ESPE Abstracts

Introduction: The Androgen Receptor (AR) is a steroid hormone receptor. Upon activation by its potent ligand, 5-alpha-dihydrotestosterone (DHT), it regulates the expression of target genes leading to the development of the outer male genitalia. Mutations in the AR gene are associated with Androgen Insensitivity Syndrome (AIS), characterized by the presence of testes and varying degrees of undervirilization of the external genitalia. Since many individuals diagnosed with AIS do not show mutations in the AR gene, it is plausible that changes in AR target genes might contribute to the phenotype. Till date only Apolipoprotein D (APOD) was identified as bona fide target of the AR in genital tissue. Thus, identifying additional target genes could aid in elucidating the etiology of AR-mutation-negative AIS and androgen-dependent signaling pathways in general.

Objective: To identify and characterize genes activated by the AR in response to DHT.

Methodology: Primary genital skin fibroblasts (GSF) (6 scrotum derived, 6 foreskins derived) were cultured and treated for 48 and 72 hours with ethanol (EtOH) as control or 10 nM DHT. RNA was isolated and subjected to Illumina mRNA sequencing. Reads were quality-checked, principal component analysis (PCA) was performed and differential gene expression analysis was carried out using DESeq2 to identify genes significantly correlated with AR induction. Functional enrichment analysis on those genes was conducted to elucidate the biological processes and pathways associated with AR activation. Validation of the top differentially androgen-regulated genes was performed by qRT-PCR.

Results: The PCA analysis separated scrotum and foreskin, and to a lesser extent EtOH and DHT treatment. We detected 144 common significantly differentially expressed genes between DHT- and control-treated cells in both scrotum and foreskin derived cells. Among them, 97 were upregulated and 47 were downregulated. Gene ontology enrichment analysis (GOEA) of upregulated genes, revealed significant enrichments in biological processes related to extracellular matrix (ECM) organization. The ECM plays an important role in cell adhesion, cell communication, and differentiation. The most enriched biological hallmark was epithelial-mesenchymal transition (EMT). EMT is integral to the formation of many tissues and organs during development.

Conclusion: Our study provides insights into the genes activated by AR upon DHT treatment in GSF, deepening our understanding of androgen signaling pathways. Although GSF are terminally differentiated, GOEA revealed enrichment in developmental processes, making these cells a good tool to study androgen action in vitro. These findings might also help improve the diagnostic yield for AR-mutation-negative AIS.