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Okamura D, Kohara A, Chigi Y, Katayama T, Sharif J, Wu J, Ito-Matsuoka Y, Matsui Y. p38 MAPK as a gatekeeper of reprogramming in mouse migratory primordial germ cells. Front Cell Dev Biol 2024; 12:1410177. [PMID: 38911025 PMCID: PMC11191381 DOI: 10.3389/fcell.2024.1410177] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2024] [Accepted: 05/06/2024] [Indexed: 06/25/2024] Open
Abstract
Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.
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Affiliation(s)
- Daiji Okamura
- Department of Advanced Bioscience, Faculty of Agriculture, Kindai University, Nara, Japan
| | - Aoi Kohara
- Department of Advanced Bioscience, Faculty of Agriculture, Kindai University, Nara, Japan
| | - Yuta Chigi
- Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Tomoka Katayama
- Department of Advanced Bioscience, Faculty of Agriculture, Kindai University, Nara, Japan
| | - Jafar Sharif
- Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, United States
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Yumi Ito-Matsuoka
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
| | - Yasuhisa Matsui
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
- Graduate School of Life Sciences, Tohoku University, Sendai, Japan
- Graduate School of Medicine, Tohoku University, Sendai, Japan
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2
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Deng X, Ning Z, Li L, Cui Z, Du X, Amevor FK, Tian Y, Shu G, Du X, Han X, Zhao X. High expression of miR-22-3p in chicken hierarchical follicles promotes granulosa cell proliferation, steroidogenesis, and lipid metabolism via PTEN/PI3K/Akt/mTOR signaling pathway. Int J Biol Macromol 2023; 253:127415. [PMID: 37848113 DOI: 10.1016/j.ijbiomac.2023.127415] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 10/09/2023] [Accepted: 10/10/2023] [Indexed: 10/19/2023]
Abstract
MicroRNAs (miRNAs) are a class of RNA macromolecules that play regulatory roles in follicle development by inhibiting protein translation through binding to the 3'UTR of its target genes. Granulosa cell (GC) proliferation, steroidogenesis, and lipid metabolism have indispensable effect during folliculogenesis. In this study, we found that miR-22-3p was highly expressed in the hierarchical follicles of the chickens, which indicated that it may be involved in follicle development. The results obtained suggested that miR-22-3p promoted proliferation, hormone secretion (progesterone and estrogen), and the content of lipid droplets (LDs) in the chicken primary GC. The results from the bioinformatics analysis, luciferase reporter assay, qRT-PCR, and Western blotting, confirmed that PTEN was directly targeted to miR-22-3p. Subsequently, it was revealed that PTEN inhibited proliferation, hormone secretion, and the content of LDs in GC. Therefore, this study showed that miR-22-3p could activate PI3K/Akt/mTOR pathway via targeting PTEN. Taken together, the findings from this study indicated that miR-22-3p was highly expressed in the hierarchical follicles of chickens, which promotes GC proliferation, steroidogenesis, and lipid metabolism by repressing PTEN to activate PI3K/AKT/mTOR pathway.
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Affiliation(s)
- Xun Deng
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, PR China; Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology (Institute of Animal Genetics and Breeding), Sichuan Agricultural University, PR China
| | - Zifan Ning
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, PR China; Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology (Institute of Animal Genetics and Breeding), Sichuan Agricultural University, PR China
| | - Liang Li
- Institute of Animal Husbandry and Veterinary Medicine, Guizhou Academy of Agricultural Sciences, Guiyang, PR China; Guizhou Hongyu Animal Husbandry Technology Development Co., Ltd, Guiyang, PR China
| | - Zhifu Cui
- College of Animal Science and Technology, Southwest University, Chongqing, PR China
| | - Xiaxia Du
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, PR China; Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology (Institute of Animal Genetics and Breeding), Sichuan Agricultural University, PR China
| | - Felix Kwame Amevor
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, PR China; Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology (Institute of Animal Genetics and Breeding), Sichuan Agricultural University, PR China
| | - Yaofu Tian
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, PR China; Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology (Institute of Animal Genetics and Breeding), Sichuan Agricultural University, PR China
| | - Gang Shu
- Department of Basic Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, China
| | - Xiaohui Du
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, PR China; Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology (Institute of Animal Genetics and Breeding), Sichuan Agricultural University, PR China
| | - Xue Han
- Institute of Animal Husbandry and Veterinary Medicine, Guizhou Academy of Agricultural Sciences, Guiyang, PR China; Guizhou Hongyu Animal Husbandry Technology Development Co., Ltd, Guiyang, PR China.
| | - Xiaoling Zhao
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, PR China; Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology (Institute of Animal Genetics and Breeding), Sichuan Agricultural University, PR China.
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3
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Kalous J, Aleshkina D, Anger M. A Role of PI3K/Akt Signaling in Oocyte Maturation and Early Embryo Development. Cells 2023; 12:1830. [PMID: 37508495 PMCID: PMC10378481 DOI: 10.3390/cells12141830] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 06/24/2023] [Accepted: 07/06/2023] [Indexed: 07/30/2023] Open
Abstract
A serine/threonine-specific protein kinase B (PKB), also known as Akt, is a key factor in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway that regulates cell survival, metabolism and proliferation. Akt phosphorylates many downstream specific substrates, which subsequently control the nuclear envelope breakdown (NEBD), centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. In vertebrates, Akt is also an important player during oogenesis and preimplantation development. In the signaling pathways regulating mRNA translation, Akt is involved in the control of mammalian target of rapamycin complex 1 (mTORC1) and thereby regulates the activity of a translational repressor, the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In this review, we summarize the functions of Akt in mitosis, meiosis and early embryonic development. Additionally, the role of Akt in the regulation of mRNA translation is addressed with respect to the significance of this process during early development.
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Affiliation(s)
- Jaroslav Kalous
- Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic
| | - Daria Aleshkina
- Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic
- Department of Cell Biology, Faculty of Science, Charles University, Albertov 6, 128 00 Praha, Czech Republic
| | - Martin Anger
- Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic
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4
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Liu C, Moten A, Ma Z, Lin HK. The foundational framework of tumors: Gametogenesis, p53, and cancer. Semin Cancer Biol 2022; 81:193-205. [PMID: 33940178 PMCID: PMC9382687 DOI: 10.1016/j.semcancer.2021.04.018] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 04/20/2021] [Accepted: 04/26/2021] [Indexed: 12/20/2022]
Abstract
The completion-of-tumor hypothesis involved in the dynamic interplay between the initiating oncogenic event and progression is essential to better recognize the foundational framework of tumors. Here we review and extend the gametogenesis-related hypothesis of tumors, because high embryonic/germ cell traits are common in tumors. The century-old gametogenesis-related hypothesis of tumors postulated that tumors arise from displaced/activated trophoblasts, displaced (lost) germ cells, and the reprogramming/reactivation of gametogenic program in somatic cells. Early primordial germ cells (PGCs), embryonic stem (ES) cells, embryonic germ cells (EGCs), and pre-implantation embryos at the stage from two-cell stage to blastocysts originating from fertilization or parthenogenesis have the potential to develop teratomas/teratocarcinomas. In addition, the teratomas/teratocarcinomas/germ cells occur in gonads and extra-gonads. Undoubtedly, the findings provide strong support for the hypothesis. However, it was thought that these tumor types were an exception rather than verification. In fact, there are extensive similarities between somatic tumor types and embryonic/germ cell development, such as antigens, migration, invasion, and immune escape. It was documented that embryonic/germ cell genes play crucial roles in tumor behaviors, e.g. tumor initiation and metastasis. Of note, embryonic/germ cell-like tumor cells at different developmental stages including PGC and oocyte to the early embryo-like stage were identified in diverse tumor types by our group. These embryonic/germ cell-like cancer cells resemble the natural embryonic/germ cells in morphology, gene expression, the capability of teratoma formation, and the ability to undergo the process of oocyte maturation and parthenogenesis. These embryonic/germ cell-like cancer cells are derived from somatic cells and contribute to tumor formation, metastasis, and drug resistance, establishing asexual meiotic embryonic life cycle. p53 inhibits the reactivation of embryonic/germ cell state in somatic cells and oocyte-like cell maturation. Based on earlier and our recent studies, we propose a novel model to complete the gametogenesis-related hypothesis of tumors, which can be applied to certain somatic tumors. That is, tumors tend to establish a somatic asexual meiotic embryonic cycle through the activation of somatic female gametogenesis and parthenogenesis in somatic tumor cells during the tumor progression, thus passing on corresponding embryonic/germ cell traits leading to the malignant behaviors and enhancing the cells' independence. This concept may be instrumental to better understand the nature and evolution of tumors. We rationalize that targeting the key events of somatic pregnancy is likely a better therapeutic strategy for cancer treatment than directly targeting cell mitotic proliferation, especially for those tumors with p53 inactivation.
