Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_20_plus_E_070916.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_D_plus_E_070916.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_20_plus_E_070916.mzid_HF1_ZRO_20_plus_E_070916.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_20_plus_E_070916.mzid_HF1_ZRO_20_plus_E_070916.MGFftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_D_plus_E_070916.mzid_HF1_ZRO_D_plus_E_070916.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_D_plus_E_070916.mzid_HF1_ZRO_D_plus_E_070916.MGFftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_20_plus_E_070916.mzid.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_D_plus_E_070916.mzid.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/Bovine_UP_crap_2016_02.fastaftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_20_plus_E_070916.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/HF1_ZRO_D_plus_E_070916.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/05/PXD012092/Samplesnames.docxprimaryOK2000000z.roth@mail.huji.ac.ilMeital KupervaserMass SpectrometryShotgun proteomicsScaffold Scaffold_4.8.2Mascot 2.5.1MehpOocyte developmental competenceTranscriptomePhthalateGene expressionhttp://www.ebi.ac.uk/pride/archive/projects/PXD012092OocyteOocyte collection Bovine ovaries were collected at a local slaughterhouse from multiparous Holstein cows, and transported to the laboratory within 60 to 90 min in physiological saline solution (0.9% w/v NaCl at 38.5 °C with 50 µg/mL penicillin–streptomycin). Cumulus oocyte complexes (COCs) were aspirated from 3- to 8-mm follicles with an 18-gauge needle attached to a 10-mL syringe. COCs were collected into HEPES–Tyrode's lactate (HEPES–TL) supplemented with 0.3% (w/v) bovine serum albumin (BSA), 0.2 mM sodium pyruvate and 0.75 mg/mL gentamicin at 38.5 °C (HEPES–TALP). Then, COCs with at least three layers of cumulus surrounding a homogeneous cytoplasm were selected for in vitro maturation. Proteomic analysis was conducted at the Nancy and Stephen Grand Israel National Center for Personalized Medicine at the Weizmann Institute of Science in Rehovot, Israel. A sample included a total of 200 oocytes for each group (control and MEHP). These were lysed by dissolving in 8 M urea and 0.1 M Tris–HCl pH 7.9 on ice for 10 min. Lysate was cleared by centrifugation at 14,000g for 10 min at room temperature, Proteins were then reduced by incubation with 5 mM dithiothreitol for 1 h at room temperature, and alkylated with 10 mM iodoacetamide in the dark for 45 min. Samples were diluted to 2 M urea with 50 mM ammonium bicarbonate, and 10 µg total protein was then subjected to digestion with trypsin (Promega) overnight at 37 oC at a 50:1 protein-to-trypsin ratio (v/v), followed by a second trypsin digestion for 4 h. The digestions were stopped by addition of trifluoroacetic acid (1% v/v final concentration). Peptides were then desalted using Oasis HLB, μElution format (Waters, Milford, MA, USA). The samples were vacuum-dried and stored at -80 oC until further analysis. Liquid chromatography Ultraperformance liquid chromatography–mass spectrometry (LC–MS)-grade solvents from Sigma were used for all chromatographic steps, unless otherwise indicated. Each sample was loaded using a split-less nano-ultra performance liquid chromatograph (UPLC) (10 kpsi nanoAcquity; Waters). The mobile phase was (A) H2O + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase Symmetry C18 trapping column (180 µm internal diameter, 20 mm length, 5 µm particle size; Waters). The peptides were then separated using a T3 HSS nanocolumn (75 µm internal diameter, 250 mm length, 1.8 µm particle size; Waters) at 0.35 µL/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 