PAK1-Dependent Regulation of Microtubule Organization and Spindle Migration Is Essential for the Metaphase I-Metaphase II Transition in Porcine Oocytes

P21-activated kinase 1 (PAK1) is a key downstream effector of small Rho GTPases, playing a central role in regulating cytoskeletal dynamics, cell proliferation, and migration. While PAK1 has been recognized as an important regulator of spindle assembly during the first meiotic division, its function during the transition from metaphase I (MI) to metaphase II (MII) in oocytes remains poorly understood. In this study, we investigated the role of PAK1 in microtubule organization and spindle positioning during the MI–MII transition in porcine oocytes.
Our findings revealed that activated PAK1 co-localized with α-tubulin, and its expression significantly increased from MI to MII (p < 0.001). Inhibition of PAK1 activation using the selective inhibitor IPA-3 at the MI stage led to a reduction in the first polar body (PB1) extrusion rate (p < 0.05), with the majority of oocytes arrested at the anaphase–telophase I (ATI) stage. IPA-3 treatment also resulted in diminished actin accumulation at the plasma membrane (p < 0.001) and a higher incidence of abnormal MII spindle reassembly and mispositioned spindles (p < 0.001). These defects were reversed when the disulfide bond between PAK1 and IPA-3 was reduced using dithiothreitol (DTT), confirming the specificity of IPA-3’s action.
Co-immunoprecipitation analysis demonstrated that PAK1 interacts with phosphorylated Aurora A and TACC3 to regulate spindle assembly, and associates with LIM kinase 1 (LIMK1) to facilitate actin filament-driven spindle migration. Collectively, these results highlight PAK1 as a critical regulator of microtubule organization and spindle migration during the MI–MII transition in porcine oocytes, functioning through pathways involving p-Aurora A, p-TACC3, and p-LIMK1.