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Affiliation(s)
- Chunfang Liu
- Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, 200040, China.
| | - Asad Moten
- Medical Sciences Division, University of Oxford, Oxford OX3 9DU, UK
| | - Zhan Ma
- Department of Laboratory Medicine, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China
| | - Hui-Kuan Lin
- Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC, 27157, USA.
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5
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Sekita Y, Sugiura Y, Matsumoto A, Kawasaki Y, Akasaka K, Konno R, Shimizu M, Ito T, Sugiyama E, Yamazaki T, Kanai E, Nakamura T, Suematsu M, Ishino F, Kodera Y, Kohda T, Kimura T. AKT signaling is associated with epigenetic reprogramming via the upregulation of TET and its cofactor, alpha-ketoglutarate during iPSC generation. Stem Cell Res Ther 2021; 12:510. [PMID: 34563253 PMCID: PMC8467031 DOI: 10.1186/s13287-021-02578-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Accepted: 08/31/2021] [Indexed: 12/13/2022] Open
Abstract
Background Phosphoinositide-3 kinase (PI3K)/AKT signaling participates in cellular proliferation, survival and tumorigenesis. The activation of AKT signaling promotes the cellular reprogramming including generation of induced pluripotent stem cells (iPSCs) and dedifferentiation of primordial germ cells (PGCs). Previous studies suggested that AKT promotes reprogramming by activating proliferation and glycolysis. Here we report a line of evidence that supports the notion that AKT signaling is involved in TET-mediated DNA demethylation during iPSC induction. Methods AKT signaling was activated in mouse embryonic fibroblasts (MEFs) that were transduced with OCT4, SOX2 and KLF4. Multiomics analyses were conducted in this system to examine the effects of AKT activation on cells undergoing reprogramming. Results We revealed that cells undergoing reprogramming with artificially activated AKT exhibit enhanced anabolic glucose metabolism and accordingly increased level of cytosolic α-ketoglutarate (αKG), which is an essential cofactor for the enzymatic activity of the 5-methylcytosine (5mC) dioxygenase TET. Additionally, the level of TET is upregulated. Consistent with the upregulation of αKG production and TET, we observed a genome-wide increase in 5-hydroxymethylcytosine (5hmC), which is an intermediate in DNA demethylation. Moreover, the DNA methylation level of ES-cell super-enhancers of pluripotency-related genes is significantly decreased, leading to the upregulation of associated genes. Finally, the transduction of TET and the administration of cell-permeable αKG to somatic cells synergistically enhance cell reprogramming by Yamanaka factors. Conclusion These results suggest the possibility that the activation of AKT during somatic cell reprogramming promotes epigenetic reprogramming through the hyperactivation of TET at the transcriptional and catalytic levels. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02578-1.
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Affiliation(s)
- Yoichi Sekita
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Yuki Sugiura
- Department of Biochemistry, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Akari Matsumoto
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Yuki Kawasaki
- Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Kazuya Akasaka
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Ryo Konno
- Department of Physics, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Momoka Shimizu
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Toshiaki Ito
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Eiji Sugiyama
- Department of Biochemistry, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Terushi Yamazaki
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Eriko Kanai
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Toshinobu Nakamura
- Laboratory for Epigenetic Regulation, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama-shi, Shiga, 526-0829, Japan
| | - Makoto Suematsu
- Department of Biochemistry, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Fumitoshi Ishino
- Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Yoshio Kodera
- Department of Physics, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan.,Center for Disease Proteomics, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan
| | - Takashi Kohda
- Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.,Laboratory of Embryology and Genomics, Department of Biotechnology, Faculty of Life and Environmental Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu-shi, Yamanashi, 400-8510, Japan
| | - Tohru Kimura
- Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara-shi, Kanagawa, 252-0373, Japan.
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6
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To Be or Not to Be a Germ Cell: The Extragonadal Germ Cell Tumor Paradigm. Int J Mol Sci 2021; 22:ijms22115982. [PMID: 34205983 PMCID: PMC8199495 DOI: 10.3390/ijms22115982] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 05/27/2021] [Accepted: 05/28/2021] [Indexed: 02/06/2023] Open
Abstract
In the human embryo, the genetic program that orchestrates germ cell specification involves the activation of epigenetic and transcriptional mechanisms that make the germline a unique cell population continuously poised between germness and pluripotency. Germ cell tumors, neoplasias originating from fetal or neonatal germ cells, maintain such dichotomy and can adopt either pluripotent features (embryonal carcinomas) or germness features (seminomas) with a wide range of phenotypes in between these histotypes. Here, we review the basic concepts of cell specification, migration and gonadal colonization of human primordial germ cells (hPGCs) highlighting the analogies of transcriptional/epigenetic programs between these two cell types.
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Gross-Thebing T, Raz E. Dead end and Detour: The function of the RNA-binding protein Dnd in posttranscriptional regulation in the germline. Curr Top Dev Biol 2020; 140:181-208. [DOI: 10.1016/bs.ctdb.2019.12.003] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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8
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Takehara A, Matsui Y. Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming. Dev Growth Differ 2019; 61:357-364. [PMID: 31199000 DOI: 10.1111/dgd.12621] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2019] [Revised: 04/25/2019] [Accepted: 05/09/2019] [Indexed: 01/15/2023]
Abstract
Primordial germ cells (PGCs) are reprogrammed into pluripotent embryonic germ cells (EGCs) under specific culture conditions, but the detailed mechanisms of PGC reprogramming have not yet been fully clarified. Previous studies have demonstrated that AKT, an important intracellular signaling molecule, promotes reprogramming of PGCs into EGCs. Because AKT likely inhibits p53 functions to enhance PGC reprogramming, and p53 negatively regulates cell cycle progression, we analyzed cell cycle changes in PGCs following AKT activation and found that the ratio of PGCs in the G1/G0 phase was decreased while that of PGCs in the G2/M phase was increased after AKT activation.
We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1‐S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation. The results suggested that the characteristic cell cycle changes of PGCs by AKT activation are, at least in part, due to decreased expression of p27kip1 . We also investigated changes in histone H3K27 tri-methylation (H3K27me3) by AKT activation in PGCs, because we previously found that decreased H3K27me3 was involved in PGC reprogramming via upregulation of cyclin D1. We observed that AKT activation in PGCs resulted in H3K27 hypomethylation. In addition, DZNeP, an inhibitor of the H3K27 trimethyl transferase Ezh2, stimulated EGC formation. These results together suggested that AKT activation promotes G1-S transition and downregulates H3K27me3 to enhance PGC reprogramming.
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Affiliation(s)
- Asuka Takehara
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Japan.,Graduate School of Life Sciences, Tohoku University, Sendai, Japan.,The Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Tokyo, Japan
| | - Yasuhisa Matsui
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Japan.,Graduate School of Life Sciences, Tohoku University, Sendai, Japan.,The Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Tokyo, Japan.,Graduate School of Medicine, Tohoku University, Sendai, Japan
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9
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An Y, Sekinaka T, Tando Y, Okamura D, Tanaka K, Ito-Matsuoka Y, Takehara A, Yaegashi N, Matsui Y. Derivation of pluripotent stem cells from nascent undifferentiated teratoma. Dev Biol 2018; 446:43-55. [PMID: 30529251 DOI: 10.1016/j.ydbio.2018.11.020] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2018] [Revised: 11/27/2018] [Accepted: 11/30/2018] [Indexed: 01/19/2023]
Abstract
Teratomas are tumors consisting of components of the three germ layers that differentiate from pluripotent stem cells derived from germ cells. In the normal mouse testis, teratomas rarely form, but a deficiency in Dead-end1 (Dnd1) in mice with a 129/Sv genetic background greatly enhances teratoma formation. Thus, DND1 is crucial for suppression of teratoma development from germ cells. In the Dnd1 mutant testis, nascent teratoma cells emerge at E15.5. To understand the nature of early teratoma cells, we established cell lines in the presence of serum and leukemia inhibitory factor (LIF) from teratoma-forming cells in neonatal Dnd1 mutant testis. These cells, which we designated cultured Dnd1 mutant germ cells (CDGCs), were morphologically similar to embryonic stem cells (ESCs) and could be maintained in the naïve pluripotent condition. In addition, the cells expressed pluripotency genes including Oct4, Nanog, and Sox2; differentiated into cells of the three germ layers in culture; and contributed to chimeric mice. The expression levels of pluripotency genes and global transcriptomes in CDGCs as well as these cells' adaption to culture conditions for primed pluripotency suggested that their pluripotent status is intermediate between naïve and primed pluripotency. In addition, the teratoma-forming cells in the neonatal testis from which CDGCs were derived also showed gene expression profiles intermediate between naïve and primed pluripotency. The results suggested that germ cells in embryonic testes of Dnd1 mutants acquire the intermediate pluripotent status during the course of conversion into teratoma cells.
