RESUMO
Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Drosophila melanogaster , Desenvolvimento Embrionário , Animais , Masculino , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Cromatina/metabolismo , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas Embrionárias/metabolismo , Células Germinativas Embrionárias/citologia , Células Germinativas/metabolismo , Epigênese Genética , Feminino , Nucleossomos/metabolismo , Nucleossomos/genética , Análise de Célula Única/métodosRESUMO
BMP4 is the critical gene of primordial germ cell formation in mammal, however, the mechanism of PGCs formation in chicken still unknown. In this research, we compared the evolution relationship of different species. Although the protein sequence is highly conservative between mouse, human and chicken, promotors vary among avian and mammal species. Therefore, it is easily to predict that there would be different regulation mechanism of Bmp4 expression in chicken. Here, we elucidate the function of chicken Bmp4 during PGCs formation. In vivo, Bmp4 can promote PGCs development and migration, and increase the expression of key genes (Cvh, c-kit, cxcr4, etc.). Whereas, the expression of these genes will decrease after knocking out Bmp4. After over-expression and knockout Bmp4 in vitro, we found that overexpression of Bmp4 could promote the formation of embryoid bodies (EB) and up-regulate the key genes of PGCs formation and migration, while knockout Bmp4 could inhibit the formation of embryoid bodies and decrease the expression of related genes. Flow and indirect immunofluorescence also indicated the same result. These all results proved that chicken Bmp4 could also promote the formation of PGCs. Furthermore, dual-luciferase activity detection showed that the promotor activity of Bmp4 was positively regulated by transcription factor Zeb1. Overexpression of Zeb1 can also increase the mRNA and protein expression of Bmp4. At the same time, DNA methylation inhibited Bmp4 transcription and histone methylation was able to promote its transcription. In conclusion, this study established that chicken Bmp4 can promote the formation of chicken PGCs. This gene is regulated by DNA, histone methylation and transcription factor Zeb1. These results lay a theoretical foundation for exploring the function and molecular mechanism of Bmp4 in the process of PGCs formation.
Assuntos
Proteína Morfogenética Óssea 4/genética , Células Germinativas Embrionárias/citologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Animais , Proteína Morfogenética Óssea 4/metabolismo , Movimento Celular , Células Cultivadas , Galinhas , Metilação de DNA , Corpos Embrioides/metabolismo , Células Germinativas Embrionárias/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de GenesRESUMO
The Tre1 G-protein coupled receptor (GPCR) was discovered to be required for Drosophila germ cell (GC) coalescence almost two decades ago, yet the molecular events both upstream and downstream of Tre1 activation remain poorly understood. To gain insight into these events, we describe a bona fide null allele and both untagged and tagged versions of Tre1. We find that the primary defect with complete Tre1 loss is the failure of GCs to properly navigate, with GC mis-migration occurring from early stages. We find that Tre1 localizes with F-actin at the migration front, along with PI(4,5)P2; dPIP5K, an enzyme that generates PI(4,5)P2; and dWIP, a protein that binds activated Wiskott-Aldrich syndrome protein (WASP), which stimulates F-actin polymerization. We show that Tre1 is required for polarized accumulation of F-actin, PI(4,5)P2, and dPIP5K. Smoothened also localizes with F-actin at the migration front, and Hh, through Smo, increases levels of Tre1 at the plasma membrane and Tre1's association with dPIP5K.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas Embrionárias/metabolismo , Proteínas Hedgehog/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Citoesqueleto de Actina/genética , Animais , Animais Geneticamente Modificados , Movimento Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Proteínas Hedgehog/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Fatores de Tempo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
BACKGROUND: Patients suffering from the BCR-ABL1-negative myeloproliferative disease prefibrotic primary myelofibrosis (pre-PMF) have a certain risk for progression to myelofibrosis. Accurate risk estimation for this fibrotic progression is of prognostic importance and clinically relevant. Commonly applied risk scores are based on clinical, cytogenetic, and genetic data but do not include epigenetic modifications. Therefore, we evaluated the assessment of genome-wide DNA methylation patterns for their ability to predict fibrotic progression in PMF patients. RESULTS: For this purpose, the DNA methylation profile was analyzed genome-wide in a training set of 22 bone marrow trephines from patients with either fibrotic progression (n = 12) or stable disease over several years (n = 10) using the 850 k EPIC array from Illumina. The DNA methylation classifier constructed from this data set was validated in an independently measured test set of additional 11 bone marrow trephines (7 with stable disease, 4 with fibrotic progress). Hierarchical clustering of methylation ß-values and linear discriminant classification yielded very good discrimination between both patient groups. By gene ontology analysis, the most differentially methylated CpG sites are primarily associated with genes involved in cell-cell and cell-matrix interactions. CONCLUSIONS: In conclusion, we could show that genome-wide DNA methylation profiling of bone marrow trephines is feasible under routine diagnostic conditions and, more importantly, is able to predict fibrotic progression in pre-fibrotic primary myelofibrosis with high accuracy.
