction between IMPDH and CTPS cytoophidia.

FEBS J 2018 10 31;285(20):3753-3768. Epub 2018 Aug 31.

Department of Physiology, Anatomy and Genetics, University of Oxford, UK.

Inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS) are two metabolic enzymes that perform rate-limiting steps in the de novo synthesis of purine and pyrimidine nucleotides, respectively. It has been shown that IMPDH and CTPS can comprise a filamentous macrostructure termed the cytoophidium, which may play a role in regulation of their catalytic activity. Although these two proteins may colocalise in the same cytoophidium, how they associate with one another is still elusive. As reported herein, we established a model HeLa cell line coexpressing OFP-tagged IMPDH2 and GFP-tagged CTPS1 and recorded the assembly, disassembly and movement of the cytoophidium in live cells. Moreover, by using super-resolution confocal imaging, we demonstrate how IMPDH- and CTPS-based filaments are aligned or intertwined in the mixed cytoophidium. Collectively, our findings provide a panorama of cytoophidium dynamics and suggest that IMPDH and CTPS cytoophidia may coordinate by interfilament interaction.
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http://dx.doi.org/10.1111/febs.14624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220823PMC
October 2018

IMP/GTP balance modulates cytoophidium assembly and IMPDH activity.

Cell Div 2018 15;13. Epub 2018 Jun 15.

1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT UK.

Background: Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis, plays an important role in cell metabolism and proliferation. It has been demonstrated that IMPDH can aggregate into a macrostructure, termed the cytoophidium, in mammalian cells under a variety of conditions. However, the regulation and function of the cytoophidium are still elusive.

Results: In this study, we report that spontaneous filamentation of IMPDH is correlated with rapid cell proliferation. Intracellular IMP accumulation promoted cytoophidium assembly, whereas elevated GTP level triggered disassociation of aggregates. By using IMPDH2 CBS domain mutant cell models, which are unable to form the cytoophidium, we have determined that the cytoophidium is of the utmost importance for maintaining the GTP pool and normal cell proliferation in the condition that higher IMPDH activity is required.

Conclusions: Together, our results suggest a novel mechanism whereby cytoophidium assembly upregulates IMPDH activity and mediates guanine nucleotide homeostasis.
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http://dx.doi.org/10.1186/s13008-018-0038-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004095PMC
June 2018

CTP synthase forms the cytoophidium in human hepatocellular carcinoma.

Exp Cell Res 2017 12 31;361(2):292-299. Epub 2017 Oct 31.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address:

CTP synthase (CTPS) can aggregate into an intracellular macrostructure, the cytoophidium, in various organisms including human cells. Previous studies have shown that assembly of human CTPS cytoophidia may be correlated with the cellular metabolic status, and is able to promote the activity of CTPS. A correlation between the cytoophidium and cancer metabolism has been proposed but not yet been revealed. In the current study we provide clear evidence of the presence of CTPS cytoophidia in various human cancers and some non-cancerous tissues. Moreover, among 203 tissue samples of hepatocellular carcinoma, 56 (28%) samples exhibited many cytoophidia, whereas no cytoophidia were detected in adjacent non-cancerous hepatocytes for all samples. Our findings suggest that the CTPS cytoophidium may participate in the adaptive metabolism of human hepatocellular carcinoma.
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http://dx.doi.org/10.1016/j.yexcr.2017.10.030DOI Listing
December 2017

BMP8A sustains spermatogenesis by activating both SMAD1/5/8 and SMAD2/3 in spermatogonia.

Sci Signal 2017 May 2;10(477). Epub 2017 May 2.

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan.

