RNA-seq of paired granulosa cells and morulas or blastocysts from naturally and superovulated 3M old mice
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000, NextSeq 2000
ORGANISM(S): Mus musculus
SUBMITTER: Angela Gonçalves
PROVIDER: E-MTAB-13480 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Two major factors contributing to reduced fertility is use of exogenous hormones and old age. We use mouse model to study transcriptional and cell-cell communication changes upon superovulation and ageing in female reproductive cells - oocytes (OC) - and somatic cells - granulosa (GC) - surrounding them. Here, we are validating the results obtained by a polyA-biased method. Oocytes from naturally and superovulated mice were collected as follows: mice were sacrificed by cervical dislocation and oviducts dissected. Ampullas were torn to release the COCs into a 96 µl M2 media drop under mineral oil at room temperature. Then, 4ul of pre-heated 500 µg/ml hyaluronidase diluted in M2 media (final concentration in the drop 20 µg/ml) was added to separate the COCs into single units. The COCs were incubated for 10-20 min at 37oC and then mechanically separated into individual M2 drops using 115-124 μm glass retransfer pipette. Individual COCs were then washed in M2 once and incubated for less than 5 min with enzymatic mix Accutase at 37oC to further separate granulosa cells from the oocytes. Oocytes were washed twice with M2 media and once with DPBS before collection. Cells were immediately flash frozen in liquid nitrogen in individual 0.2ml thin wall PCR tubes and stored in -80oC until library preparation. SMARTer Stranded Total RNA - Low Input Mammalian kit was used for lysis, cDNA amplification and library preparation with 10 cycles for PCR 1 and 16 cycles for PCR 2. Samples were sequenced as recommended by the manufacturer on Novaseq600 with paired-end sequencing.
2024-09-22 | E-MTAB-13474 | biostudies-arrayexpress
Project description:Two major factors contributing to reduced fertility is use of exogenous hormones and old age. In previous experiments, we used mouse model to study transcriptional and cell-cell communication changes upon superovulation and ageing in female reproductive cells - oocytes (OC) - and somatic cells - granulosa (GC) - surrounding them. Here, we use granulosa cell samples from human patients undergoing in vitro fertilization to test how aged hormonally stimulated cell transcriptional profiles correspond to ones in mice. Total RNA was extracted from granulosa cell samples, collected during IVF procedures, using TRIzol. Further, RNA was cleaned from DNA with DNA-free™ DNA Removal Kit. ~1 µg of RNA was used to construct libraries with Truseq Stranded mRNA kit and barcoded with IDT for Illumina-TruSeq DNA and RNA UD Indexes. Samples were sequenced on Nextseq200 (Illumina) with 100bp paired-end sequencing.
2024-09-22 | E-MTAB-13496 | biostudies-arrayexpress
Project description:With the exception of imprinted genes and certain repeats, DNA methylation is globally erased during pre-implantation development. Recent studies have suggested that Tet3-mediated oxidation of 5-methylcytosine (5mC) and DNA replication-dependent dilution both contribute to global paternal DNA demethylation, but demethylation of the maternal genome occurs via replication. Here we present genome-scale DNA methylation maps for both the paternal and maternal genomes of Tet3-depleted and/or DNA replication-inhibited zygotes. In both genomes, we found that inhibition of DNA replication blocks DNA demethylation independently from Tet3 function, and that Tet3 facilitates DNA demethylation by coupling with DNA replication. For both, our data indicate that replication-dependent dilution is the major contributor to demethylation, but Tet3 plays an important role, particularly at certain loci. Our study therefore both defines the respective functions of Tet3 and DNA replication in paternal DNA demethylation and reveals an unexpected contribution of Tet3 to demethylation of the maternal genome. In this data set, we include RNA-Seq data of mouse 2-cell embryos and blastocysts derived from both wildtype and Tet3-null oocytes
2014-10-21 | E-GEOD-61329 | biostudies-arrayexpress
Project description:In mammals, the capacity of the female germ cell, the oocyte, to develop into embryo is acquired throughout meiotic maturation. Immature oocyte cannot be fertilized while mature oocyte is apt to accept spermatozoa and to develop an embryo. In a follicle, the oocyte is surrounded by mural granulosa cells (GC) and is physically and metabolically coupled with specialized granulosa cumulus cells (CC) which play an important role in oocyte maturation and fertilization. Factors expressing in GC and CC during maturation may reflect the oocyte quality, i.e. its capacity to be fertilized and assure early embryo development. However, the modifications of the content and the amount of peptide/proteins in the oocyte and the surrounding CC during oocyte maturation are mostly unknown and so there is not an accurate way of evaluating/monitoring how different in vitro maturation (IVM) protocols being in use in assisted reproduction technologies, can affect the process. In this context, Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) was applied to bovine follicular cells (bovine single oocytes, cumulus cells and granulosa cells) in order to characterize proteomic changes that occur in the follicle during female gamete development. In order to characterize finely endogenous molecular species observed on ICM-MS profiles and to identify markers of interest with their post-translational modifications, we carried out top-down proteomic on the different follicular cells from oocyte, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts. Prior to top-down MS using a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos, depending on the amount of available biological material, we employed three analytic strategies as a direct infusion, a mono-dimensional liquid chromatography (µLC-1D-MS/MS) and an off-line multi-dimensional liquid chromatography combining four fractionations (based on reverse phase or gel filtration LC) to µLC-MS/MS. Here, we deposited our dataset from µLC-1D-MS/MS (analyses of oocytes, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts) and MDLC-MS/MS (analyses of granulosa cells protein extract).
