Contribution of the hedgehog-signaling pathway to Wolffian Duct development.

ABSTRACT: Introduction and objectives: Proper In utero Wolffian duct (WD) development requires androgens and is essential to epididymis formation and male fertility. However, non-hormonal factors that control WD homeostasis and development remain largely unknown. In this study, we investigated the contribution of Hedgehog signaling pathway to Wolffian duct development by combining pharmacological approaches on organotypic cultures of WD with microarray profiling. Methods: WD were collected from embryos isolated from 16.5 days post-coitum pregnant mice and cultured in air-liquid condition at 37oC during 72 hours in the presence of either: 1) Cyclopamine (25 µM): Hh inhibitor; 2) Smoothened agonist (0.5 µM ; SAG): Hh activator; 3) Culture media (control). WD pictures were taken and analyzed on ImageJ. At the end of the culture, WD were snap-frozen to perform transcriptomic microarray analyses. Three to four biological replicates per condition, i.e. control (n=3), SAG (n=4) and cyclopamine (n=3), were used for microarray analyses. The quality of the RNA was determined using an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and displayed a RIN ranging from 9.10 to 9.60 out of 10 for the different samples. Microarray analyses were carried out on Affymetrix Mouse Clariom S arrays (Thermofisher) according to the Affymetrix standard protocol. In brief, 100 ng total RNA samples were labeled using the GeneChip® WT Plus Reagent Kit protocol and hybridized to the arrays as described by the manufacturer (Affymetrix, Thermofisher). The cRNA hybridization cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After a 16-h hybridization period, the cocktail was removed and the arrays were washed and stained in an Affymetrix GeneChip fluidics station 450, according to the Affymetrix-recommended protocol. The arrays were scanned using the Affymetrix GCS 3000 7G and Gene-Chip Command Console Software (AGCC) (Affymetrix, Thermofisher) to produce the probe cell intensity data (CEL). The imaged data were then analyzed using the Affymetrix Expression Console Software to perform the quality control, background subtraction and normalization of probe set intensities using Robust Multiarray Analysis (RMA). Microarray processing was performed by the Gene Expression Core facility of the Genomic platform of the Centre Hospitalier Universitaire de Quebec Research Center. The microarray CEL files were imported and analyzed with Transcriptome Analysis Console (TAC) Software 4.0.2 (Applied Biosystems), and submitted to RMA normalization. Samples were included within three different treatment groups (control, SAG and cyclopamine) and compared by Analysis of Variance. Principal Component Analysis and heat maps were performed with Partek Pathway (Partek Incorporated). Biological pathways analyses were performed by using 5 distinct algorithms (i.e. DAVID, STRING, GOrilla, Metascape, GSEA and BLAST2GO). Conclusion: The data made available on GEO repository provide new insights regarding the mechanisms that control epididymis morphogenesis and male fertility. Financially supported by a CIHR grant to CB.

ORGANISM(S): Mus musculus

PROVIDER: GSE145816 | GEO | 2022/02/21

REPOSITORIES: GEO