20% B in 155 min, 20% to 90% B in 5 min, maintained at 90% B for 5 min and then back to initial conditions. Mass spectrometry The nano-UPLC was coupled online through a nano-ESI emitter (10 μm tip; New Objective, Woburn, MA, USA) to a quadrupole orbitrap mass spectrometer (Q Exactive HF, Thermo Fisher Scientific) using a FlexIon nanospray apparatus (Proxeon, Thermo Fisher Scientific). Data were acquired in data-dependent acquisition (DDA) mode, using a Top20 method. MS1 resolution was set to 120,000 (at 400 m/z), mass range of 300–1650 m/z, automatic gain control (AGC) of 3e6 and maximum injection time set to 20 ms. MS2 resolution was set to 60,000, quadrupole isolation 1.7 m/z, AGC of 1e6, dynamic exclusion of 60 s and maximum injection time, 60 ms.PrideNot availableCarbamidomethylOxidationDeamidatedRaw data were imported into Expressionist® software version 10.0.3 (Genedata) and processed as described at web address (https://www.genedata.com/products/expressionist/). The software was used for retention time alignment and peak detection of precursor peptides. A master peak list was generated for S/MS events and sent for database searching using Mascot v2.5.1 (Matrix Sciences). Data were searched against the bovine sequences from UniprotKB (http://www.uniprot.org/) appended with common laboratory contaminant proteins. Fixed modification was set to carbamidomethylation of cysteines and variable modifications were set to oxidation of methionines, deamidation of N or Q, and protein N-term acetylation. Search results were then filtered using the PeptodeProphet algorithm to achieve a maximum false discovery rate of 1% at the protein level (Keller et al. 2002). Peptide identifications were imported back to Expressionist to annotate identified peaks. Quantification of proteins from the peptide data was performed using an in-house script (Shalit et al. 2015). Data were normalized based on the total ion current. Protein abundance was obtained by summing the three most intense unique peptides per protein. Fold changes were calculated based on the ratio of the measured protein intensity in each of the samples. Proteins that were regarded as potentially differentially (expressed at fold change ±1.5 with at least 2 peptides were annotated using the DAVID tool.ProteomicsZvi RothQ ExactiveDepartment of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, the Hebrew University, Rehovot 76100, Israel Center of Excellence in Agriculture and Environmental Health, the Hebrew University, Rehovot 76100, IsraelCOMPLETEBos Taurus (bovine)31059758 Kalo D, Vitorino Carvalho A, Archilla C, Duranthon V, Moroldo M, Levin Y, Kupervaser M, Smith Y, Roth Z. Mono(2-ethylhexyl) phthalate (MEHP) induces transcriptomic alterations in oocytes and their derived blastocysts. Toxicology. 2019 10.1016/j.tox.2019.04.016meital.kupervaser@weizmann.ac.ilWeizmann Institute of ScienceIsrael10.6019/PXD012092Mono(2-ethylhexyl) phthalate (MEHP), the main di(2-ethylhexyl) phthalate (DEHP) metabolite, is a known reproductive toxicant. Residual levels of 20 nM MEHP have been found in follicular fluid aspirated from IVF-treated women and DEHP-treated animals. The current study examined whether these residual MEHP levels have any effect on the follicle-enclosed oocyte or developing embryo. Bovine oocytes were matured with or without 20 nM MEHP for 22 h. Microarray analysis was performed for both mature oocytes and 7-day blastocysts. A proteomic analysis was performed on mature oocytes (n = 200/group) to reveal a possible direct effect on the oocyte proteomic profile. Transcriptome analysis revealed MEHP-induced alterations in the expression of 456 and 290 genes in oocytes and blastocysts, respectively. The