Methods: Microdissected pairs of GD17 rat gubernacular bulbs were cultured on a Millicell CM membrane in Dulbecco modified Eagle medium/Ham, 2% charcoal stripped fetal bovine serum, 1× insulin-transferrin-selenium-X supplement and 1× antibiotic-antimycotic. Cultures were maintained in basal medium for 24 hrs and for an additional 6 or 24 hours in basal or dihydrotestosterone-(DHT, 1, 10 or 30 nM) supplemented medium prior to harvesting. RNA was extracted, labeled and hybridized to Affymetrix microarrays (5-6 replicates/group). Differential expression was determined by the LIMMA linear model approach with a false discovery rate of 5% using the limma package in Bioconductor. Analysis for overrepresented functional categories was performed with DAVID. Genes of interest were analyzed in independent samples using TaqMan gene expression assays and data analyzed using ANOVA after log transformation.
Results: DHT was associated with differential expression of 0 and 2533 probesets after 6 and 24 hours’ exposure, respectively; 55% were also regulated by INSL3 as noted in our previous experiments. Functional analysis of 1336 probesets upregulated by DHT (1098 DAVID IDs) showed overrepresentation of extracellular matrix (ECM) and basement membrane; of 34 ECM genes, 8 were collagens. For 1197 downregulated probesets (900 DAVID IDs), WNT signaling; biological processes related to cellular transport, RNA processing and transcription; nucleus and organelle (cellular components) and beta-catenin, SMAD and ion binding (molecular functions) were overrepresented. Using qRT-PCR we confirmed upregulation of Has2 and Adh1 (known AR-regulated genes) and neuromuscular developmental genes including Crlf1, Chrdl2 (both similarly upregulated by INSL3), Slit3 and Syne2. Transcripts confirmed as downregulated by DHT include Wnt4 (upregulated by INSL3), Ar, Myh7, Bmp4, Cxcl12 and Tgfb2. We were unable to validate a significant response of Npy or Sfrp2 transcript levels to DHT.
Conclusions: We identified WNT and BMP signaling as common targets in the DHT- and INSL3-regulated transcriptome of the fetal gubernaculum, and confirmed differential expression of transcripts known to be androgen responsive in other contexts. These observations support other data suggesting synergy between INSL3-RXFP2 and AR signaling in the gubernaculum. We expected that 6-hr exposure to DHT would identify direct transcriptional AR targets, but none were observed. This absence of effect may indicate incomplete penetration of the intact organ in vitro after limited exposure, technical variation and/or a possible role for rapid, non-genomic AR signaling in the fetal gubernaculum.