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Affiliation(s)
- Yuri An
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan; Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan
| | - Tamotsu Sekinaka
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan
| | - Yukiko Tando
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan
| | - Daiji Okamura
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, Nakamachi, Nara, Japan
| | - Keiko Tanaka
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan; Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
| | - Yumi Ito-Matsuoka
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan
| | - Asuka Takehara
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan
| | - Nobuo Yaegashi
- Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
| | - Yasuhisa Matsui
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan; Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan; The Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Chuo-ku, Tokyo, Japan; Center for Regulatory Epigenome and Diseases, Tohoku University School of Medicine, Sendai, Miyagi, Japan.
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10
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Identification of KLF9 and BCL3 as transcription factors that enhance reprogramming of primordial germ cells. PLoS One 2018; 13:e0205004. [PMID: 30286177 PMCID: PMC6171932 DOI: 10.1371/journal.pone.0205004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2018] [Accepted: 09/18/2018] [Indexed: 11/25/2022] Open
Abstract
Primordial germ cells (PGCs) are precursors of eggs and sperm. Although PGCs are unipotent cells in vivo, they are reprogrammed into pluripotent stem cells (PSCs), also known as embryonic germ cells (EGCs), in the presence of leukemia inhibitory factor and basic fibroblast growth factor (bFGF) in vitro. However, the molecular mechanisms responsible for their reprogramming are not fully understood. Here we show identification of transcription factors that mediate PGC reprogramming. We selected genes encoding transcription factors or epigenetic regulatory factors whose expression was significantly different between PGCs and PSCs with in silico analysis and RT-qPCR. Among the candidate genes, over-expression (OE) of Bcl3 or Klf9 significantly enhanced PGC reprogramming. Notably, EGC formation was stimulated by Klf9-OE even without bFGF. G-protein-coupled receptor signaling-related pathways, which are involved in PGC reprogramming, were enriched among genes down-regulated by Klf9-OE, and forskolin which activate adenylate cyclase, rescued repressed EGC formation by knock-down of Klf9, suggesting a molecular linkage between KLF9 and such signaling.
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Wang L, Huang D, Jiang Z, Luo Y, Norris C, Zhang M, Tian X, Tang Y. Akt3 is responsible for the survival and proliferation of embryonic stem cells. Biol Open 2017; 6:850-861. [PMID: 28483982 PMCID: PMC5483023 DOI: 10.1242/bio.024505] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2017] [Accepted: 04/24/2017] [Indexed: 12/21/2022] Open
Abstract
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays an important role in regulating cell proliferation, metabolism, and survival. However, the distinct roles of Akt isoforms (Akt1, Akt2, and Akt3) in pluripotent stem cell maintenance are not fully defined. Using mouse embryonic stem cells (ESCs), we show that direct inhibition of Akt activity leads to ESC apoptosis. The Akt3, but not Akt1 or Akt2, activity specifically regulates this effect. Inhibiting Akt3 also leads to a cell cycle arrest at G1 phase. These regulatory roles of Akt3 are dependent on its kinase activity. Blocking the expression of Akt1 plus Akt2 in ESCs does not affect cell survival or proliferation, although blocking Akt1 aggravates the apoptotic effect induced by depletion of Akt3. We further show that blocking Akt3 in ESCs results in significant nuclear accumulation of p53, as well as the activation of its downstream targets, such as Mdm2, p21, and Fas. Inhibiting p53 and its downstream targets partially rescued the effects caused by Akt3-depletion. Our results revealed an Akt3 isoform-specific mechanism for ESC survival and proliferation involving the control of p53 activity.
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Affiliation(s)
- Ling Wang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Delun Huang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
- Animal Reproduction Institute, Guangxi University, Nanning, 530004, People's Republic of China
| | - Zongliang Jiang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Yan Luo
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Carol Norris
- Center for Open Research Resources and Equipment, University of Connecticut, Storrs, CT 06269, USA
| | - Ming Zhang
- Animal Reproduction Institute, Guangxi University, Nanning, 530004, People's Republic of China
| | - Xiuchun Tian
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
| | - Young Tang
- Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
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12
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Insulin signalling and glucose transport in the ovary and ovarian function during the ovarian cycle. Biochem J 2017; 473:1483-501. [PMID: 27234585 PMCID: PMC4888492 DOI: 10.1042/bcj20160124] [Citation(s) in RCA: 143] [Impact Index Per Article: 17.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2015] [Accepted: 03/03/2016] [Indexed: 12/16/2022]
Abstract
Data derived principally from peripheral tissues (fat, muscle and liver) show that insulin signals via diverse interconnecting intracellular pathways and that some of the major intersecting points (known as critical nodes) are the IRSs (insulin receptor substrates), PI3K (phosphoinositide kinase)/Akt and MAPK (mitogen-activated protein kinase). Most of these insulin pathways are probably also active in the ovary and their ability to interact with each other and also with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) signalling pathways enables insulin to exert direct modulating influences on ovarian function. The present paper reviews the intracellular actions of insulin and the uptake of glucose by ovarian tissues (granulosa, theca and oocyte) during the oestrous/menstrual cycle of some rodent, primate and ruminant species. Insulin signals through diverse pathways and these are discussed with specific reference to follicular cell types (granulosa, theca and oocyte). The signalling pathways for FSH in granulosa cells and LH in granulosa and theca cells are summarized. The roles of glucose and of insulin-mediated uptake of glucose in folliculogenesis are discussed. It is suggested that glucose in addition to its well-established role of providing energy for cellular function may also have insulin-mediated signalling functions in ovarian cells, involving AMPK (AMP-dependent protein kinase) and/or hexosamine. Potential interactions of insulin signalling with FSH or LH signalling at critical nodes are identified and the available evidence for such interactions in ovarian cells is discussed. Finally the action of the insulin-sensitizing drugs metformin and the thiazolidinedione rosiglitazone on follicular cells is reviewed.
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Tagami T, Miyahara D, Nakamura Y. Avian Primordial Germ Cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1001:1-18. [PMID: 28980226 DOI: 10.1007/978-981-10-3975-1_1] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/05/2022]
Abstract
Germ cells transmit genetic information to the next generation through gametogenesis. Primordial germ cells (PGCs) are the first germ-cell population established during development, and are the common origins of both oocytes and spermatogonia. Unlike in other species, PGCs in birds undergo blood circulation to migrate toward the genital ridge, and are one of the major biological properties of avian PGCs. Germ cells enter meiosis and arrest at prophase I during embryogenesis in females, whereas in males they enter mitotic arrest during embryogenesis and enter meiosis only after birth. In chicken, gonadal sex differentiation occurs as early as embryonic day 6, but meiotic initiation of female germ cells starts from a relatively late stage (embryonic day 15.5). Retinoic acid controls meiotic entry in developing chicken gonads through the expressions of retinaldehyde dehydrogenase 2, a major retinoic acid synthesizing enzyme, and cytochrome P450 family 26, subfamily B member 1, a major retinoic acid-degrading enzyme. The other major biological property of avian PGCs is that they can be propagated in vitro for the long term, and this technique is useful for investigating proliferation mechanisms. The main factor involved in chicken PGC proliferation is fibroblast growth factor 2, which activates the signaling of MEK/ERK and thus promotes the cell cycle and anti-apoptosis. Furthermore, the activation of PI3K/Akt signaling is indispensable for the proliferation and survival of chicken PGCs.
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Affiliation(s)
- Takahiro Tagami
- Institute of Livestock Grassland Science, NARO, Ibaraki, Japan.
| | - Daichi Miyahara
- Institute of Livestock Grassland Science, NARO, Ibaraki, Japan
- Shinshu University, Ueda, Japan
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14
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Wang C, Deng Y, Chen F, Zhu P, Wei J, Luo C, Lu F, Yang S, Shi D. Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells. Theriogenology 2016; 91:112-120. [PMID: 28215675 DOI: 10.1016/j.theriogenology.2016.12.035] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2016] [Revised: 12/01/2016] [Accepted: 12/28/2016] [Indexed: 12/14/2022]
Abstract
Primordial germ cells (PGCs) are destined to form gametes in vivo, and they can be reprogrammed into pluripotent embryonic germ (EG) cells in vitro. Buffalo PGC have been reported to be reprogrammed into EG-like cells, but the identities of the major signaling pathways and culture media involved in this derivation remain unclear. Here, the effects of basic fibroblast growth factor (bFGF) and downstream signaling pathways on the reprogramming of buffalo PGCs into EG-like cells were investigated. Results showed bFGF to be critical to buffalo PGCs to dedifferentiate into EG-like cells (20 ng/mL is optimal) with many characteristics of pluripotent stem cells, including alkaline phosphatase (AP) activity, expression of pluripotency marker genes such as OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1-81, and the capacity to differentiate into all three embryonic germ layers. After chemically inhibiting pathways or components downstream of bFGF, data showed that inhibition of the PI3K/AKT pathway led to significantly lower EG cell derivation, while inhibition of P53 activity resulted in an efficiency of EG cell derivation comparable to that in the presence of bFGF. These results suggest that the role of bFGF in PGC-derived EG-like cell generation is mainly due to the activation of the PI3K/AKT/P53 pathway, in particular, the inhibition of P53 function.
