77 Notch-inhibition stimulates secondary ciliation in re-differentiated equine oviduct epithelial cell monolayers
B. Leemans A B , J. Marchand A , T. Stout A , A. Van Soom B , B. Gadella A B and H. Henning AA Departments of Clinical Sciences, Biomolecular Health Sciences, and Population Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
B Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
Reproduction, Fertility and Development 35(2) 164-165 https://doi.org/10.1071/RDv35n2Ab77
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Recently, we developed differentiated equine oviduct epithelial cell (EOEC) monolayers in Transwell inserts and microfluidic chips. EOEC monolayers were obtained by culturing EOECs to a confluent de-differentiated monolayer in conventional wells, before trypsinising and seeding onto a microporous membrane. EOEC monolayers were then triggered to re-differentiate by air-liquid interface introduction. EOEC monolayers demonstrated various in vivo morphological characteristics but lacked secondary ciliation, an important marker for differentiated status. In this study, we investigated whether exposure to Wnt or Notch pathway ligands or inhibitors would trigger ciliation in EOEC monolayers. Confluent EOEC monolayers were exposed for a week to either Notch or Wnt-agonists or inhibitors, starting 3 or 7 weeks after air-liquid-interface introduction. The following concentration ranges were investigated: 20–2-0 µM IWP-2 (Wnt-inhibitor) and 30–3-0 µM CHIR 99021 (Wnt-agonist); 10–1-0 µM DBZ or γ-secretase inhibitor (Notch-inhibitor) and 10–1-0 mM JAG-1 (Notch-agonist). After 1 and 2 months air-liquid interface, EOEC monolayers were fixed in 4% paraformaldehyde and EOEC phenotype was assessed after combined immunofluorescence staining to demonstrate the presence of cilia (mouse anti-acetylated α-tubulin antibody; Alexa 488-conjugated goat anti-mouse secondary antibody), cell nuclei (Hoechst 33342) and actin filaments (Alexa 568-conjugated phalloidin). Most individual or combined treatments had no effect on EOEC morphology, with the exception of Wnt-stimulation and Notch-inhibition. In this respect, unfavourable EOEC epithelial-mesenchymal transition was observed in EOEC monolayers exposed to 1, 3, and 5 µM CHIR 99021 (Wnt-agonist). Interestingly, exposure to 1 or 10 µM DBZ (Notch-inhibitor) supported patchy in vivo-like secondary ciliation over 50% of EOECs, but only if the EOEC monolayers showed a spontaneous diffuse ciliation pattern at the onset of exposure (>5% secondary ciliated EOECs). On the other hand, monolayers with <1% ciliated EOECs before DBZ exposure did not support EOEC ciliation. Overall, Notch-inhibition helped trigger development of EOECs that more closely resemble in vivo oviduct epithelial cells.
