Investigation Title Transcription profiling of immature germinal vesicle stage oocytes with mature metaphase II oocytes to identify and characterize the changed and stable transcripts during mouse oocyte maturation. Comment[Submitted Name] Identification and characterization of the changed and stable transcripts during mouse oocyte maturation Experimental Design development_or_differentiation_design co-expression_design transcription profiling by array Experimental Design Term Source REF mo:1.3.1.1 mo EFO Comment[AEMIAMESCORE] 5 Comment[ArrayExpressReleaseDate] 2007-11-13 Comment[SecondaryAccession] GSE5668 Comment[SecondaryAccession] GDS2300 Comment[ArrayExpressAccession] E-GEOD-5668 Comment[MAGETAB TimeStamp_Version] 2010-08-06 17:02:58 Last Changed Rev: 13058 Experimental Factor Name DevelopmentalStage Experimental Factor Type developmental_stage Experimental Factor Term Source REF Person Last Name Su Person First Name You-Qiang Person Mid Initials Person Email youqiang.su@jax.org Person Phone Person Fax Person Address The Jackson Laboratory, 600 Main Street, Bar Harbor, 04609, USA Person Affiliation The Jackson Laboratory Person Roles submitter Person Roles Term Source REF Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2007-11-13 PubMed ID 17022963 Publication DOI 17022963 Publication Author List You-Qiang Su, Koji Sugiura, Yong Woo, Karen Wigglesworth, Sonya Kamdar, Jason Affourtit, John J Eppig Publication Title Selective degradation of transcripts during meiotic maturation of mouse oocytes. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description There is massive destruction of transcripts during maturation of mouse oocytes. The objective of this project was to identify and characterize the transcripts that are degraded versus those that are stable during the transcriptionally silent germinal vesicle (GV)-stage to metaphase II (MII)-stage transition using the microarray approach. A system for oocyte transcript amplification using both internal and 3'-poly(A) priming was utilized to minimize the impact of complex variations in transcript polyadenylation prevalent during this transition. Transcripts were identified and quantified using Affymetrix Mouse Genome 430 v2.0 GeneChip. The significantly changed and stable transcripts were analyzed using Ingenuity Pathways Analysis and GenMAPP/MAPPFinder to characterize the biological themes underlying global changes in oocyte transcripts during maturation. It was concluded that the destruction of transcripts during the GV to MII transition is a selective rather than promiscuous process in mouse oocytes. In general, transcripts involved in processes that are associated with meiotic arrest at the GV-stage and the progression of oocyte maturation, such as oxidative phosphorylation, energy production, and protein synthesis and metabolism, were dramatically degraded. In contrast, transcripts encoding participants in signaling pathways essential for maintaining the unique characteristics of the MII-arrested oocyte, such as those involved in protein kinase pathways, were the most prominent among those stables. Experiment Overall Design: Comparison immature GV-stage oocyte (3 biological replicates) with mature MII-stage oocytes (3 biological replicates) Protocol Name P-G5668-2 P-G5668-1 P-G5668-11 P-G5668-9 P-G5668-4 P-G5668-6 Affymetrix:Protocol:Hybridization-Unknown P-AFFY-6 Protocol Type specified_biomaterial_action specified_biomaterial_action grow grow nucleic_acid_extraction labeling hybridization feature_extraction Protocol Description Ovarian follicular development was stimulated by intraperitoneal injection of 5 IU of eCG (EMD Biosciences, Inc., Calbiochem, La Jolla, CA). Mice were then injected with hCG (5IU/mouse), and the oviducts were removed 14 h post hCG injection. Ovulated OCCs were released by gently teasing apart the ampulla of oviducts. Denuded oocytes were obtained by incubating the mass of OCCs in medium containing 1 mg/ml of hyaluronidase for about 3-5 min at 37 °C. Mature oocytes with a visible first polar body (hereafter termed MII oocytes) and no apparent sign of degeneration were collected. Both GV and MII oocytes were washed thoroughly in the collection medium to ensure that no cumulus cells were present. Ovarian follicular development was stimulated by intraperitoneal injection of 5 IU of eCG (EMD Biosciences, Inc., Calbiochem, La Jolla, CA). Immature GV-stage oocytes were collected at 44-46 h post eCG priming by puncturing large antral follicles on ovarian surface with a pair of 26 gauge needles. MII oocytes were washed thoroughly in the collection medium to ensure that no cumulus cells were present. Medium used for oocyte collection was the same as for collection of GV-oocytes Released oocyte-cumulus cell complexes (OCCs) were collected and cumulus cells surrounding the oocyte were removed completely by passing OCCs several times through a hand-drawn small fine glass pipette with an inner diameter slightly narrower than the oocyte. Only those oocytes with an intact GV and no apparent sign of degeneration were collected. Both GV and MII oocytes were washed thoroughly in the collection medium to ensure that no cumulus cells were present. Medium used for oocyte collection was MEM-α (Invitrogen Corporation, Grand Island, NY) supplemented with 3 mg/ml of crystallized lyophilized bovine serum albumin (Sigma, St. Louis, MO), 75 mg/liter of penicillin G (Sigma) and 50 mg/liter streptomycin sulfate (Sigma). Milrinone (Sigma), a selective inhibitor of oocyte-specific phosphodiesterase (PDE3), was added into the medium at the concentration of 5 μM to prevent the GV-stage oocytes from undergoing maturation. Total RNA was extracted from 300 oocytes using the Picopure RNA isolation Kit (Arcturus, Mountain View, CA). Total RNA was extracted from oocytes using the PicoPure RNA Isolation Kit according to the manufacturer?s instruction. The RNA quality and yield of each sample were determined using the Bioanalyzer 2100 and RNA 6000 Pico LabChip assay (Agilent Technologies Inc, Palo Alto, CA) in combination with Quant-iTTM RiboGreen Reagent according to supplied protocols (Invitrogen, Carlsbad, CA). Ten nanograms of total RNA from each sample was employed in the two-round cDNA synthesis and subsequent in vitro-transcription according to the Two-Cycle Eukaryotic Target Labeling Assay (Affymetrix Expression Analysis Technical Manual: ? Section 2: Eukaryotic Sample and Array Processing? (http://www.affymetrix.com/support/technical/manual/expression_manual.affx)) with the following modifications. For the first and second cycle of cDNA synthesis steps, Full Spectrum? MultiStart Primers for in vitro transcription (SystemBiosciences, Mountain View, CA) were used, instead of Affymetrix?s standard T7-Oligo(dT) primer or random hexamers. Title: Affymetrix Generic Hybridization. Description: Title: Affymetrix CEL analysis. Description: Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF mo SDRF File E-GEOD-5668.sdrf.txt Term Source Name EFO ncbitax The MGED Ontology ArrayExpress mo:1.3.1.1 mo EFO The MGED Ontology Term Source File http://www.ebi.ac.uk/efo/ http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version 1.3.1.1