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Affiliation(s)
- Caizhu Wang
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China; Reproductive Medicine Center, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning, China
| | - Yanfei Deng
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China
| | - Feng Chen
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China
| | - Peng Zhu
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China
| | - Jingwei Wei
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China
| | - Chan Luo
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China
| | - Fenghua Lu
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China
| | - Sufang Yang
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
| | - Deshun Shi
- Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
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15
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Hersmus R, van Bever Y, Wolffenbuttel KP, Biermann K, Cools M, Looijenga LHJ. The biology of germ cell tumors in disorders of sex development. Clin Genet 2016; 91:292-301. [PMID: 27716895 DOI: 10.1111/cge.12882] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2016] [Revised: 09/29/2016] [Accepted: 09/30/2016] [Indexed: 01/01/2023]
Abstract
Development of a malignant germ cell tumor, i.e., germ cell cancer (GCC) in individuals with disorders of sex development (DSD) depends on a number of (epi-)genetic factors related to early gonadal- and germ cell development, possibly related to genetic susceptibility. Fetal development of germ cells is orchestrated by strict processes involving specification, migration and the development of a proper gonadal niche. In this review we will discuss the early (epi-)genetic events in normal and aberrant germ cell and gonadal development. Focus will be on the formation of the precursor lesions of GCC in individuals who have DSD. In our view, expression of the different embryonic markers in, and epigenetic profile of the precursor lesions reflects the developmental stage in which these cells are blocked in their maturation. Therefore, these are not a primary pathogenetic driving force. Progression later in life towards a full blown cancer likely depends on additional factors such as a changed endocrine environment in a susceptible individual. Genetic susceptibility is, as evidenced by the presence of specific risk genetic variants (SNPs) in patients with a testicular GCC, related to genes involved in early germ cell and gonadal development.
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Affiliation(s)
- Remko Hersmus
- Department of Pathology, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Yolande van Bever
- Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Katja P Wolffenbuttel
- Department of Pediatric Urology, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Katharina Biermann
- Department of Pathology, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Martine Cools
- Department of Pediatric Endocrinology, Ghent University Hospital and Ghent University, Ghent, Belgium
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Sekita Y, Nakamura T, Kimura T. Reprogramming of germ cells into pluripotency. World J Stem Cells 2016; 8:251-259. [PMID: 27621759 PMCID: PMC4999652 DOI: 10.4252/wjsc.v8.i8.251] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Revised: 06/08/2016] [Accepted: 07/13/2016] [Indexed: 02/06/2023] Open
Abstract
Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.
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17
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Tian-Zhong M, Bi C, Ying Z, Xia J, Cai-Ling P, Yun-Shan Z, Mei-Wen H, Yan-Ru N. Critical role of Emx2 in the pluripotency – differentiation transition in male gonocytes via regulation of FGF9/NODAL pathway. Reproduction 2016; 151:673-81. [DOI: 10.1530/rep-16-0022] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Accepted: 03/21/2016] [Indexed: 12/30/2022]
Abstract
Abstract
Emx2 deletion impairs the growth and maintenance of the genital ridge. However, its role in subsequent germ cell differentiation during embryonic stages is unknown. Using a tamoxifen-inducible Cre-loxP mouse model (Emx2flox/flox, Cre-ERTM, hereafter called as Emx2 knockdown), we showed that germ cell differentiation was impaired in Emx2-knockdown testes. Representative characteristics of male germ cell differentiation, including a reduced ability to form embryonic germ (EG) cell colonies in vitro, down-regulation of pluripotency markers and G1/G0 arrest, did not occur in Emx2-knockdown testes. Furthermore, FGF9 and NODAL signalling occurred at abnormally high levels in Emx2-knockdown testes. Both blocking FGF9 signalling with SU5402 and inhibiting NODAL signalling with SB431542 allowed germ cells from Emx2-knockdown testes to differentiate in vitro. Therefore, EMX2 in somatic cells is required to trigger germ cell differentiation in XY foetuses, posterior to its previously reported role in the growth and maintenance of the genital ridge.
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18
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Zhang Y, Ma J, Li H, Lv J, Wei R, Cong Y, Liu Z. bFGF signaling-mediated reprogramming of porcine primordial germ cells. Cell Tissue Res 2015; 364:429-41. [PMID: 26613602 DOI: 10.1007/s00441-015-2326-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2015] [Revised: 11/01/2015] [Accepted: 11/02/2015] [Indexed: 11/26/2022]
Abstract
Primordial germ cells (PGCs) have the ability to be reprogrammed into embryonic germ cells (EGCs) in vitro and are an alternative source of embryonic stem cells. Other than for the mouse, the systematic characterization of mammalian PGCs is still lacking, especially the process by which PGCs convert to pluripotency. This hampers the understanding of germ cell development and the derivation of authenticated EGCs from other species. We observed the morphological development of the genital ridge from Bama miniature pigs and found primary sexual differentiation in the E28 porcine embryo, coinciding with Blimp1 nuclear exclusion in PGCs. To explore molecular events involved in porcine PGC reprogramming, transcriptome data of porcine EGCs and fetal fibroblasts (FFs) were assembled and 1169 differentially expressed genes were used for Gene Ontology analysis. These genes were significantly enriched in cell-surface receptor-linked signal transduction, in agreement with the activation of LIF/Stat3 signaling and FGF signaling during the derivation of porcine EG-like cells. Using a growth-factor-defined culture system, we explored the effects of bFGF on the process and found that bFGF not only functioned at the very beginning of PGC dedifferentiation by impeding Blimp1 nuclear expression via a PI3K/AKT-dependent pathway but also maintained the viability of cultured PGCs thereafter. These results provide further insights into the development of germ cells from livestock and the mechanism of porcine PGC reprogramming.
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Affiliation(s)
- Yu Zhang
- College of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China
| | - Jing Ma
- College of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China
| | - Hai Li
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China
| | - Jiawei Lv
- College of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China
| | - Renyue Wei
- College of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China
| | - Yimei Cong
- College of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, People's Republic of China
| | - Zhonghua Liu
- College of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China.
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19
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20
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The Cannabinoid Receptor 2 Q63R Variant Modulates the Relationship between Childhood Obesity and Age at Menarche. PLoS One 2015; 10:e0140142. [PMID: 26447698 PMCID: PMC4598176 DOI: 10.1371/journal.pone.0140142] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2015] [Accepted: 09/21/2015] [Indexed: 01/08/2023] Open
Abstract
Background The ovary is an important site where gene variants modulate pubertal timing. The cannabinoid receptor 2 (CB2) is expressed in the ovary, plays a role in folliculogenesis and ovulation, and can be modulated by estrogens. Obesity is strictly associated with early menarche and is characterized by sex hormone and endocannabinoid derangement. Aim In this study, we investigated the role of the CB2 receptor in determining the age at menarche in obese girls. Methods We studied a cohort of 240 obese girls (age 11.9±3 years; BMI z-score 2.8±0.8). The age at menarche (if it had already occurred) was recorded at the time of the visit or via phonecall. The CNR2 rs35761398 polymorphism, which leads to the CB2 Q63R variant, was detected by the TaqMan assay. Results In total, 105 patients were homozygous for the R63-coding allele (RR), 113 were QR and 22 were QQ. Variance analysis revealed a significantly earlier age of menarche in subjects carrying the Q63 allele, which was also found after adjusting for BMI z-score (11±1.2 vs. 11.6±1.2 years, p = 0.0003). Logistic regression analysis demonstrated that patients homozygous for the Q allele had a 2.2-fold higher risk (odds ratio = 2.2; CI1.1–3.4; p = 0.02) of presenting with an early menarche (age at menarche <12 years). Conclusion We demonstrated for the first time the association between the CB2 Q63R functional variant and the age at menarche in a cohort of Italian obese girls.
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21
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Robert VJ, Garvis S, Palladino F. Repression of somatic cell fate in the germline. Cell Mol Life Sci 2015; 72:3599-620. [PMID: 26043973 PMCID: PMC11113910 DOI: 10.1007/s00018-015-1942-y] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2015] [Revised: 05/26/2015] [Accepted: 05/27/2015] [Indexed: 01/13/2023]
Abstract
Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types after fertilization. Preventing the activation of somatic programs is, therefore, crucial to the maintenance of germ cell identity. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse have revealed both similarities and differences in how somatic gene expression is repressed in germ cells, thereby preventing their conversion into somatic tissues. This review will focus on recent developments in our understanding of how global or gene-specific transcriptional repression, chromatin regulation, and translational repression operate in the germline to maintain germ cell identity and repress somatic differentiation programs.
