FOXL2 regulates RhoA expression to change actin cytoskeleton rearrangement in granulosa cells of chicken pre-ovulatory follicles

Forkhead box L2 (FOXL2) is an indispensable key regulator of female follicular development, and it plays important roles in the morphogenesis, proliferation, and differentiation of follicle granulosa cells, such as establishing normal estradiol signaling and regulating steroid hormone synthesis. Nevertheless, the effects of FOXL2 on granulosa cell morphology and the underlying mechanism remain unknown. Using FOXL2 ChIP-seq analysis, we found that FOXL2 target genes were significantly enriched in the actin cytoskeleton-related pathways. We confirmed that FOXL2 inhibited the expression of RhoA, a key gene for actin cytoskeleton rearrangement, by binding to TCATCCATCTCT in RhoA promoter region. In addition, FOXL2 overexpression in granulosa cells induced the depolymerization of F-actin and disordered the actin filaments, resulting in a slowdown in the expansion of granulosa cells, while FOXL2 silencing inhibited F-actin depolymerization and stabilized the actin filaments, thereby accelerating granulosa cell expansion. RhoA/ROCK pathway inhibitor Y-27632 exhibited similar effects to FOXL2 overexpression, even reversed the actin polymerization in FOXL2 silencing granulosa cells. This study revealed for the first time that FOXL2 regulated granulosa cell actin cytoskeleton by RhoA/ROCK pathway, thus affecting granulosa cell expansion. Our findings provide new insights for constructing the regulatory network of FOXL2 and propose a potential mechanism for facilitating rapid follicle expansion, thereby laying a foundation for further understanding follicular development.