differentially expressed genes are known to be involved in various biological pathways, such as transcription process, cytoskeleton regulation and metabolic pathway. Among these, the expression of 9 genes was impaired in both oocytes exposed to MEHP (i.e., direct effect) and blastocysts developed from those oocytes (i.e., carryover effect). In addition, 191 proteins were found to be affected by MEHP in mature oocytes (Data are available via ProteomeXchange with identifier PXD012092). The study explores, for the first time, the risk associated with exposing oocytes to low concentration (i.e., environmentally relevant concentration) of MEHP to the maternal transcripts. Although it was the oocytes that were exposed to MEHP, alterations carried over to the blastocyst stage, following embryonic genome activation, implying that these embryos are of low quality.Mono(2-ethylhexyl) phthalate (MEHP) induces transcriptomic alterations in oocytes and their derived blastocysts.Kalo D D, Vitorino Carvalho A A, Archilla C C, Duranthon V V, Moroldo M M, Levin Y Y, Kupervaser M M, Smith Y Y, Roth Z Z2-ethylhexyl hydrogen phthalate, 2-Ethylhexyl phthalate, 2-ethylhexyl ester, 2-([(2-ethylhexyl)oxy]carbonyl)benzoic acid, mono(2-ethylhexyl)phthalate, MEHP, Oocyte., mono-(2-ethylhexyl)phthalate, monoethylhexyl phthalate, 2-(2-ethylhexyloxycarbonyl)benzoic acid, 1, (2-ethylhexyl) hydrogen phthalate, mono(2-ethylhexyl) ester, Ovocytes, mono-2-ethylhexyl phthalate, 2-benzenedicarboxylic acid, phthalic acid, Ovocytedairy cow, Bru, IPP2A2, Raw, AW549739, AI461847, Laboratory, False, Ass-1, Addresses, Gene, protein, Computer, precursor, protein-containing complex, PHAPII, 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7-pentadeoxy-6-(methylamino)heptopyranoside, Caspase-1 subunit p10, subdivided, cobalt (+2) salt, Cytoplasms, anon-53Fa, Liquid Chromatography, Hallermann-Streiff syndrome, Longterm Effect, l(2)SH0173, tip, ANAC091, Spectrum Analysis, not genetically inherited, DmelCG7788, ACMICD, Medical Speciality, Putative MAPK-activating protein PM10, H2O, common salt, sodium (4:1:1) salt, Dm1, magnesium salt, HSS, wrapping, Sizes, TIP60, CG6829, forked, dark/hac-1/dapaf-1, Acid, divided, HLA-DR-associated protein II, gonada of genitalia of female organism, DI-2, I-2Dm, proportionality, formate, oogonium, gonad of reproductive system of female organism, Spectrometry, gentamycins, rate, cromium (+3), b-lactate, HARP, inhibitor of granzyme A-activated DNase, lysed material, septate, beta Trypsin, I-2PP1, TAF-IBETA, Slaughterhouses, HPO2, HPO1, Drice, ammonium (4:1) salt, (2-ethylhexyl) hydrogen phthalate, TAF-Ibeta, methyl cyanide, bovine, DRICE, lead (+2) salt, tlp, STARS, TIP41, Old astrocyte specifically-induced substance, Cleland Reagent, Particle Sizes, MTG10_1, nickel formate dihydrate, lead formate, Effects, fractured, DrIce, DrICE, aluminum salt, 4-dimercapto-, Carbamide, mono-2-ethylhexyl phthalate, HLHS1, R*)-, protein-containing complex, thomson, CG7826, ESA1, agua, mass-to-charge ratio, period, mono(2-ethylhexyl)phthalate, Medical Specialities, isolation, Oasis, CG7835, Gene Products, CG42273, ODDD, Carmol, 2-benzenedicarboxylic acid, Medical, Cesium Trifluoroacetate, proportion, thallium (+1) salt, Longterm, ARF1-BP, Slaughter Houses, PCE-2, beta-Trypsin, rubidium salt, 2pp2a, Sciences, Long-Term, methanoic acid, 6-diamino-3-[3-deoxy-4-C-methyl-3-(methylamino)pentopyranosyloxy]-2-hydroxycyclohexyl 2-amino-2, ESI, ORESARA 14, CG10574, CD156, Injectable, Hallermann Streiff Francois syndrome, drugs, DmelCG4579, ms(2)zk, monoethylhexyl phthalate, 2PP2A, Caspase-1 subunit p20, Kochsalz, dSET, dSet, peptidos, Insurance, female organism genitalia gonad, 