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Affiliation(s)
- Valérie J Robert
- Ecole Normale Supérieure de Lyon, Université de Lyon, 46 allée d'Italie, 69007, Lyon, France
| | - Steve Garvis
- Ecole Normale Supérieure de Lyon, Université de Lyon, 46 allée d'Italie, 69007, Lyon, France
| | - Francesca Palladino
- Ecole Normale Supérieure de Lyon, Université de Lyon, 46 allée d'Italie, 69007, Lyon, France.
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22
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Kimura T, Kaga Y, Sekita Y, Fujikawa K, Nakatani T, Odamoto M, Funaki S, Ikawa M, Abe K, Nakano T. Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds. Stem Cells 2015; 33:45-55. [PMID: 25186651 DOI: 10.1002/stem.1838] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2013] [Accepted: 08/18/2014] [Indexed: 12/12/2022]
Abstract
Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs.
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Affiliation(s)
- Tohru Kimura
- Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan; Department of Pathology, Osaka University, Suita, Osaka, Japan; Laboratory of Molecular Embryology, Kitasato University School of Science, Kitasato, Minami-ku, Sagamihara, Kanagawa, Japan; Laboratory of Stem Cell Biology, Kitasato University School of Science, Kitasato, Minami-ku, Sagamihara, Kanagawa, Japan
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23
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Gonadal development and germ cell tumors in mouse and humans. Semin Cell Dev Biol 2015; 45:114-23. [DOI: 10.1016/j.semcdb.2015.10.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Accepted: 10/01/2015] [Indexed: 12/12/2022]
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Tanaka T, Kanatsu-Shinohara M, Hirose M, Ogura A, Shinohara T. Pluripotent cell derivation from male germline cells by suppression of Dmrt1 and Trp53. J Reprod Dev 2015; 61:473-84. [PMID: 26227109 PMCID: PMC4623154 DOI: 10.1262/jrd.2015-059] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs
contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.
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Affiliation(s)
- Takashi Tanaka
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
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Integrative Analysis of the Acquisition of Pluripotency in PGCs Reveals the Mutually Exclusive Roles of Blimp-1 and AKT Signaling. Stem Cell Reports 2015; 5:111-24. [PMID: 26050930 PMCID: PMC4618250 DOI: 10.1016/j.stemcr.2015.05.007] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Revised: 05/07/2015] [Accepted: 05/07/2015] [Indexed: 12/19/2022] Open
Abstract
Primordial germ cells (PGCs) are lineage-restricted unipotent cells that can dedifferentiate into pluripotent embryonic germ cells (EGCs). Here we performed whole-transcriptome analysis during the conversion of PGCs into EGCs, a process by which cells acquire pluripotency. To examine the molecular mechanism underlying this conversion, we focused on Blimp-1 and Akt, which are involved in PGC specification and dedifferentiation, respectively. Blimp-1 overexpression in embryonic stem cells suppressed the expression of downstream targets of the pluripotency network. Conversely, Blimp-1 deletion in PGCs accelerated their dedifferentiation into pluripotent EGCs, illustrating that Blimp-1 is a pluripotency gatekeeper protein in PGCs. AKT signaling showed a synergistic effect with basic fibroblast growth factor plus 2i+A83 treatment on EGC formation. AKT played a major role in suppressing genes regulated by MBD3. From these results, we defined the distinct functions of Blimp-1 and Akt and provided mechanistic insights into the acquisition of pluripotency in PGCs.
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26
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Aponte PM. Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine. World J Stem Cells 2015; 7:669-680. [PMID: 26029339 PMCID: PMC4444608 DOI: 10.4252/wjsc.v7.i4.669] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Revised: 03/13/2015] [Accepted: 04/14/2015] [Indexed: 02/06/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.
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Kimura T, Kaga Y, Ohta H, Odamoto M, Sekita Y, Li K, Yamano N, Fujikawa K, Isotani A, Sasaki N, Toyoda M, Hayashi K, Okabe M, Shinohara T, Saitou M, Nakano T. Induction of primordial germ cell-like cells from mouse embryonic stem cells by ERK signal inhibition. Stem Cells 2015; 32:2668-78. [PMID: 24989326 DOI: 10.1002/stem.1781] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2013] [Revised: 05/27/2014] [Accepted: 06/06/2014] [Indexed: 12/12/2022]
Abstract
Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by extracellular signal-regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification.
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Affiliation(s)
- Tohru Kimura
- Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan; Department of Pathology, Medical School, Osaka University, Suita, Osaka, Japan; Laboratory of Molecular Embryology, Department of Biosciences, Kitasato University School of Science, Kitasato, Minami-ku, Sagamihara, Kanagawa, Japan; Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, Kitasato, Minami-ku, Sagamihara, Kanagawa, Japan
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El-Badawy A, El-Badri N. Regulators of pluripotency and their implications in regenerative medicine. STEM CELLS AND CLONING-ADVANCES AND APPLICATIONS 2015; 8:67-80. [PMID: 25960670 PMCID: PMC4410894 DOI: 10.2147/sccaa.s80157] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The ultimate goal of regenerative medicine is to replace damaged tissues with new functioning ones. This can potentially be accomplished by stem cell transplantation. While stem cell transplantation for blood diseases has been increasingly successful, widespread application of stem cell therapy in the clinic has shown limited results. Despite successful efforts to refine existing methodologies and to develop better ones for reprogramming, clinical application of stem cell therapy suffers from issues related to the safety of the transplanted cells, as well as the low efficiency of reprogramming technology. Better understanding of the underlying mechanism(s) involved in pluripotency should accelerate the clinical application of stem cell transplantation for regenerative purposes. This review outlines the main decision-making factors involved in pluripotency, focusing on the role of microRNAs, epigenetic modification, signaling pathways, and toll-like receptors. Of special interest is the role of toll-like receptors in pluripotency, where emerging data indicate that the innate immune system plays a vital role in reprogramming. Based on these data, we propose that nongenetic mechanisms for reprogramming provide a novel and perhaps an essential strategy to accelerate application of regenerative medicine in the clinic.
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Affiliation(s)
- Ahmed El-Badawy
- Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt
| | - Nagwa El-Badri
- Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt
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29
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López-Iglesias P, Alcaina Y, Tapia N, Sabour D, Arauzo-Bravo MJ, Sainz de la Maza D, Berra E, O'Mara AN, Nistal M, Ortega S, Donovan PJ, Schöler HR, De Miguel MP. Hypoxia induces pluripotency in primordial germ cells by HIF1α stabilization and Oct4 deregulation. Antioxid Redox Signal 2015; 22:205-23. [PMID: 25226357 DOI: 10.1089/ars.2014.5871] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
AIMS To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). RESULTS We found 11 genes ≥1.5-fold overexpressed in EGCs. None of the genes identified was the Yamanaka genes but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. Hypoxia induced a glycolytic program in PGCs in detriment of mitochondrial oxidative phosphorylation. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGLs), which differentiate into cells of the three germ layers in vitro and contribute to the internal cell mass of the blastocyst in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1α stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to the lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signaling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. INNOVATION AND CONCLUSION Our data suggest that the two main properties of stemness, pluripotency and self-renewal, are differentially regulated in PGC reprogramming induced by hypoxia.
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Affiliation(s)
- Pilar López-Iglesias
- 1 Cell Engineering Laboratory, IdiPaz, La Paz Hospital Research Institute , Madrid Spain
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30
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Makker A, Goel MM, Mahdi AA. PI3K/PTEN/Akt and TSC/mTOR signaling pathways, ovarian dysfunction, and infertility: an update. J Mol Endocrinol 2014; 53:R103-18. [PMID: 25312969 DOI: 10.1530/jme-14-0220] [Citation(s) in RCA: 167] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Abnormalities in ovarian function, including defective oogenesis and folliculogenesis, represent a key female reproductive deficiency. Accumulating evidence in the literature has shown that the PI3K/PTEN/Akt and TSC/mTOR signaling pathways are critical regulators of ovarian function including quiescence, activation, and survival of primordial follicles, granulosa cell proliferation and differentiation, and meiotic maturation of oocytes. Dysregulation of these signaling pathways may contribute to infertility caused by impaired follicular development, intrafollicular oocyte development, and ovulation. This article reviews the current state of knowledge of the functional role of the PI3K/PTEN/Akt and TSC/mTOR pathways during mammalian oogenesis and folliculogenesis and their association with female infertility.
