Expression And Function Of Plk1 During Porcine Oocyte-to-Embryo Developmental Transition

Mammalian oocyte-to-embryo transition is a crucial process in reproductive development.It involves several important cellular biological events and is tightly regulated by a series of protein kinases.Polo-like kinase(Plk1)is a serine/threonine protein kinase that represents a mitotic target due to its important role in multiple stages of cell division.Some studies have shown that Plk1's functions involves multiple links on somatic mitosis,but the precise underlying mechanism of Plkl regulation during the oocyte-to-embryo transition of mammalian oocytes has not been thoroughly characterized.In this study,indirect immunofluorescence,confocal microscopy imaging techniques combined with western blot analyses and specific inhibitor treatment were used to study the expression and function of Plkl protein during porcine oocyte-to-embryo transition.Based on the detection of the dynamic expression of ?-Tubulin and F-actin in oocyte maturation and the first mitotic division,indirect immunofluorescence and confocal microscopy combined with western blotting analyses were used to study the dynamic expression and subcellular localization of Plkl protein during porcine meiosis and the first mitosis in early porcine embryos.To evaluate the potential function of Plkl,a selective Plk1 inhibitor,GSK461364,was used to reveal the regulatory role of Plkl during oocyte-embryo transition.The main experimental results are as follows:Experiment 1 Dynamic expression and subcellular localization of Plk1 during porcine oocyte-to-embryo transitionInitially,the cytoskeletal changes in oocytes at different stages were systematically examined by imunofluorescence techniques,immunofluorescence staining showed that a-Tubulin was organized to form bipolar spindles that were symmetrical and barrel shaped at M? and M? stages;The F-actin becomes rich to form an actin cap in the cell cortex region;At TI stage,the ?-Tubules are distributed between two sets of the segregated chromosomes,microfilaments form a contractile ring and promote pbI extrusion in the cell cortex region.The results of immunoblotting showed that Plkl was expressed at different stages in porcine oocytes and showed a significantly higher level in MI stage;Immunofluorescence staining showed that Plkl was enriched at spindle poles at MI and M? stages,which leads to the barrel-shaped spindle.At TI stage,Plkl was accumulated at the midzone region.These morphological results showed that Plkl has close spatial and temporal correlation with the distribution of a-tubulin during the MI-to-MII meiotic progression in porcine oocytes.Experiment 2 Effect of Plkl inhibition on oocyte maturation,the morphology of spindle and chromosomes,the expression of Mad2 and BubRl proteinThe MI oocytes were treated with GSK461364 to inhibit endogenous Plkl activity during the MI-to-MII stage,the results showed that a significantly larger proportion of the treated oocytes failed to extrude the pbI,which led to a failure of oocyte maturation;Both Plkl and phosphoPlkl(p-Plkl)expression of oocytes were examined using western blots,it was shown that GSK461364 treatment significantly eliminated the phosphorylation of Plkl in porcine oocytes,suggesting that Plkl may play a crucial role during the MI-to-MII transition in porcine oocytes.Then,the proportions of the Plkl-inhibited oocytes stuck at different meiotic stages were determined,the majority of the control oocytes extruded pbI and reached M? stage,whereas most of the inhibited-oocytes were arrested at ATI stage.Finally,oocytes were treated after MI with 1.2 ?M of GSK461364 for 36 h that should reach TI stage.The results showed that a significantly larger percentage of the inhibited-oocytes exhibited normal spindle structures and severe homologous chromosome segregation defects.These data showed that Plkl inhibition led to a failure of homologous chromosome segregation,thus blocking the meiotic cell cycle from progressing to M?stage and remaining in ATI stage.In addition,we selected two kinetochore proteins Mad2 and BubRl,oocytes were treated after MI with 1.2 ?M of GSK461364 for 36 h to examine the expression levels of Mad2 and BubRl proteins.The results showed that GSK461364 treatment did not affect the protein level of BubRl,but indeed obviously reduced the expression level of Mad2 protein.It was shown that the treated oocytes which exhibited chromosome segregation defects were accompanied by a significant reduction in the level of Mad2 protein.The above result indicated that Plkl might involve in correct chromosome segregation through its regulatory function on the expression of the Mad2 protein during ATI stage in porcine oocytes.Experiment 3 Dynamic expression and subcellular localization of Plkl in porcine embryos undergoing the first mitotic divisionWe systematically examined the dynamic distribution of cytoskeleton at different mitotic stages of the porcine embryo using immunofluorescent staining techniques.The data showed that at the prometaphase,?-Tubulin gathered to form a network structure;In metaphase,a-Tubulin was observed as organized mitotic bipolar spindles that were symmetrical and barrel in shape;In anaphase,microtubules were observed around the chromosomal sets which were in the two polar regions;At telophase,a-tubulin was assembled as a midbody in the cleavage furrow.Microfilaments concentrated at the cortical cytoplasmic region before telophase.Based on these,we examined the expression of Plkl at different stages of the first mitotic cycle using western blots.The results showed that the Plkl protein showed a significantly higher level in prometaphase,immunofluorescence staining showed that after prometaphase,Plkl was associated significantly with the spindles;In metaphase,Plkl was concentrated at the central region and symmetrically distributed around the chromosome;In anaphase,Plkl was moved to the polar sites,Plkl accumulated at the midbody in telophase.This expression and localization pattern indicated that possibly Plkl may be involved in regulating spindle assembling processes during the first mitotic division in porcine embryos.Experiment 4 Effect of GSK461364 treatment on the cleavage of porcine embryo and the morphology of spindles and chromosomesEmbryos were treated with a Plkl specific inhibitor,GSK461364,for 48 h to inhibit Plkl activity,a significantly large proportion of the treated embryos failed to complete the first cleavage.The results suggested that Plkl may play a critical role during the first mitotic division in porcine embryos.The proportions of porcine embryos at different mitotic stages were stuck after inhibiting Plkl activity using Hoechst 33342,after culturing for 48 h with GSK461364,most inhibitor-treated embryos failed to complete the first cleavage and were arrested at the prometaphase stage.After treatment with 100 nM of GSK461364,a large proportion of the inhibited embryos showed abnormal spindle structures and chromosome misalignments.These above results suggested that Plkl might be involved in the regulation of the first mitotic division in porcine embryos through spindle assembly and chromosome alignment in the prometaphase stage.