2017-04-10 | PXD004892 | Pride
Project description:Human granulosa cells (GCs), obtained during medical reproductive procedures can be cultured and are a cellular model for the human ovary. Oxygen concentrations are regarded as important regulators of GCs. We examined consequences of low (1%) O2 versus standard atmospheric conditions in cultured human GCs for four days. A proteomic analysis of three pools of human GCs was performed. While differences between the pools of GCs were noted, the abundance of 133 proteins was significantly increased in hypoxia in all samples, whereas the abundance of 391 proteins was decreased.
2023-12-20 | PXD042934 | Pride
Project description:Separate transcription profiling of oocytes and granulosa cells for each follicle stage: primordial (PD), primary (PM), secondary (SC) follicles and the small antral stage (SA) obtained by Laser Capture Microdissection (LCM) and RNAseq. The purpose of this study was to describe global gene expression during early ovarian folliculogenesis for each follicular compartment, to identify differential and specific gene expression between the 2 follicular compartments and during follicular development, to investigate specific function and pathways and to explore bi-directional communication between oocytes and GC.
2014-01-29 | E-MTAB-1587 | biostudies-arrayexpress
Project description:Oocytes and granulosa cells were collected after natural and superovulation in young (3 month-old) and old (12 month-old) mice while conserving pairing information between oocyte and granulosa cells from each cumulus-oocyte-complex (COC). This experimental design allows for the analysis of the effects of ageing and / or superovulation on the transcriptome of COCs. The naming convention for samples is as follows: [ovulation, NO/SO][age, 3M/12M][cell, OC/GC][COC number], meaning that NO12MOC01 and NO12MGC01 are from the same COC. Oocyte and granulosa cell samples were processed for full-length cytoplasmic RNA amplification using slightly modified SMART-seq2 protocol. After amplification of cDNA and library preparation, samples were sequenced on NextSeq 550 using Single-Ended 75bp High-Output kit.
2024-09-22 | E-MTAB-13479 | biostudies-arrayexpress
Project description:Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5.
2010-06-13 | E-MEXP-2457 | biostudies-arrayexpress
Project description:Goal of the experiment: To examine differential gene expression in granulosa cells of women undergoing ovarian stimulation with either recombinant human follicle stimulating hormone or purified human menopausal gonadotropin for in vitro fertilization. Brief description of the experiment: The study was designed to test the hypothesis that human granulosa cell (GC) gene expression response differs between pure recombinant FSH and human menopausal gonadotropin (hMG) stimulation regimens. Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Total RNA was extracted and stored at -70C. Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays). Unnamed genes and genes with <2-fold difference in expression were excluded from further analysis. After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11), bone morphogenic protein receptor II (BMPR2), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-4, IGFBP-5, basigin, and hypoxia-inducible factor (HIF)-1 alpha. The results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence. Experimental design: Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Total RNA was extracted and stored at -70C. Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays Quality control steps: The cRNA that was synthesized from the granulosa cells from individual patients was used for hybridization to a single CodeLink (Applied Microarrays) Whole Human Genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: Granulosa cells from women < 35 years-old undergoing in vitro fertilization. Manipulations of biological samples and protocols used: Female patients were treated with gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, granulosa cells were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Experimental factor: treatment with recombinant FSH versus purified human menopausal gonadotropin Technical protocols: Granulosa cells were collected into RNAlater (Ambion) and stored at -80C until processed. The granulosa cells were pelleted by centrifugation to remove the RNAlater. Each cell pellet was homogenized in TRI reagent (Molecular Research Centers). Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the manufacturer's Instruction Protocol (www1.amershambiosciences.com, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation). The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise. 0.285 micrograms of total granulosa cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen). The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and once in 0.05% Tween 20 for 5 sec. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
2010-03-22 | E-GEOD-16523 | biostudies-arrayexpress
Project description:Four groups of human oocytes from in vitro fertilization programme were analysed using microarrays: 1.) oocytes matured in vivo (retrieved as mature by ultrasound follicular aspiration but not fertilized after in vitro fertilization), 2.) oocytes matured in vitro in a conventional way (in a maturation medium with added gonadotropins FSH and HCG), 3.) oocytes matured in vitro by a new approach (in a maturation medium with added gonadotropins FSH and HCG, and in a co-culture with cumulus cells from mature oocytes of the same patient), and 4.) not matured oocytes which did not mature in spite of in vitro maturation procedure. Each group of oocytes consisted of three biological replicates with 10 oocytes per replicate therefore 12 oocyte samples and altogether 120 human oocytes were analyzed by microarrays.
2017-03-05 | E-MTAB-5400 | biostudies-arrayexpress