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Affiliation(s)
- Annu Makker
- Post-Graduate Department of PathologyDepartment of BiochemistryKing George's Medical University, Lucknow 226003, Uttar Pradesh, India
| | - Madhu Mati Goel
- Post-Graduate Department of PathologyDepartment of BiochemistryKing George's Medical University, Lucknow 226003, Uttar Pradesh, India
| | - Abbas Ali Mahdi
- Post-Graduate Department of PathologyDepartment of BiochemistryKing George's Medical University, Lucknow 226003, Uttar Pradesh, India
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31
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Matsui Y, Takehara A, Tokitake Y, Ikeda M, Obara Y, Morita-Fujimura Y, Kimura T, Nakano T. The majority of early primordial germ cells acquire pluripotency by AKT activation. Development 2014; 141:4457-67. [PMID: 25359722 DOI: 10.1242/dev.113779] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency.
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Affiliation(s)
- Yasuhisa Matsui
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan CREST, JST, Kawaguchi, Saitama 332-0012, Japan
| | - Asuka Takehara
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan
| | - Yuko Tokitake
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan CREST, JST, Kawaguchi, Saitama 332-0012, Japan
| | - Makiko Ikeda
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan
| | - Yuka Obara
- Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan
| | - Yuiko Morita-Fujimura
- Frontier Research Institute of Interdisciplinary Sciences (FRIS), Tohoku University, Sendai, Miyagi 980-8578, Japan
| | - Tohru Kimura
- School of Science, Kitasato University, Sagamihara, Kanagawa 252-0373, Japan
| | - Toru Nakano
- CREST, JST, Kawaguchi, Saitama 332-0012, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
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32
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Yu Y, Liang D, Tian Q, Chen X, Jiang B, Chou BK, Hu P, Cheng L, Gao P, Li J, Wang G. Stimulation of somatic cell reprogramming by ERas-Akt-FoxO1 signaling axis. Stem Cells 2014; 32:349-63. [PMID: 23765875 DOI: 10.1002/stem.1447] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2012] [Revised: 04/28/2013] [Accepted: 05/15/2013] [Indexed: 12/19/2022]
Abstract
Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) shares much similarity to the cancer initiation process, and the molecular mechanisms underlying both processes remain to be elucidated. Here, we report that a tumor- or embryonic stem cell-specific Ras gene ERas, which encodes a constitutively active form of GTPase, and its downstream Phosphoinositide-3 kinase/Akt signaling pathway are important facilitators for the somatic reprogramming process. We found that overexpression of ERas retrovirally enhanced mouse iPSC induction while ERas knockdown repressed it. Modulation of Akt signaling by genetic or chemical means greatly impacted the reprogramming efficiency. Forced expression of a constitutively active Akt1 gene could rescue the reduced efficiency resulting from ERas knockdown, and point-mutation analyses further revealed that ERas is tightly coupled with Akt signaling to enhance reprogramming. Mechanistically, the forkhead transcription factor FoxO1 can function as a barrier to the iPSC induction, and the inactivation of FoxO1 by Akt-dependent phosphorylation largely accounts for the enhancing effect of ERas-Akt signaling on reprogramming. Collectively, these results unravel the significance of the ERas-Akt-FoxO1 signaling axis in iPSC generation, suggesting a possibly shared molecular basis for both somatic reprogramming and cancer initiation.
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Affiliation(s)
- Yong Yu
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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33
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Tang Y, Jiang Z, Luo Y, Zhao X, Wang L, Norris C, Tian XC. Differential effects of Akt isoforms on somatic cell reprogramming. J Cell Sci 2014; 127:3998-4008. [PMID: 25037569 DOI: 10.1242/jcs.150029] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Akt plays an important role in cell growth, proliferation and survival. The specific roles of the three Akt isoforms in somatic cell reprogramming have not been investigated. Here we report that, during iPSC generation, enhanced Akt1 activity promotes complete reprogramming mainly through increased activation of Stat3 in concert with leukemia inhibitory factor (LIF) and, to a lesser extent, through promotion of colony formation. Akt1 augments Stat3 activity through activation of mTOR and upregulation of LIF receptor expression. Similarly, enhanced Akt2 or Akt3 activation also promotes reprogramming and coordinates with LIF to activate Stat3. Blocking Akt1 or Akt3 but not Akt2 expression prohibits cell proliferation and reprogramming. Furthermore, the halt in cell proliferation and reprogramming caused by mTOR and Akt inhibitors can be reversed by inhibition of GSK3. Finally, we found that expressing the GSK3β target Esrrb overrides inhibition of Akt and restores reprogramming. Our data demonstrated that during reprogramming, Akt promotes establishment of pluripotency through co-stimulation of Stat3 activity with LIF. Akt1 and Akt3 are essential for the proliferation of reprogrammed cells, and Esrrb supports cell proliferation and complete reprogramming during Akt signaling.
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Affiliation(s)
- Yong Tang
- Center for Regenerative Biology, Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA
| | - Zongliang Jiang
- Center for Regenerative Biology, Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA
| | - Yan Luo
- Center for Regenerative Biology, Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA
| | - Xueming Zhao
- Center for Regenerative Biology, Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA
| | - Ling Wang
- Center for Regenerative Biology, Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA
| | - Carol Norris
- Department of Molecular and Cellular Biology/Biotechnology and Bioservices Center, University of Connecticut, Storrs, CT 06269, USA
| | - Xiuchun Cindy Tian
- Center for Regenerative Biology, Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA
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34
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Dai X, Liu P, Lau AW, Liu Y, Inuzuka H. Acetylation-dependent regulation of essential iPS-inducing factors: a regulatory crossroad for pluripotency and tumorigenesis. Cancer Med 2014; 3:1211-24. [PMID: 25116380 PMCID: PMC4302671 DOI: 10.1002/cam4.298] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2014] [Revised: 06/04/2014] [Accepted: 06/10/2014] [Indexed: 12/26/2022] Open
Abstract
Induced pluripotent stem (iPS) cells can be generated from somatic cells by coexpression of four transcription factors: Sox2, Oct4, Klf4, and c-Myc. However, the low efficiency in generating iPS cells and the tendency of tumorigenesis hinder the therapeutic applications for iPS cells in treatment of human diseases. To this end, it remains largely unknown how the iPS process is subjected to regulation by upstream signaling pathway(s). Here, we report that Akt regulates the iPS process by modulating posttranslational modifications of these iPS factors in both direct and indirect manners. Specifically, Akt directly phosphorylates Oct4 to modulate the Oct4/Sox2 heterodimer formation. Furthermore, Akt either facilitates the p300-mediated acetylation of Oct4, Sox2, and Klf4, or stabilizes Klf4 by inactivating GSK3, thus indirectly modulating stemness. As tumorigenesis shares possible common features and mechanisms with iPS, our study suggests that Akt inhibition might serve as a cancer therapeutic approach to target cancer stem cells.
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Affiliation(s)
- Xiangpeng Dai
- Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
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35
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Leitch HG, Tang WWC, Surani MA. Primordial germ-cell development and epigenetic reprogramming in mammals. Curr Top Dev Biol 2014; 104:149-87. [PMID: 23587241 DOI: 10.1016/b978-0-12-416027-9.00005-x] [Citation(s) in RCA: 93] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Primordial germ cells (PGCs) are the embryonic precursors of the gametes and represent the founder cells of the germline. Specification of PGCs is a critical divergent point during embryogenesis. Whereas the somatic lineages will ultimately perish, cells of the germline have the potential to form a new individual and hence progress to the next generation. It is therefore critical that the genome emerges intact and carrying the appropriate epigenetic information during its passage through the germline. To ensure this fidelity of transmission, PGC development encompasses extensive epigenetic reprogramming. The low cell numbers and relative inaccessibility of PGCs present a challenge to those seeking mechanistic understanding of the crucial developmental and epigenetic processes in this most fascinating of lineages. Here, we present an overview of PGC development in the mouse and compare this with the limited information available for other mammalian species. We believe that a comparative approach will be increasingly important to uncover the extent to which mechanisms are conserved and reveal the critical steps during PGC development in humans.
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Affiliation(s)
- Harry G Leitch
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, United Kingdom
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36
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Testicular cancer: biology and biomarkers. Virchows Arch 2014; 464:301-13. [DOI: 10.1007/s00428-013-1522-1] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2013] [Accepted: 11/25/2013] [Indexed: 12/13/2022]
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37
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PARK SHINHYUNG, KIM JEONGHWAN, NAM SOOWAN, KIM BYUNGWOO, KIM GIYOUNG, KIM WUNJAE, CHOI YUNGHYUN. Selenium improves stem cell potency by stimulating the proliferation and active migration of 3T3-L1 preadipocytes. Int J Oncol 2013; 44:336-42. [DOI: 10.3892/ijo.2013.2182] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2013] [Accepted: 10/23/2013] [Indexed: 11/05/2022] Open
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38
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Glover JD, Taylor L, Sherman A, Zeiger-Poli C, Sang HM, McGrew MJ. A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration. PLoS One 2013; 8:e77222. [PMID: 24223709 PMCID: PMC3817190 DOI: 10.1371/journal.pone.0077222] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Accepted: 09/09/2013] [Indexed: 01/15/2023] Open
Abstract
In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.
