Comment[ArrayExpressAccession]	E-GEOD-44859
MAGE-TAB Version	1.1							
Public Release Date	2013-03-06
Investigation Title	Anchoring ethinylestradiol induced gene expression changes with testicular morphology and reproductive function in the medaka							
Comment[Submitted Name]	Anchoring ethinylestradiol induced gene expression changes with testicular morphology and reproductive function in the medaka
Experiment Description	This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 M-BM-5g/L EE2) on male medaka fertility, testicular histology and testicular gene expression. The findings demonstrate that a 14 day exposure to EE2 induced impaired male reproductive capacity and time- and dose-dependent alterations in testicular morphology and gene expression. The average fertilization rate/day following the exposure for control, 1.0 and 10.0 M-BM-5g/L EE2 was 91.3% (M-BM-14.4), 62.8% (M-BM-18.3) and 28.8% (M-BM-15.8), respectively. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. The morphologic changes observed were highly associated with gene expression changes observed using a medaka-specific microarray. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell cycle and proliferation, collagen production/extracellular matrix organization, hormone signaling, male reproduction and protein ubiquitination among others. Six month old male medaka were exposed to ethinyl estradiol (EE2) for a 14 day time period. Treatment exposures were completed in triplicate including DMSO (vehicle control), 1.0 M-BM-5g/L EE2, and 10.0 M-BM-5g/L EE2. Fish were sampled for gene expression on days 1, 7 and 14 of exposure. Five male fish were placed in 2-liter beaker replicates for each treatment and sampling time point.							
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl						
Person Last Name	Miller	Miller	Clark	Hinton	Whitehead	Martin	Kwok	Kullman
Person First Name	Hilary	Hilary	Bryan	David	Andrew	Stan	Kevin	Seth
Person Mid Initials		D	W	E			W	W
Person Email	hilary.miller@duke.edu							
Person Affiliation	Duke University							
Person Address	Duke University, LSRC Science Drive Rm A305, Durham, NC, USA							
Person Roles	submitter							
Protocol Name	P-GSE44859-1	P-GSE44859-6	P-GSE44859-5	P-GSE44859-2	P-GSE44859-3	P-GSE44859-4	P-GSE44859-7	
Protocol Description	ID_REF =  VALUE = normalized signal	The arrays were scanned on an Agilent G2565BA Microarray Scanner.	Cy3-labeled cRNA (1.65 mg) from each sample was hybridized to an Agilent Custom Medaka Microarray, AMADID 022364. Control or experimental cRNA (0.75 M-NM-<g) was hybridized to each array as a single channel hybridization. Hybridization was conducted on a custom 15K x 8 Agilent medaka array using the M-bM-^@M-^\In situ hybridization Kit-PlusM-bM-^@M-^] (Agilent) at 60 M-0C for 17 h. The arrays were washed according to Agilent's SSPE wash protocol using a solution of 6M-CM-^W SSPE, 0.005% N-lauroylsarcosine, followed by a solution of 0.06M-CM-^W SSPE, 0.005% N-lauroylsarcosine, and Agilent's Stabilization and Drying Solution.	Six month old male medaka were exposed to ethinyl estradiol (EE2; Control (DMSO), 1.0ug/L EE2 or 10.0ug/L EE2) for a 14 day time period. Fish were sampled for gene expression on days 1, 7 and 14 of exposure. Five male fish were placed in 2-liter beaker replicates for each treatment and sampling time point. ERM was spiked with the appropriate EE2 stock (0.0025% of total volume) and equally distributed between the 2-L beakers for a total of 2 L spiked ERM per beaker with a 50% renewal of spiked ERM every other day for 14 days. The fish were maintained under a 16:8 hour light:dark cycle and fed ad libitum the dry diet as above on alternate days. On each sampling day, the testis of three fish per replicate beaker (n=3) were removed, pooled and immediately frozen in liquid nitrogen for RNA isolation.	Each sample (comprised of 3 pooled testes) were homogenized with 1 ml RNA Bee (TelTest, Friendswood, Texas) using a Polytron homogenizer (Kinematica, Bohemia, New York) cleaned with RNaseZAP (Sigma, St. Louis, Missouri), diethylpyrocarbonate (DEPC) treated water, and sterile de-ionized water. Total RNA was purified from the homogenate using RNeasy Mini Kit (Qiagen, Valencia, California) followed by an on-column-digest with DNase to eliminate DNA contamination using RNase-free DNase Set according to manufacturerM-bM-^@M-^Ys instructions (Qiagen, Valencia, California). The sample was then eluted with 30 M-5l RNase-free water (52M-0C). Total RNA samples were stored at -80M-0C.The quantity of each of the total RNA samples associated with this project was determined by spectrophotometry and the size distribution was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California).	Two hundred nanograms of total RNA was converted into labeled cRNA with nucleotides coupled to fluorescent dye Cy3 using the Quick Amp Kit (Agilent Technologies, Palo Alto, CA) following the manufacturerM-bM-^@M-^Ys protocol. The quality of the labeled cRNA was verified and concentration was measured spectrophotometrically.	Data from the scans were compiled with Agilent Feature Extraction Software 8.1 Analysis of the microarray data was performed using JMP Genomics 4.1(SAS Institute Inc, Cary, North Carolina). Data was log2 transformed during the import process and normalized using the standard normalization routine implemented in JMP Genomics 4.1. A distribution analysis was conducted for quality control purposes prior- and subsequent to normalization and alignment of the overlay plots was used as an indicator of high quality data. Data analysis was performed by conducting a Principal components analysis (PCA) by day and treatment using time-matched treatment-to-control differences calculated from standard least-square mean. An ANOVA was performed to test for statistical differences between treatment and control groups on a day by day basis. The False Discovery Rate (FDR) at alpha 0.05 was used to account for the multiple testing problem. Hierarchical clustering was performed using the significant gene sets derived from the ANOVA analysis data set.	
Protocol Type	bioassay_data_transformation	image_aquisition	hybridization	specified_biomaterial_action	nucleic_acid_extraction	labeling	feature_extraction	
Experimental Factor Name	TREATMENT DOSE	TREATMENT	TESTICULAR HISTOLOGY	REPLICATE	FERTILIZATION RATE	EXPOSURE DAY		
Experimental Factor Type	treatment dose	treatment	testicular histology	replicate	fertilization rate	exposure day		
Comment[SecondaryAccession]	GSE44859							
Comment[GEOReleaseDate]	2013-03-06							
Comment[ArrayExpressSubmissionDate]	2013-03-05							
Comment[GEOLastUpdateDate]	2013-03-07							
Comment[AEExperimentType]	transcription profiling by array							
SDRF File	E-GEOD-44859.sdrf.txt