Intercellular bridges coordinate the transition from pluripotency to meiosis in mouse fetal oocytes
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PROVIDER: PRJNA699296 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: Investigate the transcriptomes of Tex14 heterogygous sample (with intercellular bridges intact) compared to Tex14 homozygous mutant (lacking intracellular bridges) to ascertain what effects the loss of cytoplasmic sharing has on mitotic to meiotic mouse ovary Methods: Single cell RNA sequencing libaries were created from embryonic day 13.5 mouse ovaries from Tex14 +/- and Tex14 -/- samples. Three Tex14+/- and two Tex14-/- E13.5 ovaries were pooled. Libaries were creating using 10X Chromium Single Cell 3' (v2 Chemistry) library kits per 10X posted protocols. Samples were sequenced on HiSeq 4000 (Illumina) with paired-end sequencing parameters: Read1, 98 cycles; Index1, 14 cycles; Index2, 8 cycles; and Read2, 10 cycles. Tex14+/- sample had 23,567 mean reads per cell, and Tex14-/- had 19,735 mean reads per cell. Since 10X Genomics recommends an average of 60k raw reads per cell, we submitted our two libaries for deeper sequencing. Our second sequencing run was done on a Novaseq S2 flowcell with the following parameters: Read 1 Length 26bp; Index 1 Length 8bp; Index 2 Length 0bp; Read 2 Length: 98 bp. Follwing deeper sequencing, Tex14+/- sample had 55,744 mean reads per cell, and Tex14-/- had 41,117 mean reads per cell. Cells which passed quality control metrics were further analyzed. Results: We used CellRanger v2.1.0 to align the sequenced data to the 10X Genomics pre-built mm10 genome. We mapped about 300,000,000 sequence reads for each shallowly sequenced library, and 750-800,000,000 reads for the deeply sequenced libraries. We used Seurat v3 to keep high quality cells, excluding cells which did not all of the following metrics: genes expressed in at least 3 cells, fewer than 25,000 unique fragments (to exclude cell doublets), greater than 200 genes expressed (to exclude GEMS with no cells), and cells with 7.0% or less mitochondrial gene expression. After quality control exclusion, cell counts for the gene by cell matrices are as follows: tex14 het shallow, 12260 cells; tex14 ko shallow, 14715 cells; tex14 het deep, 11794 cells; tex14 ko deep, 13982 cells. Conclusions: Our study represents the first single cell RNA sequencing experiment conducted on fetal mouse ovaries lacking intracellular bridges. We observed that the absence of Tex14 did not alter the transcriptional composition of ovarian somatic cells, but specifically attenuated the level of pluripotency-associated transcripts as germ cells enter meiosis.
2021-04-06 | GSE166121 | GEO
Project description:Meiosis is critical to generating oocytes however, the mechanisms regulating the switch from mitotic primordial germ cells (PGCs) to meiotic germ cells are poorly understood in females. We showed that the onset of meiosis in the fetal murine ovary propagates via intercellular bridges between developing germ cells by using Tex14 mutant (Tex14-/-) mice. Tex14-/- germ cells, which lack intercellular bridges, initiates meiosis prematurely and they more rapidly extinguish pluripotency-associated transcripts (such as Dppa3) upon entering meiosis. Considering the involvement of Dppa3 in methylation process as well as the importance of epigenetic reprogramming for gamete formation and meiosis, we isolated Tex14 wild-type, heterozygous and mutant germ cells from E13.0 and E13.5 ovaries for whole genome bisulfite sequencing. We compared how methylation differs between age-matched wild-type/heterozygous controls and mutants. We find that DNA methylation across the genome is not affected in Tex14 mutant germ cells however, promoters of Germline Reprogramming Responsive (GRR) genes and younger L1 genes are hypomethylated. These results suggest that cytoplasmic sharing via intercellular bridges slows and coordinates the cell state transition from pluripotency to meiosis potentially by diluting regulatory factors of epigenetic reprogramming.
2021-04-06 | GSE165010 | GEO
Project description:Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in sea star oocytes from prophase I through the first embryonic cleavage. Although protein levels are broadly stable, dynamic waves of phosphorylation underlie each meiotic stage. We find that the phosphatase PP2A-B55 is reactivated at the Meiosis I/II transition resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI / MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after meiosis I. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.
2020-08-14 | MSV000085949 | MassIVE
Project description:RNA-Seq can comprehensively and quickly obtain almost all transcripts and information of specific cells in rustic states.Here,we compared up-regulated genes in GV and MII oocytes from LSM14b deficient mice and wild-type mice by RNA-Seq,highlighting the important role of LSM14b in meiosis of mouse oocytes.