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Affiliation(s)
- James D Glover
- The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, United Kingdom
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39
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Li Y, Hong WX, Lan B, Xu X, Liu Y, Kong L, Li Y, Zhou S, Liu Y, Feng R, Jiang S, He Q, Tan J. PDGF mediates derivation of human embryonic germ cells. Differentiation 2013; 86:141-8. [DOI: 10.1016/j.diff.2013.11.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2013] [Revised: 11/22/2013] [Accepted: 11/29/2013] [Indexed: 12/25/2022]
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40
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Leitch H, Nichols J, Humphreys P, Mulas C, Martello G, Lee C, Jones K, Surani M, Smith A. Rebuilding pluripotency from primordial germ cells. Stem Cell Reports 2013; 1:66-78. [PMID: 24052943 PMCID: PMC3757743 DOI: 10.1016/j.stemcr.2013.03.004] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Revised: 03/22/2013] [Accepted: 03/23/2013] [Indexed: 02/02/2023] Open
Abstract
Mammalian primordial germ cells (PGCs) are unipotent progenitors of the gametes. Nonetheless, they can give rise directly to pluripotent stem cells in vitro or during teratocarcinogenesis. This conversion is inconsistent, however, and has been difficult to study. Here, we delineate requirements for efficient resetting of pluripotency in culture. We demonstrate that in defined conditions, routinely 20% of PGCs become EG cells. Conversion can occur from the earliest specified PGCs. The entire process can be tracked from single cells. It is driven by leukemia inhibitory factor (LIF) and the downstream transcription factor STAT3. In contrast, LIF signaling is not required during germ cell ontogeny. We surmise that ectopic LIF/STAT3 stimulation reconstructs latent pluripotency and self-renewal. Notably, STAT3 targets are significantly upregulated in germ cell tumors, suggesting that dysregulation of this pathway may underlie teratocarcinogenesis. These findings demonstrate that EG cell formation is a robust experimental system for exploring mechanisms involved in reprogramming and cancer.
A defined system for generation of pluripotent EG cells at high efficiency 20% of single primordial germ cells become EG cells Stimulation with LIF but not FGF drives conversion to pluripotency LIF/STAT3 targets are upregulated in pluripotent germ cell tumors
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Affiliation(s)
- Harry G. Leitch
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Jennifer Nichols
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Physiology, Development, and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Peter Humphreys
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Carla Mulas
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Graziano Martello
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Caroline Lee
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Ken Jones
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - M. Azim Surani
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Physiology, Development, and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Austin Smith
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
- Corresponding author
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Chen L, Faire M, Kissner MD, Laird DJ. Primordial germ cells and gastrointestinal stromal tumors respond distinctly to a cKit overactivating allele. Hum Mol Genet 2013; 22:313-27. [PMID: 23077213 PMCID: PMC3526162 DOI: 10.1093/hmg/dds430] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2012] [Revised: 10/01/2012] [Accepted: 10/08/2012] [Indexed: 12/31/2022] Open
Abstract
KitL, via its receptor cKit, supports primordial germ cell (PGC) growth, survival, migration and reprogramming to pluripotent embryonic germ cells (EGCs). However, the signaling downstream of KitL and its regulation in PGCs remain unclear. A constitutively activating mutation, cKit(V558Δ), causes gain-of-function phenotypes in mast cells and intestines, and gastrointestinal stromal tumors (GISTs) when heterozygous. Unexpectedly, we find that PGC growth is not significantly affected in cKit(V558Δ) heterozygotes, whereas in homozygotes, increased apoptosis and inefficient migration lead to the depletion of PGCs. Through genetic studies, we reveal that this oncogenic cKit allele exhibits loss-of-function behavior in PGCs distinct from that in GIST development. Examination of downstream signaling in GISTs from cKit(V558Δ/+) mice confirmed hyperphosphorylation of AKT and ERK, but both remain unperturbed in cKit(V558Δ/+) PGCs and EGCs. In contrast, we find reduced activation of ERK1/2 and JNK1 in cKit(V558Δ) homozygous PGCs and EGCs. Inhibiting JNK, though not ERK1/2, increased apoptosis of wild-type PGCs, but did not further affect the already elevated apoptosis of cKit(V558Δ)(/V558Δ) PGCs. These results demonstrate a cell-context-dependent response to the cKit(V558Δ) mutation. We propose that AKT overload protection and JNK-mediated survival comprise PGC-specific mechanisms for regulating cKit signaling.
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Affiliation(s)
| | | | | | - Diana J. Laird
- Department of Obstetrics/Gynecology and Reproductive Sciences, Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94143-0667, USA
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42
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Retinoic acid promotes proliferation of chicken primordial germ cells via activation of PI3K/Akt-mediated NF-κB signalling cascade. Cell Biol Int 2012; 36:705-12. [PMID: 22548360 DOI: 10.1042/cbi20110542] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
As embryonic progenitors for the gametes, PGCs (primordial germ cells) proliferate and develop under strict regulation of numerous intrinsic and external factors. As the most active natural metabolite of vitamin A, all-trans RA (retinoic acid) plays pivotal roles in regulating development of various cells. The proliferating action of RA on PGCs was investigated along with the intracellular PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B; also known as Akt)-mediated NF-κB (nuclear factor κB) signalling cascade. The results show that RA significantly promoted PGC proliferation in a dose- and time-dependent manner, confirmed by BrdU (bromodeoxyuridine) incorporation and cell cycle analysis. However, this promoting effect was attenuated by sequential inhibitors of LY294002 for PI3K, KP372-1 for Akt and SN50 for NF-κB respectively. Western blot analysis showed increased Akt phosphorylation (Ser473) of PGCs after stimulation with RA, but this was abolished by LY294002 or KP372-1. Treatment with RA increased expression of NF-κB and decreased IκBα (inhibitory κBα) expression, which were inhibited by SN50. Blockade of PI3K or Akt activity inhibited NF-κB translocation from the cytoplasm to the nucleus. Finally, mRNA expression of cell cycle regulating genes [cyclin D1 and E, CDK6 (cyclin-dependent kinase 6) and CDK2] was up-regulated in the RA-treated cells. This stimulation was also markedly retarded by combined treatment with LY294002, KP372-1 and SN50. These results suggest that RA activates the PI3K/Akt and NF-κB signalling cascade to promote proliferation of the cultured chicken PGCs.
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Abstract
Germ cell development creates totipotency through genetic as well as epigenetic regulation of the genome function. Primordial germ cells (PGCs) are the first germ cell population established during development and are immediate precursors for both the oocytes and spermatogonia. We here summarize recent findings regarding the mechanism of PGC development in mice. We focus on the transcriptional and signaling mechanism for PGC specification, potential pluripotency, and epigenetic reprogramming in PGCs and strategies for the reconstitution of germ cell development using pluripotent stem cells in culture. Continued studies on germ cell development may lead to the generation of totipotency in vitro, which should have a profound influence on biological science as well as on medicine.
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Affiliation(s)
- Mitinori Saitou
- Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Japan.
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Lim J, Maher GJ, Turner GDH, Dudka-Ruszkowska W, Taylor S, Meyts ERD, Goriely A, Wilkie AOM. Selfish spermatogonial selection: evidence from an immunohistochemical screen in testes of elderly men. PLoS One 2012; 7:e42382. [PMID: 22879958 PMCID: PMC3412839 DOI: 10.1371/journal.pone.0042382] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2012] [Accepted: 07/04/2012] [Indexed: 01/26/2023] Open
Abstract
The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia–caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively–represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection.
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Affiliation(s)
- Jasmine Lim
- Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Geoffrey J. Maher
- Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Gareth D. H. Turner
- Department of Cellular Pathology, NIHR Biomedical Research Centre, Oxford University Hospitals NHS Trust, Oxford, United Kingdom
| | - Wioleta Dudka-Ruszkowska
- Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Stephen Taylor
- Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Ewa Rajpert-De Meyts
- University Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark
| | - Anne Goriely
- Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
| | - Andrew O. M. Wilkie
- Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
- * E-mail:
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Imamura M, Lin ZYC, Okano H. Cell-intrinsic reprogramming capability: gain or loss of pluripotency in germ cells. Reprod Med Biol 2012; 12:1-14. [PMID: 29699125 DOI: 10.1007/s12522-012-0131-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2012] [Accepted: 05/30/2012] [Indexed: 12/23/2022] Open
Abstract
In multicellular organisms, germ cells are an extremely specialized cell type with the vital function of transmitting genetic information across generations. In this respect, they are responsible for the perpetuity of species, and are separated from somatic lineages at each generation. Interestingly, in the past two decades research has shown that germ cells have the potential to proceed along two distinct pathways: gametogenesis or pluripotency. Unequivocally, the primary role of germ cells is to produce gametes, the sperm or oocyte, to produce offspring. However, under specific conditions germ cells can become pluripotent, as shown by teratoma formation in vivo or cell culture-induced reprogramming in vitro. This phenomenon seems to be a general propensity of germ cells, irrespective of developmental phase. Recent attempts at cellular reprogramming have resulted in the generation of induced pluripotent stem cells (iPSCs). In iPSCs, the intracellular molecular networks instructing pluripotency have been activated and override the exclusively somatic cell programs that existed. Because the generation of iPSCs is highly artificial and depends on gene transduction, whether the resulting machinery reflects any physiological cell-intrinsic programs is open to question. In contrast, germ cells can spontaneously shift their fate to pluripotency during in-vitro culture. Here, we review the two fates of germ cells, i.e., differentiation and reprogramming. Understanding the molecular mechanisms regulating differentiation versus reprogramming would provide invaluable insight into understanding the mechanisms of cellular reprogramming that generate iPSCs.
