Conserved function of WNT4 in reptilian granulosa cell proliferation: insights from the Chinese soft-shelled turtle

The continuous process of folliculogenesis involves precise coordination between the oocyte and surrounding somatic cells, especially the granulosa cells. However, the roles of granulosa cells and associated gene expression changes during vitellogenesis remain poorly understood. In the Chinese soft-shelled turtle, our study found that granulosa cells undergo a morphological change from flattened to dense cuboidal during the white-to-yellow follicle transition, accompanied by increased relative expression of WNT4, as a WNT ligand, as confirmed through RNA-seq analysis and RT-qPCR. Notably, in situ hybridization revealed that WNT4 was expressed in granulosa cells, as further demonstrated by its co-localization with FOXL2 immunofluorescence signaling, a granulosa cell-specific transcription factor. Furthermore, we successfully established a functional primary culture of granulosa cell, characterized by the expression of key markers FOXL2 and CYP19A1 (aromatase). Treatment with IWP-2, a specific inhibitor of WNT4, significantly reduced both EdU signal and the phosphorylation level of JNK in cultured granulosa cells. Together, these results shed light on the regulation of granulosa cell proliferation, and highlight a conserved role for WNT4 in this process.