Subcellular Localization And Function Of Plk1 During Meiotic Division Of Porcine Oocytes

Oocyte meiosis is an important foundation for subsequent embryonic mitosis and differentiation development.The meiosis involves orchestrated and exquisite events,is precisely and orderly adjusted by a series of cell cycle regulators in both time and space.Plk1(Polo-like kinase 1),as a highly conserved ser/threonine protein kinase that regulates cell division,is not known to be involved in the regulation of meiosis in pig oocytes.Plkl and p-Plkl(Thr-210)were observed via western blot during the whole meiotic division of porcine oocytes,coupled indirect immunofluorescent detection with Immunofluorescence laser scanning confocal microscope found that dynamic expression of Plkl;GSK461364,the Plkl selective inhibitor,used to test potential role of Plkl during the meiosis of pig oocytes.In order to explore the underlying reasons of Plkl inhibited cell in GVBD,causing the failure of progression to MI,the relative expression of p-Aurora A(Thr-288)and p-Plkl(Thr-210)were detected and effect of spindle organization after Aurora A inhibition.The results are as follows:Experiment 1.Expression and subcellular localization of Polo-like kinase 1 during oocyte meiosisPlkl and p-Plkl(Thr-210)were examined in pig oocytes during meiotic progression by western blot analysis.Plkl relative intensity significantly increased at the beginning of GVBD and reached maximum ATI,the expression of Plkl was not significant between GV stage and GVBD stage.The expression of p-Plkl(Thr-210)was stable during whole meiotic maturation stages in porcine oocytes,and lower than Plkl.Combined indirect immunofluorescent detection with Immunofluorescence laser scanning confocal microscope found that,chromatin was gathered ring-shape,Plk1 was observed amorphously outside germinal vesicles at GV;later germinal vesicles breakout,Plk 1 was accumulated round the chromosomes at GVBD;when the cell progressed to MI,the chromosomes was gathered into line-like,Plkl was enriched at both side of chromosomes.At ATI,chromosomes was extend to spindle poles and showed furrow between two sets of the chromosomes,Plkl spreads between the two sets of the chromosomes.At MII,the first polar body was extruded,Plkl was enriched at both side of the line-like chromosomes.Little was gathered around the first polar body.Based on the dynamic subcellular localization examined above,Plkl and a-tubulin appeared to have a similar distribution pattern during meiosis in pig oocytes.To confirm this hypothesis,Plkl and a-tubulin were subjected for co-staining via immunoluorescent staining show that,a yellow colour emerged where their fluorescence signals overlapped.This dynamic subcellular localization pattern indicated the possibility that Plkl may be involved in regulating porcine oocyte meiotic maturation,which is associated with the spindle organization processesExperiment 2.Effects of GSK461364 on oocytes maturation,spindle structure and chromosome alignment during MI stageTo investigate the potential role of Plkl during meiotic maturation,oocytes were treated with a Plk 1-specific inhibitor,GSK461364,for 44 hr to inhibit Plk 1 activity during cultivation,after which the pbI extrusion was examined.A large percentage of the GSK461364-treated oocytes failed to extrude the pbI in a concentration-dependent manner,the percentage of cells was arrested at the GVBD stage was increased significantly.Most of treated oocytes showed misaligned chromosomes and aberrant spindles.The rate of embryos cleavage and blastocyst was significant lower between control group and GSK461364-treated group.This results showed Plkl has a role in the porcine oocytes meiotic division through spindle organization and chromosome alignment;the potential development of embryo was significant inhibited after Plkl was inhibited.Experiment 3.Mechanism of Plkl regulation during oocyte meiosisTo further investigated the role of Plk1 during the meiotic division of porcine oocytes and to analyze whether Aurora A is upstream regulator of Plkl in porcine oocyte meiotic.Cell was exposed to Aurora A inhibitor(MLN8054)and the results showed meiotic division failed,misalignment chromosome and aberrant spindles,similar to the result of Plkl inhibitor.Cells were exposed to Aurora A inhibitor,we detected the relative expression of p-Plkl(Thr-210)in treatment group decreased,accompany by p-Aurora A(Thr-288)decreasing.p-Aurora A(Thr-288)and p-Plkl(Thr-210)was significantly decreased,compared the control group at MI stage.These results suggest that the fuction of Plkl during the meiosis of procine oocytes is regulated by its upstream Aurora A activity.