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Affiliation(s)
- Masanori Imamura
- Department of Physiology, School of Medicine Keio University 35 Shinanomachi 160-8582 Shinjuku-ku Tokyo Japan
| | - Zachary Yu-Ching Lin
- Department of Physiology, School of Medicine Keio University 35 Shinanomachi 160-8582 Shinjuku-ku Tokyo Japan
| | - Hideyuki Okano
- Department of Physiology, School of Medicine Keio University 35 Shinanomachi 160-8582 Shinjuku-ku Tokyo Japan
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Nagamatsu G, Kosaka T, Saito S, Takubo K, Akiyama H, Sudo T, Horimoto K, Oya M, Suda T. Tracing the conversion process from primordial germ cells to pluripotent stem cells in mice. Biol Reprod 2012; 86:182. [PMID: 22423052 DOI: 10.1095/biolreprod.111.096792] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
To understand mechanisms underlying acquisition of pluripotency, it is critical to identify cells that can be converted to pluripotent stem cells. For this purpose, we focused on unipotent primordial germ cells (PGCs), which can be reprogrammed into pluripotent embryonic germ (EG) cells under defined conditions. Treatment of PGCs with combinations of signaling inhibitors, including inhibitors of MAP2K (MEK), GSK3B (GSK-3beta), and TGFB (TGFbeta) type 1 receptors, induced cells to enter a pluripotent state at a high frequency (12.1%) by Day 10 of culture. When we employed fluorescence-activated cell sorting to monitor conversion of candidate cells to a pluripotent state, we observed a cell cycle shift to S phase, indicating enrichment of pluripotent cells, during the early phase of EG formation. Transcriptome analysis revealed that PGCs retained expression of some pluripotent stem cell-associated genes, such as Pou5f1 and Sox2, during EG cell formation. On the other hand, PGCs lost their germ lineage characteristics and acquired expression of pluripotent stem cell markers, such as Klf4 and Eras. The overall gene expression profiles revealed by this system provide novel insight into how pluripotency is acquired in germ-committed cells.
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Affiliation(s)
- Go Nagamatsu
- Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan.
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Boztug K, Rosenberg PS, Dorda M, Banka S, Moulton T, Curtin J, Rezaei N, Corns J, Innis JW, Avci Z, Tran HC, Pellier I, Pierani P, Fruge R, Parvaneh N, Mamishi S, Mody R, Darbyshire P, Motwani J, Murray J, Buchanan GR, Newman WG, Alter BP, Boxer LA, Donadieu J, Welte K, Klein C. Extended spectrum of human glucose-6-phosphatase catalytic subunit 3 deficiency: novel genotypes and phenotypic variability in severe congenital neutropenia. J Pediatr 2012; 160:679-683.e2. [PMID: 22050868 DOI: 10.1016/j.jpeds.2011.09.019] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2011] [Revised: 08/09/2011] [Accepted: 09/08/2011] [Indexed: 11/29/2022]
Abstract
OBJECTIVE To delineate the phenotypic and molecular spectrum of patients with a syndromic variant of severe congenital neutropenia (SCN) due to mutations in the gene encoding glucose-6-phosphatase catalytic subunit 3 (G6PC3). STUDY DESIGN Patients with syndromic SCN were characterized for associated malformations and referred to us for G6PC3 mutational analysis. RESULTS In a cohort of 31 patients with syndromic SCN, we identified 16 patients with G6PC3 deficiency including 11 patients with novel biallelic mutations. We show that nonhematologic features of G6PC3 deficiency are good predictive indicators for mutations in G6PC3. Additionally, we demonstrate genetic variability in this disease and define novel features such as growth hormone deficiency, genital malformations, disrupted bone remodeling, and abnormalities of the integument. G6PC3 mutations may be associated with hydronephrosis or facial dysmorphism. The risk of transition to myelodysplastic syndrome/acute myeloid leukemia may be lower than in other genetically defined SCN subgroups. CONCLUSIONS The phenotypic and molecular spectrum in G6PC3 deficiency is wider than previously appreciated. The risk of transition to myelodysplastic syndrome or acute myeloid leukemia may be lower in G6PC3 deficiency compared with other subgroups of SCN.
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Affiliation(s)
- Kaan Boztug
- Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Vienna, Austria.
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The proto-oncogene Ret is required for male foetal germ cell survival. Dev Biol 2012; 365:101-9. [PMID: 22360967 DOI: 10.1016/j.ydbio.2012.02.014] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2011] [Revised: 01/20/2012] [Accepted: 02/10/2012] [Indexed: 01/08/2023]
Abstract
The spermatogenic and oogenic lineages originate from bipotential primordial germ cells in response to signalling in the foetal testis or ovary, respectively. The signals required for male germ cell commitment and their entry into mitotic arrest remain largely unknown. Recent data show that the ligand GDNF is up regulated in the foetal testis indicating that it may be involved in male germ cell development. In this study genetic analysis of GDNF-RET signalling shows that RET is required for germ cell survival. Affected germ cells in Ret-/- mice lose expression of key germ cell markers, abnormally express cell cycle markers and undergo apoptosis. Surprisingly, a similar phenotype was not detected in Gdnf-/- mice indicating that either redundancy with a Gdnf related gene might compensate for its loss, or that RET operates in a GDNF independent manner in mouse foetal germ cells. Either way, this study identifies the proto-oncogene RET as a novel component of the foetal male germ cell development pathway.
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Notch and the p53 clan of transcription factors. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2012; 727:223-40. [PMID: 22399351 DOI: 10.1007/978-1-4614-0899-4_17] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Notch 1 to 4 and the p53 clan, comprising p53, p63 and p73 plus numerous isoforms thereof, are gene transcription regulators that are critically involved in various aspects of cell differentiation, stem cell maintenance and tumour suppression. It is thus perhaps no surprise that extensive crosstalk between the Notch and p53 pathways is implemented during these processes. Typically, Notch together with p53 and even more so with transactivation competent p63 or p73, drives differentiation, whereas Notch combined with transactivation impaired p63 or p73 helps maintain undifferentiated stem cell compartments. With regard to cancer, it seems that Notch acts as a tumour suppressor in cellular contexts where Notch signalling supports p53 activation and both together can bring on its way an anti-proliferative programme of differentiation, senescence or apoptosis. In contrast, Notch often acts as an oncoprotein in contexts where it suppresses p53 activation and activity and where differentiation is unwanted. It is no accident that the latter pathways-the inhibition by Notch of p53 and differentiation-are operative in somatic stem cells as well as in tumour cells.
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De Felici M. Nuclear reprogramming in mouse primordial germ cells: epigenetic contribution. Stem Cells Int 2011; 2011:425863. [PMID: 21969835 PMCID: PMC3182379 DOI: 10.4061/2011/425863] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2011] [Accepted: 07/11/2011] [Indexed: 12/17/2022] Open
Abstract
The unique capability of germ cells to give rise to a new organism, allowing the transmission of primary genetic information from generation to generation, depends on their epigenetic reprogramming ability and underlying genomic totipotency. Recent studies have shown that genome-wide epigenetic modifications, referred to as “epigenetic reprogramming”, occur during the development of the gamete precursors termed primordial germ cells (PGCs) in the embryo. This reprogramming is likely to be critical for the germ line development itself and necessary to erase the parental imprinting and setting the base for totipotency intrinsic to this cell lineage. The status of genome acquired during reprogramming and the associated expression of key pluripotency genes render PGCs susceptible to transform into pluripotent stem cells. This may occur in vivo under still undefined condition, and it is likely at the origin of the formation of germ cell tumors. The phenomenon appears to be reproduced under partly defined in vitro culture conditions, when PGCs are transformed into embryonic germ (EG) cells. In the present paper, I will try to summarize the contribution that epigenetic modifications give to nuclear reprogramming in mouse PGCs.
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Affiliation(s)
- Massimo De Felici
- Section of Histology and Embryology, Department of Public Health and Cell Biology, University of Rome "Tor Vergata," 00173 Rome, Italy
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