Comment[ArrayExpressAccession] E-GEOD-10367 Public Release Date 2008-02-08 Investigation Title Gene expression data throughout the normal lifespan of rhesus macaque corpora lutea during natural menstrual cycles. Comment[Submitted Name] Gene expression data throughout the normal lifespan of rhesus macaque corpora lutea during natural menstrual cycles. Experiment Description The molecular and cellular processes required for development, function, and regression of the primate corpus luteum (CL) are poorly defined. We hypothesized that there are dynamic changes in gene expression occurring during the CL lifespan, which represent proteins and pathways critical to its regulation. Therefore, a genomic approach was utilized to systematically identify differentially expressed genes in the rhesus macaque CL during the luteal phase of natural menstrual cycles. CL were collected between days 3-5 (early stage), 7-8 (mid), 10-12 (mid-late), 14-16 (late), or 18-19 (very-late) after the midcycle LH surge. From the early through very-late stages, 3234 transcripts were differentially expressed, with 879 occurring from the early through late stages that encompass the processes of luteinization, maintenance, and functional regression. To characterize gene changes most relevant to these processes, ontology analysis was performed using the list of 879 differentially expressed transcripts. Four main groups of related genes were identified with relevance to luteal physiology including: 1) immune function; 2) hormone and growth factor signaling; 3) steroidogenesis; and 4) prostaglandin biosynthesis, metabolism, and signaling. A subset of genes representing each of the four major categories was selected for validation of microarray results by quantitative real-time PCR. Results in mRNA levels were similar between the two methodologies for 17 of 18 genes. Additionally, protein levels for 3 genes were determined by Western blot analysis to parallel mRNA levels. This database will facilitate the identification of many novel or previously underappreciated pathways that regulate the structure and function of the primate CL. Keywords: time course The sole experimental factor is the stage of the luteal phase when the CL was collected. There were five stages: 1) between days 3-5 post LH-surge (early stage), 2) 7-8 (mid stage), 3) 10-12 (mid-late stage), 4) 14-16 (late stage), and 5) 18-19 (very-late stage). The early CL are undergoing luteinization, the mid are fully functional CL that are at their peak progesterone producing capacity, the mid-late stage is a transitionary period where CL are still producing significant quantities of progesterone but are nearing the time when luteolysis initiates in non-conception cycles, the late stage corresponds to CL undergoing functional regression (cessation of progesterone secretion), and the very-late stage (menses) is when the structural remodeling and apoptosis associated with luteolysis is occurring. Measuring mRNA levels in CL collected during these periods provides a comprehensive analysis of the primate transcriptome throughout CL lifespan. There were 20 biological replicates: n = 4 per stage, 5 stages in total. The early CL were used as the baseline reference for gene expression. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Bogan Bogan Murphy Stouffer Hennebold Person First Name Randy Randy Melinda Richard Jon Person Mid Initials Lloyd L J L D Person Email boganr@ohsu.edu Person Affiliation Oregon National Primate Research Center Person Phone (503)614-3751 Person Fax (503)690-5563 Person Address Reproductive Sciences, Oregon National Primate Research Center, 505 NW 185th Ave, Beaverton, OR, USA 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-GSE10367-1 P-GSE10367-6 P-GSE10367-5 P-GSE10367-2 P-GSE10367-3 P-GSE10367-4 P-GSE10367-7 Protocol Description ID_REF =
VALUE = Quantification GeneChips were scanned using the GeneChip Scanner 3000 with the 7G upgrade (Affymetrix) and GCOS version 1.4.0 software (Affymetrix), yielding cell fluorescence intensity (.CEL files). Image inspection was performed manually immediately following each scan. Labeled target is fragmented at 95C in the presence of high magnesium concentration. The fragmented material is combined with biotinylated hybridization control oligomer and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Ten micrograms of target is hybridized for 16 hours at 45C to the GeneChip Rhesus Macaque Genome array (Affymetrix), followed by washing, staining with streptavidin-phycoerythrin (Molecular Probes), signal amplification with biotinylated anti-streptavidin antibody (Vector Labs), and a final staining step on the Fluidics Station 450 (Affymetrix). Blood samples were collected daily by saphenous venipuncture starting six days after onset of menses. Serum was separated and samples were assayed daily for estradiol concentrations. Following the midcycle estradiol surge, the first day of low serum estradiol (< 100pg/ml) corresponds with the day after the LH surge (LH surge = day 0). On the appropriate day following the midcycle LH surge, the CL was collected during aseptic midline laparotomy, immediately flash frozen in liquid nitrogen, and stored at -80°C until total RNA was extracted. Trizol (Invitrogen) extraction of total RNA was performed according to the manufacturer's instructions, and total RNA obtained by Trizol extraction was further purified using RNeasy spin columns (Qiagen) following manufacturer instructions. Messenger RNA is amplified and labeled from 2 micrograms of total RNA in two steps according to the standard Affymetrix one-cycle cDNA protocol (Expression Analysis Technical Manual Rev.4). In the first step, mRNA is converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix). In the second step, amplified and labeled cRNA (the target) is produced in an in vitro transcription reaction using T7 RNA polymerase and biotin-UTP (Affymetrix). Following removal of free nucleotides, target yield is measured by UV260 absorbance. The processed image files (.CEL) were normalized across arrays using the robust multichip average (RMA) algorithm and log-transformed (base 2).After normalization, GeneSifter (VizX Labs) microarray expression analysis software was used to analyze the resultant data. Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation image_aquisition hybridization specified_biomaterial_action nucleic_acid_extraction labeling feature_extraction Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Systematic determination of differential gene expression in the primate corpus luteum during the luteal phase of the menstrual cycle. Publication Author List Bogan RL, Murphy MJ, Stouffer RL, Hennebold JD PubMed ID 18258683 Publication DOI 10.1210/me.2007-0484 Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE10367 Comment[GEOLastUpdateDate] 2012-03-20 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2008-02-08 Comment[ArrayExpressSubmissionDate] 2008-02-03 SDRF File E-GEOD-10367.sdrf.txt