MeRIP sequencing analysis of female germline stem cells (FGSCs)
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ABSTRACT:
PROVIDER: PRJEB77835 | ENA |
REPOSITORIES: ENA
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Project description:Alternative polyadenylation (APA) is an important post-transcriptional modification implicated in development. Female germline stem cell (FGSC) is unipotent and capable of giving rise to oocyte. However, whether alternative polyadenylation plays a role in self-renew and cell fate determination of FGSCs remain elusive. Here, we used 3T-Seq developed in our lab to profile genome-wide 3a termini of transcripts and delineate APA sites in mouse FGSCs and explored the biological significance of APA modulation in FGSC identity.
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Project description:Next-Generation Sequencing of Usp7 Overexpression and Knockdown Female Germline Stem Cells (FGSCs) Transcriptomes
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Project description:Female germline stem cells (FGSCs) are adult stem cells capable of self-renewal and differentiation into mature oocytes. AKT3, a member of the AKT kinase family, plays crucial roles in multiple cellular processes, such as proliferation, migration, and apoptosis. However, the mechanism by which AKT3 affects the development of FGSCs is poorly understood. We performed liquid chromatography (LC)-mass spectrometry (MS) on mouse FGSCs in which AKT3 was knocked down using a lentivirus and on control FGSCs to investigate how AKT affects the development of FGSCs. Based on the raw LC-MS data and database searches and data filtering, we identified 46,260 peptides, including 45,821 unique peptides, corresponding to 6849 identified proteins and 6697 comparable proteins. These identified proteins were functionally annotated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Protein Domain, Clusters of Orthologous Genes (COG)/Eukaryotic Orthologous Groups (KOG), STRING database, Reactome, WikiPathways, HallMark, and transcription factor (TF) analyses. Fisher’s exact test was used to assess the significance of functional enrichment of the differentially abundant proteins. We identified 281 differentially abundant proteins between AKT3 knockdown and control FGSCs, comprising 229 upregulated and 52 downregulated proteins. We performed clustering analysis on these differentially abundant proteins based on functional enrichment using GO, Domain, KEGG, Reactome and WikiPathways platforms. A protein–protein interaction network was constructed to demonstrate interactions between proteins. These datasets will facilitate future investigations into the mechanisms governing FGSC self-renewal and differentiation and will provide a foundation for understanding diseases related to abnormal germ cell development.
2024-08-15 | PXD054663 | Pride
Project description:Germ stem cells are the only stem cells which can produce gametes in vivo. Recently, accumulating evidence demonstrates the isolation and culture of female germline stem cells (FGSCs) from adult mice and humans. Here, we established FGSCs line from single EGFP transgenetic mouse, and traced their differentiation in vivo. Then we compared the follile development from transplanted FGSCs mice (F-TF) with that from wide type mice (F-WT) by RNA-Seq. qRT–PCR validation was performed using SYBR Green assays. Using Illumina HiSeq2500 sequencer, we generated 51.5 Gb of sequencing data from the 8 samples. On average, we detected expression of 20 966 transcripts. Under the criteria fold change > 2 or < 0.5, we obtained 7005 and 5764 differential expressed genes (DEGs) from F-TF or F-WT,respectively. Weighted correlation network analysis (WGCNA) analysis identified core genes and two potential networks during folliculogenesis which were mainly down-regulated after preantral follicle. This study was the first to analyze the development of transplanted FGSCs progressively and would provide the theoretical basis for clinical translational research of FGSCs.
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Project description:We used the microarray analysis to compare the globe gene expression profiles of female germ line stem cells (FGSCs) and spermatogonial stem cells (SSCs).
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Project description:Next-generation sequencing (NGS) was performed to analyze the usp7 effects on FGSCs.
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Project description:Female germline stem cells (FGSCs) have been successfully isolated from postnatal mammalian and human ovarian tissues. However, the effects and mechanisms of action of natural small-molecule compounds on FGSCs are largely unknown. Here, we determined the effects of daidzein, a phytoestrogen present in soybean , on cell viability and proliferation using Cell Counting Kit-8 (CCK8) and 5-ethynyl-2-deoxyuridine (EdU). We found that daidzein promoted the viability and proliferation of FGSCs in a concentration-dependent manner. To elucidate the mechanism underlying this, we performed RNA-Seq in daidzein-treated FGSCs and controls. The results showed that there were 153 upregulated and 156 downregulated genes after daidzein treatment. We confirmed the expression of some genes related to cell proliferation in the sequencing results by RT-PCR, such as Type C lectin domain family 11 member a (Clec11a), Mucin1 (Muc1), Glutathione peroxidase 3 (Gpx3), and Tet methylcytosine dioxygenase 1(Tet1). The high expression of Clec11a at the protein level after daidzein treatment was also confirmed by western blotting. Furthermore, recombinant mouse Clec11a (rmClec11a) protein was shown to promote the viability and proliferation of FGSCs. However, knockdown of Clec11a inhibited the viability and proliferation of FGSCs, which could not be rescued by the administration of daidzein. These results indicate that daidzein promoted the viability and proliferation of FGSCs through Clec11a. In addition, both daidzein and rmClec11a activated the Akt signaling pathway in FGSCs. However, Clec11a knockdown inhibited this pathway, which could not be rescued by daidzein administration. Taken together, our findings revealed that daidzein activates the Akt signaling pathway to promote cell viability and proliferation through upregulating Clec11a. This study should deepen our understanding of the developmental mechanism of FGSCs and female infertility.
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Project description:ObjectivesExistence of germline stem cells (GSCs) in juvenile mammalian female ovaries has been drastically debated recently since reports that adult mouse ovaries still have mitotically active germ cells have been proposed. In addition, definitive location of such female germline stem cells (FGSCs) had not been demonstrated.Materials and methodsWe segregated porcine FGSCs mechanically from ovary cortex, and tested our hypotheses by utilizing immunofluorescent staining, qRT-PCR and western blotting.ResultsWe attached emphasis to unambiguous location of FGSCs, which settle simultaneously in the theca. Dissected cells from porcine thecal layers maintained similar characteristics to mouse FGSCs and ESCs over 4-months in vitro culture.ConclusionThese results may provide a new resource for the study of oogenesis and therapy for ovarian sterility.
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Project description:Activation of JAK-STAT3 signaling by leukemia inhibitory factor (LIF) is required for maintaining self-renewal of mouse embryonic stem cells (mESCs). STAT3 perform cell type-specific roles in different cell type, here we revisit the role of STAT3 using mouse female germ stem cell (mFGSCs). We applied CRISPR/Cas9 system to generate Stat3 knockout FGSCs and then observed cell growth inhibition and cell cycle arrest in KO cell line. By combining genome wide ChIP-Seq and RNA-Seq, we identified 5990 STAT3 binding sites and discovered serval genes specific regulated by STAT3 that were involved in stem cell proliferation and female gonad development in FGSCs. In general, we identify key roles of STAT3 for sustains self-renewal and proliferation for FGSCs in this study.
2017-12-20 | E-MTAB-6163 | biostudies-arrayexpress