Mutation in either of the genes encoding bone morphogenetic protein (BMP) 8A or 8B ( or ) causes postnatal depletion of spermatogonia in mice. We found that , but not , was expressed predominantly in the neonatal mouse spermatogonia. Although most BMPs induce activation of SMADs 1, 5, and 8 (SMAD1/5/8), but not SMADs 2 and 3 (SMAD2/3), we found that BMP8A induced signaling through both sets of transcription factors. In undifferentiated mouse spermatogonia, BMP8A activated SMAD1/5/8 through receptor complexes formed by ALK3 and either ACVR2A or BMPR2 and activated SMAD2/3 through receptor complexes formed by ALK5 and ACVR2A, ACVR2B, or TGFBR2. Signaling through SMAD2/3 promoted the proliferation of germ cells, whereas that through SMAD1/5/8 directed the subsequent differentiation of spermatogonia. BMP8A promoted spermatogenesis in cultured mouse testis explants, and the resulting spermatids were functionally competent for fertilization. These results suggest that the dual role of BMP8A in promoting proliferation and differentiation of spermatogonia may be exploited clinically to treat male infertility.
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http://dx.doi.org/10.1126/scisignal.aal1910DOI Listing
May 2017

Derivation of Patient Specific Pluripotent Stem Cells Using Clinically Discarded Cumulus Cells.

PLoS One 2016 1;11(11):e0165715. Epub 2016 Nov 1.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.

Induced pluripotent stem cells (iPSCs) are powerful tools for basic and translational research, as well as regenerative medicine. In routine human in vitro fertilization (IVF) practices, cumulus cells (CCs) are discarded, representing a potential source of biological materials for regenerative medicine. In this study, we derived patient-specific iPSCs using CCs from human infertility clinics for the first time. The human cumulus cell derived iPSCs (hc-iPSCs) were characterized for growth, karyotype, expression of pluripotency genes, and were subjected to embryoid bodies (EBs) and teratoma assays to evaluate their differentiation capacity. Hc-iPSCs display typical iPSC characteristics, and are capable of differentiating into all germ layers in vitro and in vivo. We further show that putative primordial germ cell like cells (PGCLCs) can be derived using hc-iPSCs. Our data demonstrate the feasibility of deriving patient-specific pluripotent stem cells using CCs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165715PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089679PMC
June 2017

Production of Live Offspring from Vitrified-Warmed Oocytes Collected at Metaphase I Stage.

PLoS One 2016 22;11(6):e0157785. Epub 2016 Jun 22.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.

Vitrification of matured oocytes is widely adopted in human clinics and animal research laboratories. Cryopreservation of immature oocytes, particularly those at metaphase I (MI), remains a challenge. In the present work, mouse MI oocytes denuded of cumulus cells were vitrified and warmed (V/W) either prior to (V/W-BEFORE-IVM, n = 562) or after (V/W-AFTER-IVM, n = 664) in vitro maturation (IVM). Derivative metaphase II (MII) oocytes were then used for intracytoplasmic sperm injection (ICSI). In the control groups, in vivo matured MII oocytes were used freshly (FRESH-MII, n = 517) or after V/W (MII-V/W, n = 617). In vitro and in vivo developmental competencies were compared among groups. Satisfactory blastocyst rates were achieved in V/W-BEFORE-IVM (27.5%) and V/W-AFTER-IVM (32.4%) groups, albeit as expected still lower than those from fresh-MII (56.1%) or MII-V/W (45.6%) oocytes. Similarly, the term development rates from V/W-BEFORE-IVM and V/W-AFTER-IVM were 12.4% and 16.7% respectively, acceptable but lower than those of the fresh-MII (41.2%) and MII-V/W (23.3%) groups. These data demonstrate that oocytes collected at MI stage are amenable to V/W, which can be performed before or after IVM with acceptable development rates including production of healthy pups. These findings provide useful knowledge to researchers and clinical practitioners for preservation and use of the otherwise discarded MI oocytes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157785PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917218PMC
July 2017

Paracrine regulation of matrix metalloproteinases contributes to cancer cell invasion by hepatocellular carcinoma-secreted 14-3-3σ.

Oncotarget 2016 Jun;7(24):36988-36999

Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan 350, Taiwan.