Assuntos
Impressões Digitais de DNA/métodos , Fibrose/genética , Estudo de Associação Genômica Ampla/métodos , Mielofibrose Primária/genética , Experimentação Animal , Medula Óssea/metabolismo , Competição entre as Células/genética , Técnicas de Reprogramação Celular/métodos , Ilhas de CpG/genética , Metilação de DNA , Progressão da Doença , Células Germinativas Embrionárias/metabolismo , Epigenômica/métodos , Feminino , Fibrose/patologia , Proteínas de Fusão bcr-abl/genética , Ontologia Genética , Humanos , Masculino , Valor Preditivo dos Testes , Mielofibrose Primária/patologia , Prognóstico , Fatores de RiscoRESUMO
Knockout CRISPR screening enables the unbiased discovery of genes with a functional role in almost any cellular or molecular process of interest. The approach couples a genome-scale library of guide RNA (gRNA), the Cas9 endonuclease, and a faithful phenotypic read-out to systematically identify candidate genes via their loss-of-function effect. Here we provide a detailed description of the CRISPR screen protocol and outline how to apply it to decipher the gene networks that underlie developmental cell fate decisions. As a paradigm we use the in vitro model of cell state transition(s) from naive pluripotency to primordial germ cell (PGC) fate, exploiting the Stella-GFP:Esg1-tdTomato (SGET) mouse ESC line. The principles in this protocol can be readily adapted to characterize lineage regulators for other cell fate models and/or for other species.
Assuntos
Sistemas CRISPR-Cas , Células Germinativas Embrionárias/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Diferenciação Celular , Linhagem Celular , Células Germinativas Embrionárias/metabolismo , Redes Reguladoras de Genes , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , RNA Guia de Cinetoplastídeos/genética , Transdução GenéticaRESUMO
Cleavage under targets and release using nuclease (CUT&RUN) allows the chromatin profiling of proteins of interest for which specific antibodies are available. Because it is performed on intact chromatin in situ, CUT&RUN provides exceptional signal over background, making it an ideal choice for chromatin profiling on primary cells available at limited numbers. Here, we describe its application to the profiling of histone post-translational modifications in germ cells isolated from mouse embryos from 12.5 up to 18.5 days postfertilization. This approach can be applied to as low as 100 isolated germ cells, allowing the generation of multiple genome-wide profiles from the cells obtained from a single embryo.
Assuntos
Cromatina/genética , Células Germinativas Embrionárias/metabolismo , Código das Histonas , Camundongos/genética , Animais , Separação Celular/métodos , Células Cultivadas , Células Germinativas Embrionárias/citologia , Biblioteca Gênica , Camundongos/embriologia , Camundongos Transgênicos , Processamento de Proteína Pós-TraducionalRESUMO
Arginine methylation is one of the most important post-translational modifications which is catalyzed by protein arginine methyltransferases (PRMTs). Previous studies have demonstrated that Prmt5 plays important role in germ cell development. Prmt7 is the only family member responsible for mono-methylation of arginine residue. However, whether Prmt7 is also involved in germ cell development remains unclear. In this study, we find that PRMT7 is abundantly expressed in the male germ cells during embryonic stage (from E10.5). Depletion of Prmt7 results in the defect of germ cell proliferation during embryonic stage and the number of primordial germ cells is significantly reduced in Prmt7-/- mice at E11.5. We also find that the size of testes is reduced in Prmt7-/- mice at P5 with reduced germ cell number and the diameter of seminiferous tubules. Further study reveals that the expression of BMPs and TGF-ß singling pathway is significantly changed in germ cells of Prmt7-/- mice at E12.5. However, no defect of testes development is observed in adult Prmt7-/flox; Mvh-Cre mice. Collectively, this study demonstrates that Prmt7 plays roles in male germ cell proliferation during embryonic stages and it is not required for germ cell development postnatally.
