Abstract
LIMKi 3 is a specific selective LIMK inhibitor against LIMK1 and LIMK2, while LIMK1 and LIMK2 are the main regulators of actin cytoskeleton to participate in many cell activities. However, the effect of LIMKi 3 in porcine oocyte meiosis is still unclear. The present study was designed to investigate the effects of LIMKi 3 and potential regulatory role of LIMK1/2 on porcine oocyte meiotic maturation. Immunofluorescent staining of p-LIMK1/2 antibody showed that LIMK1/2 was localized mainly to the cortex of porcine oocyte, which co-localized with actin. After LIMKi 3 treatment, the diffusion of COCs became weak and the rate of polar body extrusion was decreased. This could be rescued by moving oocytes to fresh medium. After prolonging the culture time of oocytes, the maturation rate of porcine oocyte increased in LIMKi 3 groups, indicating that LIMKi 3 may suppress the cell cycle during porcine oocyte maturation. We also found that after LIMKi 3 treatment actin distribution was significantly disturbed at porcine oocyte membranes and cytoplasm, indicating the conserved roles of LIMK1/2 on actin dynamics. Next we examined the meiotic spindle positioning in porcine oocyte, and the results showed that a majority of spindles were not attached to the cortex of porcine oocyte, indicating that LIMKi 3 may affect actin-mediated spindle positioning. Taken together, these results showed that LIMK1/2 inhibitor LIMKi 3 had a repressive role on porcine oocyte meiotic maturation.
Highlights
The meiosis in mammalian oocyte is arrested at diplotene stage of first meiotic prophase, manifested by the germinal vesicle (GV) located at the center of the oocyte
We investigated the potential roles of LIMK1/2 inhibitor LIMKi 3 during porcine oocyte maturation
We first examined LIMK1/2 expression in porcine oocyte, and we found that LIMK1/2 existed at the cortex of oocyte in porcine during meiosis, which was similar with actin filament
Summary
The meiosis in mammalian oocyte is arrested at diplotene stage of first meiotic prophase, manifested by the germinal vesicle (GV) located at the center of the oocyte. Actin is enriched to form an actin cap over the spindle, and cortical granules (CGs) become redistributed to form a CG-free domain (CGFD) where microvilli are lost (Deng et al, 2003). These events are regarded as cortical reorganization. HeLa cell as a model cancer cell has been used to investigate the roles of LIMK, and results showed that LIMK-mediated cofilin phosphorylation was required for precise spindle positioning to ensure the achievement of division during mitosis (Kaji, Muramoto & Mizuno, 2008). We showed that LIMKi 3 treatment caused aberrant actin distribution and spindle positioning, which might have contributed to a failure of porcine oocyte maturation
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