14-3-3σ overexpression results in enhanced hepatocellular carcinoma (HCC) cell migration and HCC tumor vascular-invasion is significantly associated with 14-3-3σ expression. However, increased expression of 14-3-3σ paradoxically suppresses in vitro cell invasion of HCC. We hypothesize that surrounding tumor-associated stromal cells play a crucial role in 14-3-3σ-regulated HCC cell invasion. In this study, H68 fibroblasts, THP-1 and phorbol-12-myristate-13-acetate (PMA)-treated THP-1 (PMA-THP-1) cells were incubated with conditioned media of control (control-CM) and 14-3-3σ-overepxressing cells (14-3-3σ-CM), followed by co-culture with HCC cells. Invasiveness of HCC cells was examined by a Boyden chamber assay. HCC cells co-cultured with 14-3-3σ-CM treated cells significantly enhanced their invasive ability compared with control-CM treated cells. Moreover, incubation with 14-3-3σ-CM induced differential expression profiles of matrix metalloproteinases (MMPs) in fibroblasts (MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14), THP-1 (MMP-1 and MMP-12) and PMA-THP-1 cells (MMP-2, MMP-12 and MMP-14). In contrast, silencing of 14-3-3σ by siRNA significantly abolished 14-3-3σ-CM induced MMPs. In addition, treatment with recombinant 14-3-3σ (r14-3-3σ) protein exhibits a similar expression profile of MMPs induced by 14-3-3σ-CM in fibroblasts, THP-1 and PMA-THP-1 cells. Finally, knockdown of aminopeptidase N (APN) significantly abrogated r14-3-3σ induced expression of MMPs in HS68 fibroblasts. These results suggest that HCC-secreted 14-3-3σ promotes expression of MMPs in cancerous surrounding cells via an APN dependent mechanism. 14-3-3σ has a paracrine effect in educating stromal cells in tumor-associated microenvironment.
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http://dx.doi.org/10.18632/oncotarget.9234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095053PMC
June 2016

Rho-associated kinase inhibitors promote the cardiac differentiation of embryonic and induced pluripotent stem cells.

Int J Cardiol 2015 Dec 15;201:441-8. Epub 2015 Aug 15.

Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan; Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan. Electronic address:

Background: Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation.

Methods: Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography.

Results: Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function.

Conclusions: These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases.
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http://dx.doi.org/10.1016/j.ijcard.2015.08.118DOI Listing
December 2015

Cytoophidium assembly reflects upregulation of IMPDH activity.

J Cell Sci 2015 Oct 24;128(19):3550-5. Epub 2015 Aug 24.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan, Republic of China Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, Republic of China

Cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) (both of which have two isoforms) can form fiber-like subcellular structures termed 'cytoophidia' under certain circumstances in mammalian cells. Although it has been shown that filamentation of CTPS downregulates its activity by disturbing conformational changes, the activity of IMPDH within cytoophidia is still unclear. Most previous IMPDH cytoophidium studies were performed under conditions involving inhibitors that impair GTP synthesis. Here, we show that IMPDH forms cytoophidia without inhibition of GTP synthesis. First, we find that an elevated intracellular CTP concentration or treatment with 3'-deazauridine, a CTPS inhibitor, promotes IMPDH cytoophidium formation and increases the intracellular GTP pool size. Moreover, restriction of cell growth triggers the disassembly of IMPDH cytoophidia, implying that their presence is correlated with active cell metabolism. Finally, we show that the presence of IMPDH cytoophidia in mouse pancreatic islet cells might correlate with nutrient uptake in the animal. Collectively, our findings reveal that formation of IMPDH cytoophidia reflects upregulation of purine nucleotide synthesis, suggesting that the IMPDH cytoophidium plays a role distinct from that of the CTPS cytoophidium in controlling intracellular nucleotide homeostasis.
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http://dx.doi.org/10.1242/jcs.175265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610212PMC
October 2015

Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency.

Reproduction 2015 Oct 9;150(4):245-56. Epub 2015 Jul 9.