Assuntos
Células Germinativas Embrionárias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Células Germinativas Embrionárias/citologia , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Histonas/metabolismo , Masculino , Metilação , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteína-Arginina N-Metiltransferases/deficiência , Proteína-Arginina N-Metiltransferases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Testículo/citologia , Testículo/embriologia , Fator de Crescimento Transformador beta/genéticaRESUMO
PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked-in (gene-trapped) PGCs into the blood vessels of Hamburger-Hamilton stages (HH-stages) 13-16 chicken embryos. Gene-trapped chickens were established by crossing a chimeric chicken with a wild-type hen with very high efficiency. Heterozygous gene-trapped chickens grew normally and SSEA-1-positive cells expressed eGFP during HH-stages 13-30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene-trapped embryos obtained by crossing heterozygous gene-trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.
Assuntos
Células Germinativas Embrionárias/metabolismo , Marcação de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Células Cultivadas , Embrião de Galinha , Proteínas de Fluorescência Verde/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransgenesRESUMO
Being infected by SARS-CoV-2 may cause damage to multiple organs in patients, such as the lung, liver and heart. Angiotensin-converting enzyme 2 (ACE2), reported as a SARS-CoV-2 receptor, is also expressed in human male testes. This suggests a potential risk in human male reproductive system. However, the characteristics of ACE2-positive cells and the expression of other SARS-CoV-2 process-related genes are still worthy of further investigation. Here, we performed singlecell RNA seq (scRNA-seq) analysis on 853 male embryo primordial germ cells (PGCs) and 2,854 normal testis cells to assess the effects of the SARS-CoV-2 virus on the male reproductive system from embryonic stage to adulthood. We also collected and constructed the scRNA-seq library on 228 Sertoli cells from three non-obstructive azoospermia (NOA) patients to assess the effects at disease state. We found that ACE2 expressing cells existed in almost all testis cell types and Sertoli cells had highest expression level and positive cells ratio. Moreover, ACE2 was also expressed in human male PGCs. In adulthood, the level of ACE2 expression decreased with the increase of age. We also found that ACE2 positive cells had high expressions of stress response and immune activation-related genes. Interestingly, some potential SARS-CoV-2 process-related genes such as TMPRSS2, BSG, CTSL and CTSB had different expression patterns in the same cell type. Furthermore, ACE2 expression level in NOA donors' Sertoli cells was significantly decreased. Our work would help to assess the risk of SARS-CoV-2 infection in the male reproductive system.
Assuntos
Azoospermia/genética , Betacoronavirus/patogenicidade , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral , Testículo/metabolismo , Testículo/virologia , Adulto , Enzima de Conversão de Angiotensina 2 , Azoospermia/complicações , Azoospermia/metabolismo , Betacoronavirus/metabolismo , COVID-19 , Infecções por Coronavirus/complicações , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Células Germinativas Embrionárias/metabolismo , Células Germinativas Embrionárias/virologia , Expressão Gênica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Pneumonia Viral/fisiopatologia , Pneumonia Viral/virologia , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2 , Células de Sertoli/metabolismo , Células de Sertoli/virologia , Análise de Célula Única , Espermatogênese/genética , Espermatogênese/fisiologia , Testículo/citologiaRESUMO
DNA methylation is a major silencing mechanism of transposable elements (TEs). Here we report that TEX15, a testis-specific protein, is required for TE silencing. TEX15 is expressed in embryonic germ cells and functions during genome-wide epigenetic reprogramming. The Tex15 mutant exhibits DNA hypomethylation in TEs at a level similar to Mili and Dnmt3c but not Miwi2 mutants. TEX15 is associated with MILI in testis. As loss of Tex15 causes TE desilencing with intact piRNA production, our results identify TEX15 as a new essential epigenetic regulator that may function as a nuclear effector of MILI to silence TEs by DNA methylation.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Elementos de DNA Transponíveis/genética , Inativação Gênica/fisiologia , Células Germinativas/metabolismo , Animais , Metilação de DNA , Células Germinativas Embrionárias/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , MutaçãoRESUMO
