17β-Estradiol Promotes Goat Granulosa Cell Proliferation via the ERα/NO/Wnt/β-Catenin Signaling Axis

Abstract

Ovarian granulosa cells (GCs) are central to follicular development in goats, and their proliferation is tightly regulated by estrogen, yet the downstream signaling cascade mediating this effect in ruminants remains undefined. This study aimed to investigate whether estrogen regulates goat ovarian GC proliferation through the nitric oxide (NO) signaling pathway and to further elucidate the molecular mechanisms by which NO modulates GC proliferation. Primary goat GCs were treated with 17β-estradiol (17β-E2), the NO donor sodium nitroprusside (SNP), the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), the Wnt inhibitor FH535, and the Wnt agonist SKL2001. Cell viability, proliferation, and protein expression were assessed by CCK‑8, EdU, and Western blotting. 17β-E2 at concentrations of 1, 2, and 10 μg/mL all promoted GC viability (P < 0.05), with maximal effect at 1 μg/mL (P < 0.01), and simultaneously upregulated inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) protein expression via estrogen receptor α (ERα). The NO donor SNP enhanced GC proliferation, whereas the NOS inhibitor L‑NMMA suppressed it; co‑treatment with SNP partially rescued the L‑NMMA‑induced inhibition. Mechanistically, SNP increased β‑catenin and proliferating cell nuclear antigen (PCNA) protein levels, an effect reversed by FH535, whereas L‑NMMA‑induced NO depletion reduced their expression, which was restored by SKL2001, positioning NO upstream of the Wnt/β‑catenin pathway. Collectively, these findings establish that 17β-E2 promotes goat GC proliferation through a signaling cascade involving ERα, NOS, NO, and the Wnt/β‑catenin pathway, providing the functional evidence for this axis in a ruminant model and offering a mechanistic framework for understanding ovarian follicular dynamics with implications for improving reproductive efficiency in goats.