2023-12-01 | GSE198414 | GEO
Project description:Metastasis is a major cause of mortality, and remains a final frontier in the search for a cure for cancer. While there has been much research on the ‘seed’ (metastatic tumor cells) and the ‘soil’ (colonized host tissue), interactions between metastatic cancer cells and stromal endothelial cells, which occur at multiple stages during metastasis, are less well understood. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for intercellular communication in vitro and in vivo, including horizontal transfer of microRNAs (miRNA). The communication between the tumor cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of “metastatic hijack”: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in syngeneic- and human xenograft-breast cancer models. Targeting the formation of these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer. A miRNA microarray was used to evaluate the transport of endogenous microRNAs. The intercellular transfer-ve and intercellular transfer+ve samples were sorted from the same endothelial cell population with the only difference being the occurrence of intercellular transport. The heat map shows potential miRNA candidates for exogenous transfer on two independent biological replicates. These miRNA candidates were significantly up-regulated in the cells receiving transfer of intercellular contents. HUVECs that were not exposed to cancer cells were used as a baseline control.
2015-09-04 | E-GEOD-72679 | biostudies-arrayexpress
Project description:Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization, and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 down-regulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a novel meiotic and embryonic competence factor in meiosis and mitosis, safeguarding genome integrity at the onset of life. We performed expression profiling on pools of 16 denuded GV-oocytes isolated per mouse. We used oocytes from 4 Setdb1 f/+; Zp3-cre mice and 2 Setdb1 f/- mice as controls and oocytes from 4 Setdb1 f/-; Zp3-cre mice as mutant.
2016-06-27 | E-GEOD-82002 | biostudies-arrayexpress
Project description:Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization, and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 down-regulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a novel meiotic and embryonic competence factor in meiosis and mitosis, safeguarding genome integrity at the onset of life.
2016-06-27 | GSE82002 | GEO
Project description:Human meiosis in oocytes entails a fine regulation of the transcriptome to support late oocyte growth and early embryo development, both key to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic passages, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 oocytes at different meiotic maturation stages, both in vivo ovulated and matured in vitro. We found abundant UTR processing, mostly at the 3’-, of meiosis-related genes between the stages of GV and MII, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very little differences among GV oocytes across several lengths of in vitro maturation, as long as they did not reach MII, suggesting a specific association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, though specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3’-UTR shortening, drives a selective translational regulation of transcripts necessary to reach the final meiotic maturation.
2023-05-27 | GSE213267 | GEO
Project description:Maternal RNAs are stored from minutes to decades in oocytes throughout meiotic arrest. The nature and dynamics of maternal RNAs during this arrest remain uncharted. We address this by performing single-oocyte RNA sequencing on arrested and maturing C. elegans oocytes. We discovered a population of transcripts that increases as the arrested meiosis I oocyte ages, but ruled out ERK signaling and nascent transcription as a mechanism for this increase. Instead, we report extracellular communication from neighboring somatic cells as a mechanism for the increase in transcripts during meiosis I arrest. These analyses provide a single-cell resolution of the RNA landscape during the developmental history of a meiosis I arrested oocyte and as it prepares for oocyte maturation and embryonic progression.
2023-05-24 | GSE209988 | GEO
Project description:In vertebrates, sexual reproduction depends on the precisely temporally controlled translation of mRNAs stockpiled in the oocyte. The RNA-binding proteins Zar1 and Zar2 have been implicated in translational control in oocytes of several vertebrate species, but how they act mechanistically is still incompletely understood. Here, we investigate the function of Zar1l, a so far uncharacterized member of the Zar protein family, in Xenopus laevis oocytes. By combining biochemical assays and mass spectrometry, we reveal that Zar1l is a constituent of a known large ribonucleoparticle containing the translation repressor 4E-T and the central polyadenylation regulator CPEB1. Employing TRIM-Away, we show that depletion of Zar1l from prophase-I arrested oocytes results in premature meiotic maturation upon hormone treatment. We provide evidence that this is based on the precocious expression of the kinase cMos, a key promotor of meiotic resumption. Based on our data, we propose a model according to which degradation of Zar1l results in dissociation of 4E-T from CPEB1, thus weakening translation inhibition imposed by the mRNA 3’UTR of cMos and probably also other M-phase promoting regulators. Thus, our detailed characterization of the function of Zar1l reveals novel mechanistic insights into the regulation of maternal mRNA translation during vertebrate meiosis.
2022-09-22 | PXD032170 | Pride