Spontaneous testicular teratomas (STTs) derived from primordial germ cells (PGCs) in the mouse embryonic testes predominantly develop in the 129 family inbred strain. Ter (spontaneous mutation) is a single nucleotide polymorphism that generates a premature stop codon of Dead end1 (Dnd1) and increases the incidence of STTs in the 129 genetic background. We previously found that DND1 interacts with NANOS2 or NANOS3 and that these complexes play a vital role in male embryonic germ cells and adult spermatogonia. However, the following are unclear: (a) whether DND1 works with NANOS2 or NANOS3 to regulate teratoma incidence, and (b) whether Ter simply causes Dnd1 loss or produces a short mutant DND1 protein. In the current study, we newly established a conventional Dnd1-knockout mouse line and found that these mice showed phenotypes similar to those of Ter mutant mice in spermatogenesis, oogenesis, and teratoma incidence, with a slight difference in spermiogenesis. In addition, we found that the amount of DND1 in Dnd1+/Ter embryos decreased to half of that in wild-type embryos, while the expression of the short mutant DND1 was not detected. We also found that double mutants for Dnd1 and Nanos2 or Nanos3 showed synergistic increase in the incidence of STTs. These data support the idea that Ter causes Dnd1 loss, leading to an increase in STT incidence, and that DND1 acts with NANOS2 and NANOS3 to regulate the development of teratoma from PGCs in the 129 genetic background. Thus, our results clarify the role of Dnd1 in the development of STTs and provide a novel insight into its pathogenic mechanism.
Assuntos
Células Germinativas Embrionárias/patologia , Proteínas de Neoplasias/fisiologia , Proteínas de Ligação a RNA/metabolismo , Teratoma/etiologia , Neoplasias Testiculares/etiologia , Testículo/patologia , Animais , Células Germinativas Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Oogênese , Proteínas de Ligação a RNA/genética , Espermatogênese , Teratoma/metabolismo , Teratoma/patologia , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Testículo/metabolismoRESUMO
Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1-mCherry fusion protein under the control of the Oct4ΔPE embryonic germ cell-specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA DSB-inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose- and time-dependent manner. The dynamic time-sensitive and dose-sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells.
Assuntos
Dano ao DNA , Células Germinativas Embrionárias/metabolismo , Genes Reporter , Engenharia Genética/métodos , Animais , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Feminino , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: In order to prepare the genome for gametogenesis, primordial germ cells (PGCs) undergo extensive epigenetic reprogramming during migration toward the gonads in mammalian embryos. This includes changes on a genome-wide scale and additionally in females the remodeling of the inactive X-chromosome to enable X-chromosome reactivation (XCR). However, if global remodeling and X-chromosomal remodeling are related, how they occur in PGCs in vivo in relation to their migration progress and which factors are important are unknown. RESULTS: Here we identify the germ cell determinant PR-domain containing protein 14 (PRDM14) as the first known factor that is instrumental for both global reprogramming and X-chromosomal reprogramming in migrating mouse PGCs. We find that global upregulation of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark is PRDM14 dosage dependent in PGCs of both sexes. When focusing on XCR, we observed that PRDM14 is required for removal of H3K27me3 from the inactive X-chromosome, which, in contrast to global upregulation, takes place progressively along the PGC migration path. Furthermore, we show that global and X-chromosomal reprogramming of H3K27me3 are functionally separable, despite their common regulation by PRDM14. CONCLUSIONS: In summary, here we provide new insight and spatiotemporal resolution to the progression and regulation of epigenome remodeling along mouse PGC migration in vivo and link epigenetic reprogramming to its developmental context.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Germinativas Embrionárias/metabolismo , Histonas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Cromossomo X/metabolismo , Animais , Movimento Celular/fisiologia , Reprogramação Celular , Metilação de DNA , Proteínas de Ligação a DNA/genética , Células Germinativas Embrionárias/citologia , Epigênese Genética , Feminino , Histonas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Cromossomo X/genética , Inativação do Cromossomo XRESUMO
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.
