RESUMEN
Sex-specific metabolic characteristics emerge in the mouse germ line after reaching the genital ridges around embryonic day 10.5, coinciding with sexual differentiation. However, the impact of such metabolic characteristics on germ cell development remains unclear. In this study, we observed the specific upregulation in male fetal germ cells of D-3-phosphoglycerate dehydrogenase (PHGDH), the primary enzyme in the serine-glycine-one-carbon metabolism, along with an increase in a downstream metabolite, S-adenosylmethionine (SAM), crucial for protein and nucleic acid methylation. Inhibiting PHGDH in fetal testes resulted in reduced SAM levels in germ cells, accompanied by increases in the number of mouse vasa homolog (MVH/VASA)-positive germ cells and the promyelocytic leukemia zinc finger (PLZF)-positive undifferentiated spermatogonia ratio. Furthermore, PHGDH inhibition led to a decrease in the methylation of histone H3 and DNA, resulting in aberrations in gene expression profiles. In summary, our findings underscore the significant role of certain metabolic mechanisms in the development of male germ cells.
RESUMEN
Fate determination of primordial germ cells (PGCs) is regulated in a multi-layered manner, involving signaling pathways, epigenetic mechanisms, and transcriptional control. Chemical modification of macromolecules, including epigenetics, is expected to be closely related with metabolic mechanisms but the detailed molecular machinery linking these two layers remains poorly understood. Here, we show that the hexosamine biosynthetic pathway controls PGC fate determination via O-linked ß-N-acetylglucosamine (O-GlcNAc) modification. Consistent with this model, reduction of carbohydrate metabolism via a maternal ketogenic diet that decreases O-GlcNAcylation levels causes repression of PGC formation in vivo. Moreover, maternal ketogenic diet intake until mid-gestation affects the number of ovarian germ cells in newborn pups. Taken together, we show that nutritional and metabolic mechanisms play a previously unappreciated role in PGC fate determination.
Asunto(s)
Acetilglucosamina , Transducción de Señal , Recién Nacido , Humanos , Transducción de Señal/fisiología , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Regulación de la Expresión Génica , Epigénesis Genética , Células Germinativas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Exposure to environmental factors during fetal development may lead to epigenomic modifications in fetal germ cells, altering gene expression and promoting diseases in successive generations. In mouse, maternal exposure to di(2-ethylhexyl) phthalate (DEHP) is known to induce defects in spermatogenesis in successive generations, but the mechanism(s) of impaired spermatogenesis are unclear. Here, we showed that maternal DEHP exposure results in DNA hypermethylation of promoters of spermatogenesis-related genes in fetal testicular germ cells in F1 mice, and hypermethylation of Hist1h2ba, Sycp1, and Taf7l, which are crucial for spermatogenesis, persisted from fetal testicular cells to adult spermatogonia, resulting in the downregulation of expression of these genes. Forced methylation of these gene promoters silenced expression of these loci in a reporter assay. These results suggested that maternal DEHP exposure-induced hypermethylation of Hist1h2ba, Sycp1, and Taf7l results in downregulation of these genes in spermatogonia and subsequent defects in spermatogenesis, at least in the F1 generation.
Asunto(s)
Dietilhexil Ftalato/farmacología , Exposición Materna/efectos adversos , Mutación , Ácidos Ftálicos/efectos adversos , Efectos Tardíos de la Exposición Prenatal/genética , Espermatogénesis/efectos de los fármacos , Espermatogénesis/genética , Animales , Metilación de ADN , Regulación hacia Abajo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ácidos Ftálicos/química , Plastificantes/efectos adversos , Embarazo , Espermatogonias/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacosRESUMEN
Fetal ovarian germ cells show characteristic energy metabolism status, such as enhanced mitochondrial metabolism as well as glycolysis, but their roles in early folliculogenesis are unclear. We show here that inhibition of pyruvate uptake to mitochondria by UK5099 in organ cultures of fetal mouse ovaries resulted in repressed early folliculogenesis without affecting energy production, survival of oocytes, or meiosis. In addition, the abnormal folliculogenesis by UK5099 was partially rescued by α-ketoglutarate and succinate, intermediate metabolites in the TCA cycle, suggesting the importance of those metabolites. The expression of TGFß-related genes Gdf9 and Bmp15 in ovarian germ cells, which are crucial for folliculogenesis, was downregulated by UK5099, and the addition of recombinant GDF9 partially rescued the abnormal folliculogenesis induced by UK5099. We also found that early folliculogenesis was similarly repressed, as in the culture, in the ovaries of a germ cell-specific knockout of Mpc2, which encodes a mitochondria pyruvate carrier that is targeted by UK5099. These results suggest that insufficient Gdf9 expression induced by abnormal pyruvate metabolism in oocytes results in early follicular dysgenesis, which is a possible cause of defective folliculogenesis in humans.
Asunto(s)
Acrilatos/farmacología , Proteína Morfogenética Ósea 15/genética , Factor 9 de Diferenciación de Crecimiento/genética , Oocitos/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Ácido Pirúvico/metabolismo , Animales , Transporte Biológico , Proteína Morfogenética Ósea 15/metabolismo , Ciclo del Ácido Cítrico , Femenino , Regulación de la Expresión Génica , Factor 9 de Diferenciación de Crecimiento/metabolismo , Ratones , Mitocondrias/metabolismo , Oocitos/metabolismoRESUMEN
Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.
