Inhibitory effect of SLIT2 on granulosa cell proliferation mediated by the CDC42-PAKs-ERK1/2 MAPK pathway in the prehierarchical follicles of the chicken ovary.

The SLIT2 ligand and ROBO receptors of the SLIT/ROBO pathway are expressed in hen ovarian follicles and have been shown to play critical roles in ovary development, cell proliferation and apoptosis in mammals. However, the exact roles of SLIT2 and the molecular mechanisms of chicken follicle development remain poorly understood. Here, we discovered that high levels of SLIT2 suppress FSHR, GDF9, STAR and CYP11A1 mRNA and protein expression in granulosa cells (GCs) and cell proliferation (p < 0.01). However, these inhibitory effects can be abolished by the siRNA-mediated knockdown of the ROBO1 and ROBO2 receptors. Furthermore, the activity of CDC42, which is a key Rho GTPase in the SLIT/ROBO pathway, is regulated by the ligand SLIT2 because the intrinsic GTPase activation activity of CDC42 is activated or repressed by regulating SRGAP1 expression (p < 0.01). The effects of the SLIT2 overexpression on GC proliferation and phosphorylation of the B-RAF, RAF1 and ERK1/2 kinases were completely abrogated by knocking down endogenous PAK1 and partially abrogated by the knockdown of PAK2 and PAK3 in the GCs. Collectively, our findings indicate that SLIT2 suppresses GC proliferation, differentiation and follicle selection mainly by a mechanism involving ROBO1 and ROBO2 and that this suppression is mediated by the CDC42-PAKs-ERK1/2 MAPK signaling cascade in the prehierarchical follicles of the chicken ovary.