Dynamic Expression And Function Of Aurora A During Porcine Oocyte-to-Embryo Transition

Oocyte-to-embryo transition is an important biological process that transfers from asymmetrical meiosis to symmetrical mitosis in mammals.Aurora A,one of evolutionarily conserved serine/threonine kinases,little information is available about the inportant role of Aurora A in the regulation of cell division and molecular mechanism.In the present study,Western blot and indirect immunofluorescence were used to test the dynamic expression and subcellular localization of Aurora A during oocyte-to-embryo transition.The potential role of Aurora A was analyzed using a selective Aurora A inhibitor,MLN8054,during oocyte-to-embryo transition.Base on above analysis and for the purpose to expand the understanding of related molecular mechanism,the expression of Aurora A and p-TACC3 were also assessed after MLN8054 treatment.The experimental results are as follows:Experiment 1.Dynamic expression and distribution of Aurora A during porcine oocyte-to-embryo transitionAurora A and p-Aurora A(T288)were expressed in M??M? and each phase of porcine embryo mitosis.Indirect immunofluorescence and laser confocal microscopy analysis showed that,when oocytes developed at the MI and M? phase,Aurora A was distributed a symmetrical pattern around the chromosome in the cortex of oocyte.At the pronuclear stage,Aurora A expressed a diffuse distribution in the cytoplasm.During the prophase,a reticulated pattern of Aurora A was gradually expressed.Afterwards,Aurora A appeared in a symmetrical pattern on both sides of the chromosome and then migrated toward opposite poles of embryo while sister chromatids segregated,which was similar with the distribution of a-tubulin.From what we had observed,it's speculated that Aurora A and a-tubulin had the similar distribution.Co-localization of Aurora A and a-tubulin was performed in order to test hypothesis.A yellow color emerged when their fluorescence signals overlapped,showing that Aurora A co-localized with a-tubulin during the oocyte to embryo transition,suggested that Aurora A was closely related to the dynamic distribution and function of spindle.Experiment 2.Effects of Aurora A inhibition on oocyte maturation,spindle and chromosomeTo further investigate the potential role of Aurora A during the oocyte-to-embryo transition in pigs,the inhibitior of Aurora A MLN8054 was used for MI oocytes.It was shown that first polar body extrusion rate which means the percentage of oocytes maturation was significantly reduced with the increase of MLN8054.The percentage of oocytes with abnormal spindle morphology and improper chromosome alignment increased significantly.But the treatement of MLN8054 had no significant effect on the transition of porcine M? oocyte to embryo.Aurora A participated in the porcine oocyte meiosis via the regulation of spindle and chromosome.Experiment 3.Effects of Aurora A inhibition on the regulation of the pronuclear to division stage transition and the mechanism during the first embryonic mitisisTo further detected the role of Aurora A during the first embryonic mitosis of porcine.Cells were treated with MLN8054 after activation and results showed that cleavage rate of embryos gradually decreased with the increasing concentrations of MLN8054.Cell cycle progression was altered with Aurora A inhibition,and embryos mainly arrested at pronuclear stage,suggested that Aurora A perhaps play an essential role in the regulation of pronuclear to division stage transition for embryo.For the reason of Aurora A's role in the regulation of this transition.Embryos developed to 18 and 12 h exposed to MLN8054 were collected,and we found that the difference of pronuclear morphology induced by MLN8054 treatment befor 12 h,had no effect on embryo cleavage,while the mistakes in chromosome condensation and a-tubulin assembly due to Aurora A inhibition from 12 to 18 h affected the pronuclear to division stage transition.Cells exposed to MLN8054 after activation for 18 h were collected to understand the regulatory mechanism of Aurora A for the failure of transition.Western blot analysis showed that the expression of p-Aurora A(T288)and p-TACC3(S558)were decreased significantly following MLN8054 treatment.These results suggested that TACC3 may be involved in the regulation of Aurora A in the pronuclear to division stage transition of pig embryos.