Asunto(s)
Diferenciación Celular/fisiología , Células Germinales Embrionarias/fisiología , Metabolómica , Proteómica , Animales , ADN/genética , ADN/metabolismo , Metilación de ADN , Embrión de Mamíferos/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Transgénicos , Diferenciación Sexual , Factores Sexuales , Factores de TranscripciónRESUMEN
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 G1S 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.
Asunto(s)
Reprogramación Celular/fisiología , Ciclina D1/metabolismo , Células Germinales Embrionarias/citología , Células Germinales Embrionarias/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Fase G1 , Fase G2 , Histonas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Activación Enzimática , Fase G1/fisiología , Fase G2/fisiología , Masculino , Metilación , Ratones , Ratones Transgénicos , Transducción de SeñalRESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias de Ratones/metabolismo , Proteínas de Neoplasias/genética , Células Madre Pluripotentes/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Ratones Transgénicos , Células Madre Embrionarias de Ratones/citología , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Proteínas de Neoplasias/deficiencia , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Teratoma/genética , Teratoma/metabolismo , Teratoma/patología , Testículo/citología , Testículo/embriología , Testículo/metabolismoRESUMEN
In mouse embryos, primordial germ cells (PGCs) are fate-determined from epiblast cells. Signaling pathways involved in PGC formation have been identified, but their epigenetic mechanisms remain poorly understood. Here, we show that the histone methyltransferase SETDB1 is an epigenetic regulator of PGC fate determination. Setdb1-deficient embryos exhibit drastic reduction of nascent PGCs. Dppa2, Otx2 and Utf1 are de-repressed whereas mesoderm development-related genes, including BMP4 signaling-related genes, are downregulated by Setdb1 knockdown during PGC-like cell (PGCLC) induction. In addition, binding of SETDB1 is observed at the flanking regions of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs, and trimethylation of lysine 9 of histone H3 is reduced by Setdb1 knockdown at those regions. Furthermore, DPPA2, OTX2 and UTF1 binding is increased in genes encoding BMP4 signaling-related proteins, including SMAD1. Finally, overexpression of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs results in the repression of BMP4 signaling-related genes and PGC determinant genes. We propose that the localization of SETDB1 to Dppa2, Otx2 and Utf1, and subsequent repression of their expression, are crucial for PGC determination by ensuring BMP4 signaling.
Asunto(s)
Proteína Morfogenética Ósea 4/metabolismo , Linaje de la Célula , Células Germinativas/citología , Células Germinativas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Transducción de Señal , Animales , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
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.
Asunto(s)
Reprogramación Celular , Células Germinales Embrionarias/citología , Factores de Transcripción de Tipo Kruppel/metabolismo , Óvulo/citología , Proteínas Proto-Oncogénicas/metabolismo , Espermatozoides/citología , Factores de Transcripción/metabolismo , Animales , Proteínas del Linfoma 3 de Células B , AMP Cíclico/metabolismo , Células Germinales Embrionarias/metabolismo , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Primordial germ cells (PGCs) are fate determined from pluripotent epiblasts. Signaling pathways and transcriptional regulators involved in PGC formation have been identified, but detailed molecular mechanisms of PGC fate determination remains poorly understood. Using RNAi screening, we identified histone deacetylase 3 (HDAC3) as a regulator of PGC formation. Hdac3 deficiency resulted in decreased nascent PGCs in vitro and in vivo, and somatic developmental genes were de-repressed by Hdac3 knockdown during PGC induction. We also demonstrated BLIMP1-dependent enrichment of HDAC3 and deacetylation of H3 and H4 histones in the somatic developmental genes in epiblast-like cells. In addition, the HDAC3/BLIMP1-targeted somatic gene products were enriched in PGC determinant genes; overexpression of these gene products in PGC-like cells in culture resulted in repression of PGC determinant genes. We propose that selective recruitment of HDAC3 to somatic genes by BLIMP1 and subsequent repression of these somatic genes are crucial for PGC fate determination.
Asunto(s)
Células Germinativas/metabolismo , Histona Desacetilasas/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Acetilación , Animales , Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasas/genética , Histonas/metabolismo , Ratones , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Spontaneous testicular teratoma develops from primordial germ cells (PGCs) in embryos; however, the molecular mechanisms underlying teratoma formation are not fully understood. Mutation of the dead-end 1 (Dnd1) gene, which encodes an RNA-binding protein, drastically enhances teratoma formation in the 129/Sv mouse strain. To elucidate the mechanism of Dnd1 mutation-induced teratoma formation, we focused on histone H3 lysine 27 (H3K27) trimethylation (me3), and found that the levels of H3K27me3 and its responsible methyltransferase, enhancer of zeste homolog 2 (Ezh2), were decreased in the teratoma-forming cells of Dnd1 mutant embryos. We also showed that Dnd1 suppressed miR-26a-mediated inhibition of Ezh2 expression, and that Dnd1 deficiency resulted in decreased H3K27me3 of a cell-cycle regulator gene, Ccnd1 In addition, Ezh2 expression or Ccnd1 deficiency repressed the reprogramming of PGCs into pluripotent stem cells, which mimicked the conversion of embryonic germ cells into teratoma-forming cells. These results revealed an epigenetic molecular linkage between Dnd1 and the suppression of testicular teratoma formation.
RESUMEN
Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.
Asunto(s)
Células Germinales Embrionarias/metabolismo , Células Madre Embrionarias/metabolismo , Metabolismo Energético/fisiología , Glucólisis/fisiología , Fosforilación Oxidativa , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Células Germinales Embrionarias/citología , Células Madre Embrionarias/citología , Regulación de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteoma/análisisRESUMEN
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 â¼60% 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.