Identification of WNT/β-CATENIN signaling pathway components in human cumulus cells

Signaling via the conserved WNT/b-CATENIN pathway controls diverse developmental processes. To explore its potential role in the ovary, we investigated the expression of WNTs, frizzled (FZD) receptors and other pathway components in human cumulus cells obtained from oocytes collected for in vitro fertilization. Proteins were detected in cultured cells using immunofluorescence microscopy. Protein-protein interactions were analyzed by means of immunoprecipitation. WNT2, FZD2, FZD3 and FZD9 were identified but WNT1, WNT4 and FZD4 were not detected. WNT2 is co-expressed with FZD2, FZD3 and FZD9. Co-immunoprecipitation using WNT2 antibody demonstrated that WNT2 interacts with both FZD3 and FZD9, but only FZD9 antibody precipitated WNT2. We also identified DVL (disheveled), AXIN, GSK-3b (glycogen synthase kinase-3b) and b-CATENIN. b-CATENIN is concentrated in the plasma membranes. DVL co-localizes with FZD9 and AXIN in the membranes, but GSK-3b has little co-localization with AXIN and b- CATENIN. Interestingly, b-CATENIN is highly co-localized with FZD9 and AXIN. CDH1 (E-cadherin) was also detected in the plasma mem- branes and cytoplasm, co-localized with b-CATENIN, and CDH1 antibody precipitated b-CATENIN. The results suggest that WNT2 could act through its receptor FZD9 to regulate the b-CATENIN pathway in cumulus cells, recruiting b-CATENIN into plasma membranes and promoting the formation of adherens junctions involving CDH1.