LIMK1/2 Regulates Porcine Oocyte Meiosis

Mammalian oocyte maturation begins with the diplotene stage of the first meiosis,and ends with the metaphase of the second meiosis.Unlike the symmetric division of somatic cell,oocyte meiosis is characterized by an asymmetric division form.The key to the asymmetry of oocyte meiosis lies in the positioning of meiotic spindles near the cortex.Another symbolic feature of oocyte asymmetrical division is the formation of an "actin cap"which presents a thick layer of microfilaments enriched on the cortex above the spindle.Thus,microfilaments and microtubules play an irreplaceable role in the process of asymmetric division of female mammals.LIMK1/2,as an actin regulatory factor,regulates the actin cytoskeleton reorganization via the phosphorylation of cofilin,which makes it participate in many cell activities.Moreover,LIMK1/2 has been found to participate in microtubule depolymerization.Although LIMK1/2 has an important regulatory effect on microfilaments and microtubules,its role in porcine oocytes meiosis is unclear.Therefore,it is necessary to explore the role of LIMK1/2 in porcine oocyte maturation,which has a far-reaching effect on the study of LIMK1/2 biological function and the complementarity of oocytes maturation mechanism.In this study,porcine oocytes were employed as the experimental object.We used western blot,immunofluorescence staining and laser confocal microscopy,to investigate the potential role of LIMK1/2 in porcine oocytes meiotic maturation.First,the localization and expression of LIMK1/2 in porcine oocyte meiotic maturation were detected by immunofluorescence staining and western blot.Then,we selected specific LIMK 1/2 inhibitor LIMKi 3 treatment to disrupt protein activity or function and further explored its effects on the assembly and distribution of actin and spindle organization and positioning in the process of porcine oocyte meiosis,which helped us to understand how LIMK1/2 regulate polar body extrusion.The main results of this experiment are as follows:(1)Immunofluorescent staining showed that LIMK 1/2 was localized mainly to the cortex of porcine oocyte,which co-localized with actin.(2)After LIMKi 3 treatment,the diffusion of COCs became weak together with the failure of polar body extrusion.However,the inhibitory effect of LIMKi 3 would become weaken with the prolongation of culture time.(3)LIMKi 3 treated oocytes could extrude the first polar body after rescue,indicating that the inhibition of LIMKi 3 for porcine oocytes is reversible.(4)After LIMKi 3 treatment and cultured for 26 h,actin distribution was disturbed,presenting that actin fluorescence intensity both at the membrane and cytoplasm of oocyte became much weaker.Meanwhile,a majority of the LIMKi 3-treated oocytes spindles were not attached to the cortex due to the failure of migration.Taken together,these results demonstrated that LIMKi 3 affected the distribution of actin and the positioning of spindle,which inhibited the extrusion of the first polar body and was important for porcine oocytes meiotic maturation.Therefore,we can speculate that LIMK1/2 may be involved in porcine oocyte meiotic maturation by regulating actin-dependent spindle migration.