Institute of BiotechnologyDepartment of Animal Science and TechnologyGenome and Systems Biology Degree ProgramNational Taiwan University, Taipei 106, TaiwanGenome and Systems Biology Degree ProgramAcademia Sinica, Taipei, TaiwanInstitut CurieCNRS UMR3244, Université Pierre et Marie Curie, 75248 Paris Cedex 05, FranceDepartment of Animal Science and BiotechnologyTunghai University, Taichung 407, TaiwanINSERM U934/CNRS UMR3215Institut Curie, 75005 Paris, FranceDepartment of Genetics and DevelopmentCollege of Physicians and Surgeons of Columbia University, New York, New York 10032, USAAgricultural Biotechnology Research CenterAcademia Sinica, Taipei 115, TaiwanCenter for Systems BiologyResearch Center for Developmental Biology and Regenerative MedicineNational Taiwan University, Taipei 106, Taiwan Institute of BiotechnologyDepartment of Animal Science and TechnologyGenome and Systems Biology Degree ProgramNational Taiwan University, Taipei 106, TaiwanGenome and Systems Biology Degree ProgramAcademia Sinica, Taipei, TaiwanInstitut CurieCNRS UMR3244, Université Pierre et Marie Curie, 75248 Paris Cedex 05, FranceDepartment of Animal Science and BiotechnologyTunghai University, Taichung 407, TaiwanINSERM U934/CNRS UMR3215Institut Curie, 75005 Paris, FranceDepartment of Genetics and DevelopmentCollege of Physicians and Surgeons of Columbia University, New York, New York 10032, USAAgricultural Biotechnology Research CenterAcademia Sinica, Taipei 115, TaiwanCenter for Systems BiologyResearch Center for Developmental Biology and Regenerative MedicineNational Taiwan University, Taipei 106, Taiwan Institute of BiotechnologyDepartment of Animal Science and TechnologyGenome and Systems Biology Degree ProgramNational Taiwan University, Taipei 106, TaiwanGenome and Systems Biology Degree ProgramAcademia Sinica, Taipei, TaiwanInstitut CurieCNRS UMR3244, Université Pierre et Marie Curie, 75248 Paris Cedex 05, FranceDepartment of Animal Science and BiotechnologyTunghai University, T

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.
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http://dx.doi.org/10.1530/REP-15-0031DOI Listing
October 2015

Telomere elongation and naive pluripotent stem cells achieved from telomerase haplo-insufficient cells by somatic cell nuclear transfer.

Cell Rep 2014 Dec 20;9(5):1603-1609. Epub 2014 Nov 20.

Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA. Electronic address:

Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs) have been efficiently achieved by somatic cell nuclear transfer (SCNT). We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc(+/-)) mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc(+/-) cells exhibit naive pluripotency as evidenced by generation of Terc(+/-) ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.
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http://dx.doi.org/10.1016/j.celrep.2014.10.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268138PMC
December 2014

Nucleotide synthesis is regulated by cytoophidium formation during neurodevelopment and adaptive metabolism.

Biol Open 2014 Oct 17;3(11):1045-56. Epub 2014 Oct 17.

Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom

The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. Recently, it has been demonstrated that the enzymatic activity of CTPsyn is attenuated by incorporation into cytoophidia in bacteria and yeast cells. Here we demonstrate that CTPsyn is regulated in a similar manner in Drosophila tissues in vivo. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.
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http://dx.doi.org/10.1242/bio.201410165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232762PMC
October 2014

14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma.

BMC Cancer 2014 Jun 12;14:425. Epub 2014 Jun 12.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.

Background: 14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear.

Methods: We investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay.

Results: In this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β.

Conclusions: Our findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via β-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC.
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http://dx.doi.org/10.1186/1471-2407-14-425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4061114PMC
June 2014

SMN is required for the maintenance of embryonic stem cells and neuronal differentiation in mice.

Brain Struct Funct 2015 17;220(3):1539-53. Epub 2014 Mar 17.

Institute of Biotechnology, National Taiwan University, No. 81, Chang-Xiang St., Da-an District, Taipei, 106, Taiwan, ROC.

