tissue: Testis gender: male age: 6 months treatment: EE2 treatment dose: 1.0 ug/L exposure day: Day 7 replicate: rep C testicular histology: small percent of animals with thickening of the interstitial tissue and decreased spermatozoa fertilization rate: 62.8%
Treatment protocol
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.
Extracted molecule
total RNA
Extraction protocol
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 manufacturer’s instructions (Qiagen, Valencia, California). The sample was then eluted with 30 µl RNase-free water (52°C). Total RNA samples were stored at -80°C.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).
Label
Cy3
Label protocol
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 manufacturer’s protocol. The quality of the labeled cRNA was verified and concentration was measured spectrophotometrically.
Hybridization protocol
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 μg) was hybridized to each array as a single channel hybridization. Hybridization was conducted on a custom 15K x 8 Agilent medaka array using the “In situ hybridization Kit-Plus” (Agilent) at 60 °C for 17 h. The arrays were washed according to Agilent's SSPE wash protocol using a solution of 6× SSPE, 0.005% N-lauroylsarcosine, followed by a solution of 0.06× SSPE, 0.005% N-lauroylsarcosine, and Agilent's Stabilization and Drying Solution.
Scan protocol
The arrays were scanned on an Agilent G2565BA Microarray Scanner.
Description
Testicular gene expression of adult medaka of exposure day 7 (1.0 ug/L EE2)
Data processing
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.