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dihydridooxygen, domestic cow, mono(2-ethylhexyl) ester, Medicines, dApaf1, dihydrogen oxide, GPHYSD2, Slaughter House, ammonium (2:1) salt, Long-Term Effects, Hallermann Streiff syndrome, Oocyte, ratio, phapii, Specialties, rock salt, ice, iCE, D-Apaf-1, enclosing, drice, gonada of reproductive system of female organism, gonad of female organism reproductive system, ZC2HC5, aqua, Dmel_CG7826, DFNB38, StF-IT-1, Th, HTATIP1, fragmented, ur, Spectroscopy, carbamide, C20orf48, Cleland, Ovaries, drIce, drICE, Specialty, laboratory, nickel (+2) salt, female reproductive system gonad, Dmel_CG7835, Acetic acid, Slaughter, Mnb, MNB, ammonium salt, MS/MS, cracked, P45, Trifluoroacetic, hydrogen hydroxide, cobaltous formate, 3-Butanediol, hac1, Hallermann's syndrome, CG4299, MASS, AW124434, natrii chloridum, acqua, dapaf-1S, Insurance Medicines, nanospray, H2NC(O)NH2, apaf-1, dapaf-1L, female organism genitalia gonada, Cesium, 2-(2-ethylhexyloxycarbonyl)benzoic acid, Acetamide, copper salt, p45, 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calcium salt, TCV-interacting protein, Peptid, assay, AA407739, groupe, dAPAF-1, sodium chlorideprojections, 2-Ethylhexyl phthalate, dairy cow, type 1, Preimplantation Embryo, Materials, Microtrabecular, determination, plant germ, non Brugada type, PNT-P1, DmelCG17077, metazoans, Gene Expression Profile, Ovocytes, EY3-1, Pnt, Profiles, protein, paroxysmal familial ventricular fibrillation (disorder), Ovocyte, Long Term, CDCD2, dysfunctional, Relative, protein polypeptide chains, primary metabolites, paroxysmal familial, png, Cytoskeletons, Nanoarray Analytical Devices, 1, antral fluid, Analysis, D-ets-2, 3520, protein aggregate, Effect, Antral Fluid, Microtrabecular Lattice, Follicular, Pnt-P1, Ovarian Follicle, Animalia, blastocystis, ART, me75, PFHB1, Cytoplasmic Filament, Genomes, Cytoskeletal, Di(2-ethylhexyl)phthalate, Bos bovis, Phthalic acid bis(2-ethylhexyl) ester, Microchips, ovary follicle fluid, Analytical Device, proteins, Signatures, D17Mit170, Microarray Analytical 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Ets94F, oogonium, Filaments, blastula, ICCD, lacks function of type, exposed, projection, ridge, low functionality, ptd, PntP2, paroxysmal ventricular fibrillation, hyaline, Microarray Microchips, 2-(2-ethylhexyloxycarbonyl)benzoic acid, liquor follicularis, Material, spine, PntP1, bacterial transcription, metabolites, E(E2F)3D, ovarian follicular fluid, (2-ethylhexyl) hydrogen phthalate, Cistron, PNTP2, bovine, PNTP1, time, Gruppe, 0998/12, transcription, Lattices, Microarray Analytical, Nanoarray, Transcriptome Profile, Effects, ovary follicular fluid, lamellae, Cytoplasmic Filaments, Nanoarray Analytical Device, ETS2, Liquor Folliculi, Ets2, developmental stage, bis(2-ethylhexyl) benzene-1, secondary metabolites, Gene, impaired, mono-2-ethylhexyl phthalate, CMD1E, protein-containing complex, animals, process of organ, skin follicle, protrusion, ovarian follicle, lamella, period, mono(2-ethylhexyl)phthalate, ventricular fibrillation during myocardial infarction, Filament, DMPOINT1A, pntegfr, mono-(2-ethylhexyl)phthalate, 0608/07, Nanoarray Analytical, polypeptide chain, Bis(2-ethylhexyl) o-phthalate, cattle, Girls, Cytoskeletal Filaments, Dioctyl phthalate, HH1, Gene Products, Low, Animal, Microarray Analytical Devices, 2-benzenedicarboxylic acid, Microchip, Di(ethylhexyl) phthalate, study, Microarray Microchip, Genetic, pointed-RC, Longterm, Profile, dysfunction, cutaneous appendage follicle, primitive ectoderm, DEHP, ridges, Long-Term, EK3-2, Analytical Devices, Cytoplasmic, ventricular fibrillation, l(3)07825, Follicular Fluids, extruding