Comment[ArrayExpressAccession] E-GEOD-32263 Public Release Date 2011-09-22 Investigation Title Effect of cryopreservation on rabbit late blastocyst transcriptome Comment[Submitted Name] Effect of cryopreservation on rabbit late blastocyst transcriptome Experiment Description Studies on embryo cryopreservation efficiency had been focused mainly on technical and embryo factors. While structural damages can be easily evaluated, physiological damages only can be estimated by analyzing their in vitro and in vivo development to later stages. In order to determine how cryopreservation process affect embryo pre-implantory development, a transcriptional microarray study has been performed comparing gene expression of 6 days old rabbit embryos, previously vitrified or frozen and transferred into recipients rabbit females, to their in vivo counterparts. For each experimental group, control, vitrified and frozen late blastocysts, total RNA was extracted from 3 pools of approximately 10 embryos and labeled with Cy3 or Cy5 dyes. Then, six competitive hybridizations were carried out including two dye-swaps to compensate dye-bias. A specifically microarray designed to study rabbit gene expression profiling, the Rabbit 44K oligonucleotide array (Agilent Technologies), was used in this study. Identification of differentially expressed transcripts from 6 day old blastocysts was achieved using the Limma algorithm, and functional annotation was performed by Blast2GO software. Compared to 6 day old in vivo derived embryos, viable vitrified embryos only present 3 differentially expressed genes, in contrast to frozen viable embryos with 24 genes upregulated and 46 genes downregulated. These results reveal that effects of cryopreservation still remain in late blastocyst pre-implantatory gene expression, and potential damage and alterations produced by vitrification and slow-freezing procedures are differentially resolved after 3 days of in vivo development. Transcriptional microarray study that compares gene expression of viable 6 day old rabbit embryos, previosly vitrified or frozen and tranferred into recipient rabbit females, to their in vivo counterparts. Experiment 1: Control embryos vs. Vitrified embryos and Experiment 2: Control embryos vs. Frozen embryos. Biological replicates used: 3 replicates for control embryos, 3 replicates for vitrified embryos and 3 replicates for frozen embryos. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Saenz-de-Juano Saenz-de-Juano Marco-Jiménez Vicente Person First Name M. Desemparats M F J Person Mid Initials D S Person Email masaede@upvnet.upv.es Person Affiliation Universidad Politécnica Valencia Person Phone Person Fax Person Address Animal Science, Universidad Politécnica Valencia, Camino de Vera s/n, Valencia, Valencia, Spain Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE32263-2 P-GSE32263-1 P-GSE32263-8 P-GSE32263-9 P-GSE32263-7 P-GSE32263-3 P-GSE32263-4 P-GSE32263-5 P-GSE32263-6 P-GSE32263-10 Protocol Description ID_REF =
VALUE = -INV_VALUE: normalized log10 ratio Cy3/Cy5
INV_VALUE = normalized log10 ratio Cy5/Cy3 ID_REF =
VALUE = normalized log10 ratio Cy5/Cy3 Scanned on an Agilent G2505B scanner. Images were quantified using Agilent Feature Extraction Software (version 10.10.1.1). Hibridization was done using Agilent Gene Expression Hybridization Kit, following manufacturer's instructions. After 17 hours at 65ºC on a , hybridised slides were washed sequential. Vitrification: The vitrifcation procedure was carried out in two steps at 20oC. In the frst step, embryos were placed for 2 min in a vitrifcation solution consisting of 12.5% (v/v) dimethyl sulphoxide (3.5 M DMSO, Sigma) and 12.5% (v/v) ethylene glycol (4.4 M EG, Sigma) in DPBS supplemented with 0.2% (w/v) of BSA. In the second step, embryos were suspended for 1 min in a solution of 20% (v/v) DMSO and 20% (v/v) EG in DPBS supplemented with 0.2% (w/v) of BSA. Then embryos suspended in vitrifcation medium were loaded into 0.25 ml plastic straws and two sections of DPBS were added at either end of each straw separated by air bubbles. Finally, straws were sealed and plunged directly into liquid nitrogen. Devitrifcation was performed by immersing the central and final sections of the straws in a water bath at 20oC for 10-15 sec. The vitrifcation medium was eliminated employing a solution of DPBS with 0.33 M sucrose for 5 min and a wash in a solution of DBPS for another 5 min. Slow-freezing: Embryos were placed successively for 5 min into three different solutions consisting, respectively, of 0.5M, 1M and 1.5M dimethyl sulfoxide (DMSO) in DPBS supplemented with BSA. Then, the embryos suspended in the cryopreservation medium were oaded into 0.125 mL sterile plastic straws, and submited to slow freezing 1oC/min up to -7oC. Five minutes later manual seeding were done, and then embryos were cooled at 0.3oC/min up to -35oC. After that, the straws were plunged directly into liquid nitrogen. Thawing was performed by placing the straws 10 sec in air at room temperature and then in a water bath at 20oC for 1 min. The cryopreservation medium was removed in three steps of 5 min, in which embryos were placed successively in three wash solutions consisting of 1 M, 0.5 M and 0M DMSO in DPBS supplemented with BSA. Total RNA extracted using Trizol following manufacturer's instructions Total RNA (100 ng) was labelled using QuickAmp Labelling Kit (Agilent Technologies) following manufacturer's instructions. Excess dye was removed with the RNAeasy Mini Kit (QIAGEN) and dye incorporation and concentration were determined using the microarray setting on the Nanodrop 1000. Agilent Feature Extraction Software (v 10.10.1.1) was used for background correction and LOWESS normalization. Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation bioassay_data_transformation image_aquisition image_aquisition hybridization specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction labeling feature_extraction Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name TREATMENT Experimental Factor Type treatment Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE32263 Comment[GEOLastUpdateDate] 2011-09-23 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2011-09-21 Comment[ArrayExpressSubmissionDate] 2011-09-19 SDRF File E-GEOD-32263.sdrf.txt