FSH stimulated bovine granulosa cell steroidogenesis involves both canonical and noncanonical WNT signaling

Gonadotrophins play key roles in follicular development; however, the underlying molecular mechanisms are not fully understood. Follicle stimulating hormone (FSH) regulation of aromatase and subsequent estradiol (E₂) production relies on β-catenin, a key effector of WNT signaling. We previously demonstrated that treatment with the canonical WNT inhibitor, IWR-1, reduced FSH induced bovine granulosa cell E₂ production in vitro. Here we demonstrated that intrafollicular injection in vivo with IWR-1 alters steroidogenesis and triggers a significant decrease in estrogen to progesterone ratio in the IWR-1 treated follicles compared to diluent injected control follicles. We next examined markers of canonical and noncanonical WNT signaling in dominant and subordinate follicles collected at different stages of follicular development and showed that protein for both CTNNB1 (canonical pathway) and phosphorylated (p)-LEF1 (noncanonical pathway) was significantly elevated in dominant compared to subordinate follicles at the early dominance stage of development. Therefore, we hypothesized that canonical and/or noncanonical WNT ligands modulate FSH stimulated E₂ production. Hence, we examined the effects of specific WNT ligands on FSH stimulated E₂ production in the absence of endogenous WNT production in vitro. Universal WNT signaling inhibitor, LGK-974 was able to inhibit FSH stimulation of E₂ and reduce the abundance of proteins linked to canonical and noncanonical WNT pathway activation. Supplementation with the canonical ligand WNT2b did not affect the inhibitory effects of LGK-974 on FSH stimulated E₂ production but rescued the LGK-974 mediated inhibition of CTNNB1 (canonical pathway) but not p-LEF1, p-JNK or p-P38 abundance (noncanonical pathway) abundance. In contrast, WNT5a treatment rescued FSH stimulated estradiol production and indices of activation of both the canonical (CTNNB1) and noncanonical (p-LEF1, p-JNK and p-P38) WNT signaling pathways in LGK-974 treated granulosa cells. Taken together, these results suggest that both canonical and noncanonical WNT pathways activation is linked to FSH stimulation of E₂ production by bovine granulosa cells.