from, monoethylhexyl phthalate, embryonic organism, grupos, T1., Nav1.5, CG17077, Long-Term Effect, bis(2-ethylhexyl)phthalate, laminae, l(3)j1B7, Bis(2-ethylhexyl)phthalate, VF1, Di-sec-octyl phthalate, cellular transcription, Ets, Transcriptome, grupo, cou, Liquor, cow, Women's Group, Microarray, protein complex, anatomical process, exits through, Women Groups, Proteins, Preimplantation, ectoblast, Cistrons, Woman, group, Device, Lr, Fluid, Di(2-ethylhexyl) o-phthalate, native protein, natural protein, Blastocysts, Bos primigenius taurus, Preimplantation Embryos, Embryos, Metazoa, Gene Expression Signatures, chemical analysis, Protein, Long Term Effects, idiopathic ventricular fibrillation, domestic cattle, Gene Expression Signature, blastocyst, pnt-P1, pnt-P2, phthalic acid, l(3)s118306, flange, organ process, Relative Risk, CMPD2, Embryonic, 2-dicarboxylate, Embryo, Women's Groups, Risks, Rest, metabolite, DNA-templated, Longterm Effects, Protein Gene Products, 2-ethylhexyl hydrogen phthalate, multicellular animals, 2-ethylhexyl ester, processes, process, Gene Proteins, having decreased function, Ets58AB, developmental tissue, clear, ox, Gene Expression Profiles, Lattice, Bra, processus, Phthalic acid di(2-ethylhexyl) ester, regulation, assay, Octyl phthalate, Signature, epiblastus, groupe, 2-Benzenedicarboxylic acid bis(2-ethylhexyl) ester, POINT, CG87050.00.00.00.00.00falseTranscriptomic alterations induced by physiologically relevant level of mono(2-ethylhexyl) phthalate (MEHP) in oocytes and blastocystsMono(2-ethylhexyl) phthalate (MEHP), the main di(2-ethylhexyl) phthalate (DEHP) metabolite, is a known reproductive toxicant. Residual levels of 20 nM MEHP have been found in follicular fluid aspirated from IVF-treated women and DEHP-treated animals. It is not yet clear whether these residual MEHP levels have any effect on the follicle-enclosed oocyte or developing embryo. To clarify this point, bovine oocytes were matured with or without 20 nM MEHP for 22 h. Microarray analysis was performed for both mature oocytes and 7-day blastocysts. A feasibility examination was performed on mature oocytes (n = 200/group) to reveal a possible direct effect on the oocyte proteomic profile. Transcriptome analysis revealed MEHP-induced alterations in the expression of 456 and 290 genes in oocytes and blastocysts, respectively. The differentially expressed genes are known to be involved in various biological pathways, such as transcription process, cytoskeleton regulation and metabolic pathway. Among these, the expression of 9 genes was impaired in both oocytes exposed to MEHP (i.e., direct effect) and blastocysts developed from those oocytes (i.e., carryover effect). In addition, 191 proteins were found to be affected by MEHP in mature oocytes. The study explores, for the first time, the risk associated with exposing oocytes to physiologically relevant MEHP concentrations to the maternal transcripts. Although it was the oocytes that were exposed to MEHP, alterations carried over to the blastocyst stage, following embryonic genome activation, implying that these embryos are of low quality.2019-05-092018-12-18PXD012092445441736309100906989361604889986388067825355548840194565795596157959704487574721567631351107616996345771216979623910022669157463604232135622135588334747146479297601011714594399131639101165691569313076118259040818761383803942725631459537227243230883937021000589NCBITaxon:26279343052848125541773355511935019903463475155972936329979662871882295476709111510458002317447628256228736031148173623185962982548961015109823126469049323994611676544404NCBITaxon:96152607101280321857327626326931281183657599838307972237561749200319710659222612607052607078853189606455862652831059758