Assuntos
Reprogramação Celular/fisiologia , Ciclina D1/metabolismo , Células Germinativas Embrionárias/citologia , Células Germinativas Embrionárias/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fase G1 , Fase G2 , Histonas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Ativação Enzimática , Fase G1/fisiologia , Fase G2/fisiologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Transdução de SinaisRESUMO
During the sex differentiation, the primordial germ cells (PGCs) pass through a differentiation, becoming spermatogonial cells in males and oocytes in females. In this phase, there is difference in gene expression and differentiation potency between males and females. Specific cell markers have been essential in the PGC meiosis beginning and become oocyte cells. However, there are few studies about germline in domestic animals. The domestic dog (Canis lupus familiaris) is an interesting animal model to be used in the investigation about the mammal development because it has several biochemical and physiological similarities to humans. In addition, some additional investigations about dogs may contribute to a better understanding of the biology and genetic components, improving clinical veterinary and zoological sciences. Here, we elucidated by immunofluorescence and quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), the dynamics of the expression of pluripotent (POU5F1 and NANOG) and germline (DDX4, DAZL and DPPA3) markers that are very important in the development of female canine germ cells during 35-50 days post-fertilization (dpf). The female canine germ cells were positive for pluripotent markers during middle developmental period. The number of DDX4, DAZL and DPPA3 cells increased along the germ cell maturation from 45 to 50 dpf. We provided an expression analysis of the pluripotent and germline markers in paraffin sections using the middle and later periods in female canine germ cells. The results can contribute the understanding about the timeline of each marker along the maturation of female canine germ cells. These results have a great significance to demonstrate the germ cell profile changes because it may allow the development of protocols about in vitro germ cell derivation.
Assuntos
Cães/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Animais , Diferenciação Celular/genética , RNA Helicases DEAD-box/genética , Células Germinativas Embrionárias/citologia , Células Germinativas Embrionárias/metabolismo , Feminino , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Oócitos/citologia , Ovário/citologia , Ovário/embriologia , Proteínas de Ligação a RNA/genéticaRESUMO
Germline stem cells are germ cells at an early developmental stage, so their development is key to ensuring human reproduction. There is increasing evidence that long noncoding RNA (lncRNA) and circular RNA (circRNA) play important roles in the development of germ cells. This data descriptor provides unique lncRNA and circRNA transcriptomic information for mouse germline stem cells. Using the Illumina HiSeqx 2000 system, a total of 511,836,732 raw reads were generated. High-quality transcripts, lncRNAs, and circRNAs were identificated and quantified using the reads, and more precise annotations of lncRNAs (especially 9357 novel lncRNAs) and circRNAs were performed in the germline stem cells. We then analyzed the transcript structures, genetic variants, and the interaction between circRNA and microRNA to provide the basis for subsequent functional experiments. This comprehensive dataset will help advance data sharing and deepen our understanding of mouse germline stem cells, providing a theoretical foundation for research on germ cell development and human reproduction, among others.
Assuntos
Células Germinativas Embrionárias , Células Germinativas , RNA Longo não Codificante , RNA , Animais , Células Germinativas Embrionárias/citologia , Células Germinativas Embrionárias/metabolismo , Genoma , Células Germinativas/citologia , Células Germinativas/metabolismo , Camundongos , RNA CircularRESUMO
Primordial germ cells (PGCs), the precursors of gametes, are the only cells capable of acquiring totipotency upon fertilization, but the molecular mechanisms regulating germ cell characteristics have not been fully elucidated. Although intracellular metabolic status and regulation are responsible for the control of cell function and differentiation, little is known about the metabolic features of PGCs. Here, we describe use of an integrated metabolomic, proteomic, and energy metabolic analysis method to comprehensively elucidate the metabolic characteristics of PGCs using mass spectrometry.
Assuntos
Células Germinativas Embrionárias/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Proteoma/metabolismo , Proteômica/métodos , Animais , Cromatografia Líquida , Células Germinativas Embrionárias/citologia , Células Germinativas Embrionárias/efeitos dos fármacos , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Proteoma/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
Dexamethasone (Dx) is often used in obstetric practice to promote fetal lung maturation and to prevent respiratory distress syndrome when the risk of preterm delivery persists. This therapy enables survival of the newborn, but also is associated with deleterious effects on the offspring, such as reproductive disorders. The aim of this study was to determine specifically whether prenatal exposure to Dx disturbs the physiological balance between proliferation and apoptosis of germinative cells (GC) in the ovary of 19- and 21-day-old fetuses and thus induces developmental programming of the female reproductive system. Pregnant Wistar rats (n = 10/group), separated into control (vehicle) and Dx-treated (0.5 mg/kg body mass) groups, received injections on gestational days 16, 17, and 18. Exposure to Dx lowered the volume of the fetal ovary by 30% (P < 0.05) in 21-day-old fetuses, as well as the total number of GC in the ovary by 21% (P < 0.05). When compared to the controls, in Dx-exposed fetuses, the total number of PCNA-positive GC was 27% lower at 19 days and 71% lower at 21 days old (P < 0.05), while total numbers of caspase-3-positive GC were 2.3-fold and 34% higher, respectively (P < 0.05). Our results demonstrate that prenatal exposure to Dx diminished proliferation but increased the rate of germinative cell apoptosis, with consequently reduced total germinative cell number and ovary volume. Impairment of fetal oogenesis and fewer GC in the fetal ovary compromise the oogonial stock and thus may constitute a risk of female fertility.
