ative RT-PCR and serial analysis of gene expression. Several candidate genes, such as cyclin D2, growth and differentiation factor 9 , forkhead box transcription factor , inhibina and inhibinbB were shown to be regulated by E using these approaches. However, many downstream gene targets of E were unlikely to have been discovered using this approach. The aromatase knockout female mouse is deficient in aromatase activity postnatally and therefore is an excellent model to allow us to define E-dependent ovarian genes in adulthood. Using RT-PCR we identified genes usually associated with male reproduction such as Sox9, DAX1, liver homolog-1 and Mullerian-inhibiting substance as being significantly increased in the ArKO ovary. Steroidogenic enzymes such as 17a-OHase, 17Hsd1, and 17b-Hsd3 mRNA’s were also increased and the patterns of expression of the steroidogenic enzymes responsible for androgen biosynthesis in the ArKO ovaries correlated with increased serum testosterone levels in ArKO females. The development of microarray technology now enables the simultaneous measurement of thousands of gene transcripts in a biological sample. Therefore, in order to identify E-dependent genes in the ovary, this study utilized a microarray approach profiling wildtype and ArKO ovaries. The study 3131684 aimed to 1) confirm the preliminary observations on estrogen dependent genes, 2) provide novel information about genes that can be used to unravel the mechanism of E in maintaining the female gonad, and 3) provide new insights into the regulation of ovarian follicular development. Materials and Methods Animals Wild-type and ArKO mice on a J129/C57B6 background were maintained under LBH589 specific pathogen-free conditions, on a 12L:12D regimen and fed ad libitum a soy free mouse chow. All animal procedures were approved by a Monash University Animal Ethics Committee and were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. February 2011 | Volume 6 | Issue 2 | e14672 Estrogen-Dependent Genes Experimental design ArrayExpress. The Illumina microarray was performed by 2187993 the Australian Genome Research Facility Melbourne, Australia. Statistical analysis for microarray data Prior to differential gene expression analysis, quality control diagnostic measurements were run on the raw data generated from the Illumina chips. 6 Sentrix Mouse-6 Expression Beadchip samples were normalised using lumi package in R statistical software and were subsequently imported into GeneSpring GX software for further analysis. The significant E-dependent differentially expressed genes were obtained from comparing KO samples to WT samples and were defined by a fold difference of 2.0 and a P-value cut-off of 0.05. Genes that 
met these parameters Fold Change Differential Expressed Genes Up Down 159 total 291 159 450 47,2.0 22.0,215.7 Total 291 – Up; up regulated within designated fold range. Down; down regulated within designated fold range. All E-dependent DEG were significantly differentially expressed . doi:10.1371/journal.pone.0014672.t001 February 2011 | Volume 6 | Issue 2 | e14672 Estrogen-Dependent Genes were included in the E-dependent DEG list and colour coded with blue representing down-regulation and red representing upregulation of transcript when comparing KO samples to WT samples. The data normalisation and statistical analysis to generate E-dependent DEG list was performed by the AGRF Melbourne, Australia. 3 February 201