Survival motor neuron (SMN) is the determining factor in spinal muscular atrophy, the most common genetic cause of childhood mortality. We have previously found that SMN regulates stem cell division, proliferation and differentiation in Drosophila. However, it is unknown whether a similar effect exists in vertebrates. Here, we show that SMN is enriched in highly proliferative embryonic stem cells (ESCs) in mice and reduction of SMN impairs the pluripotency of ESCs. Moreover, we find that SMN reduction activates ERK signaling and affects neuronal differentiation in vitro. Teratomas with reduced SMN grow more slowly and show weaker signals of neuronal differentiation than those with a normal level of SMN. Finally, we show that over-expression of SMN is protective for ESCs from retinoic acid-induced differentiation. Taken together, our results suggest that SMN plays a role in the maintenance of pluripotent ESCs and neuronal differentiation in mice.
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http://dx.doi.org/10.1007/s00429-014-0743-7DOI Listing
January 2016

CTP synthase forms cytoophidia in the cytoplasm and nucleus.

Exp Cell Res 2014 Apr 3;323(1):242-253. Epub 2014 Feb 3.

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK. Electronic address:

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase.
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http://dx.doi.org/10.1016/j.yexcr.2014.01.029DOI Listing
April 2014

An opposite effect of the CDK inhibitor, p18(INK4c) on embryonic stem cells compared with tumor and adult stem cells.

PLoS One 2012 26;7(9):e45212. Epub 2012 Sep 26.

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.

Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18(INK4c), is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045212PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458833PMC
April 2013

Dynamic profiles of Oct-4, Cdx-2 and acetylated H4K5 in in-vivo-derived rabbit embryos.

Reprod Biomed Online 2012 Oct 17;25(4):358-70. Epub 2012 Jul 17.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC.

This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts.
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http://dx.doi.org/10.1016/j.rbmo.2012.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465499PMC
October 2012

14-3-3σ regulates β-catenin-mediated mouse embryonic stem cell proliferation by sequestering GSK-3β.

PLoS One 2012 29;7(6):e40193. Epub 2012 Jun 29.

Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.

Background: Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear.

Methodology And Principal Findings: In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding.

Significance: Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040193PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387134PMC
November 2012

Spatial and temporal distribution of Oct-4 and acetylated H4K5 in rabbit embryos.

Reprod Biomed Online 2012 Apr 10;24(4):433-42. Epub 2012 Jan 10.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC.

Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB. Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.
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http://dx.doi.org/10.1016/j.rbmo.2012.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320679PMC
April 2012

Follicular oocytes better support development in rabbit cloning than oviductal oocytes.

Cell Reprogram 2011 Dec 26;13(6):503-12. Epub 2011 Oct 26.

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, Republic of China.

This study was conducted to determine the effect of rabbit oocytes collected from ovaries or oviducts on the developmental potential of nuclear transplant embryos. Donor nuclei were obtained from adult skin fibroblasts, cumulus cells, and embryonic blastomeres. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12 h post-hCG injection. The majority of collected oocytes were still attached to the sites of ovulation on the ovaries. We found that follicular oocytes had a significantly higher rate of fusion with nuclear donor cells than oviductal oocytes. There was no difference in the cleavage rate between follicular and oviductal groups, but morula and blastocyst development was significantly higher in the follicular group than in the oviductal group. Two live clones were produced in follicular group using blastomere and cumulus nuclear donors, whereas one live clone was produced in the oviductal group using a cumulus nuclear donor. These results demonstrate that cloned rabbit embryos derived from follicular oocytes have better developmental competence than those derived from oviductal oocytes.
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http://dx.doi.org/10.1089/cell.2011.0030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229818PMC
December 2011

Impact of phase transition on the mouse oocyte spindle during vitrification.

Reprod Biomed Online 2011 Feb 31;22(2):184-91. Epub 2010 Oct 31.

Reproductive Biology Associates, Atlanta, GA, USA.

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.
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http://dx.doi.org/10.1016/j.rbmo.2010.10.009DOI Listing
February 2011
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