Assuntos
Dexametasona/toxicidade , Células Germinativas Embrionárias/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Ovário/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Germinativas Embrionárias/metabolismo , Feminino , Idade Gestacional , Ovário/embriologia , Ovário/metabolismo , Óvulo/metabolismo , Gravidez , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos WistarRESUMO
Inheritance (sequestration of a localized determinant: germplasm) and zygotic induction are two modes of metazoan primordial germ cell (PGC) specification. vasa and nanos homologs are evolutionarily conserved germline marker genes that have been used to examine the ontogeny of germ cells in various animals. In the lepidopteran insect Bombyx mori, although the lack of vasa homolog (BmVLG) protein localization as well as microscopic observation suggested the lack of germplasm, classical embryo manipulation studies and the localization pattern of Bm-nosO (one of the four nanos genes in Bombyx) maternal mRNA in the egg raised the possibility that an inheritance mode is operating in Bombyx. Here, we generated Bm-nosO knockouts to examine whether the localized mRNA acts as a localized germ cell determinant. Contrary to our expectations, Bm-nosO knockout lines could be established. However, these lines frequently produced abnormal eggs, which failed to hatch, to various extent depending on the individuals. We also found that Bm-nosO positively regulated BmVLG expression at least during embryonic stage, directly or indirectly, indicating that these genes were on the same developmental pathway for germ cell formation in Bombyx. These results suggest that these conserved genes are concerned with stable germ cell production. On the other hand, from the aspect of BmVLG as a PGC marker, we showed that maternal Bm-nosO product(s) as well as early zygotic Bm-nosO activity were redundantly involved in PGC specification; elimination of both maternal and zygotic gene activities (as in knockout lines) resulted in the apparent lack of PGCs, indicating that an inheritance mechanism indeed operates in Bombyx. This, however, together with the fact that germ cells are produced at all in Bm-nosO knockout lines, also suggests the possibility that, in Bombyx, not only this inheritance mechanism but also an inductive mechanism acts in concert to form germ cells or that loss of early PGCs are compensated for by germline regeneration: mechanisms that could enable the evolution of preformation. Thus, Bombyx could serve as an important organism in understanding the evolution of germ cell formation mechanisms; transition between preformation and inductive modes.
Assuntos
Bombyx/embriologia , Bombyx/fisiologia , Animais , Padronização Corporal/fisiologia , RNA Helicases DEAD-box/metabolismo , Embrião não Mamífero/metabolismo , Células Germinativas Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes , Proteínas de Insetos/genética , Oogênese/genética , RNA Mensageiro/metabolismoRESUMO
The germ line is a crucial cell lineage that is distinct from somatic cells, and solely responsible for the trans-generational transmission of hereditary information in metazoan sexual reproduction. Primordial germ cells (PGCs)-the precursors to functional germ cells-are among the first cell types to be allocated in embryonic development, and this lineage commitment is a critical event in partitioning germ line and somatic tissues. Classically, mammalian PGC development has been largely informed by investigations on mouse embryos and embryonic stem cells. Recent findings from corresponding nonrodent systems, however, have indicated that murine PGC specification may not be fully archetypal. In this review, we outline the current understanding of molecular mechanisms in PGC specification, emphasizing key transcriptional events, and focus on salient differences between early human and mouse PGC commitment. Beyond these latest findings, we also contemplate the future outlook of inquiries in this field, highlighting the importance of comprehensively understanding early fate decisions that underlie the segregation of this unique lineage. This article is categorized under: Developmental Biology > Stem Cell Biology and Regeneration Biological Mechanisms > Cell Fates Physiology > Mammalian Physiology in Health and Disease.