MKlp2 And Profilin 1 Regulate Polar Body Extrusion During Mouse Oocyte Maturation

One of the most important processes during the origin of mammalians' lives is the oocyte maturation,which is a heated topic of many years scientific research.Oocyte maturation involves several complex cellular processes.The dynamics of cytoskeleton,including microtubule and actin,are essential for the maturation.Any defects during the processes may result in failure or abnormality of oocyte maturation,which can further affect the development of the zygote and early embryo and lead to the defect and malformation in infants.However,current research cannot full explain the underline mechanism of the regulation of actin and microtubule.Although many regulators and signal pathways have been demonstrated,there are still plenty of uncovered aspects in this field.Therefore,further research in molecular regulation mechanism of oocyte maturation has profound significance in consummating people's understand of mammalian reproductive mechanism and improving livestock's reproductive capacity and human's reproductive health.In these experiments,mice were employed as the experimental animals.We used in vitro culture,inhibitor treatment,morpholino injection,time-lapse microscopy,immunofluorescence staining and western blot to investigate the roles of related molecules during oocyte maturation.First,immunofluorescence staining was employed to examine the localization of MKlp2 and profilin 1 during oocyte maturation.Then,we used inhibitor treatment and morpholino injection to disrupt protein activity or function and further explored their effects on spindle formation,chromatin alignment and actin assembly,which helped us learn how they regulate polar body extrusion during oocyte maturation.Besides,with the employment of western bolt,we examined the impact of profilin 1 knockdown on actin as well as the expression of several related molecules,which further indicated the possible mechanism of profilin 1 regulation on mouse oocyte meiotic maturation.These results provide vital evidences for completing the regulation mechanism of mammalian oocyte maturation.This study consists of the following two experiments.Project 1.MKlp2 Inhibition Results in Polar Body Extrusion Failure during Mouse Oocyte Maturation Mammalian oocyte meiotic maturation involves a number of important processes,including spindle assembly and migration,cortical reorganization and polar body extrusion.Numerous proteins contribute to these processes,but it is unknown whether MKlp2(mitotic kinesin-like protein 2;also called KIF20A),a microtubule-associated protein that regulates cytokinesis during mitosis,is involved in oocyte maturation.Here we examined the role of MKlp2 in mouse oocyte.Immunostaining results showed that MKlp2 localized to oocyte microtubules.Time-lapse microscopy showed that disrupting MKlp2 expression with paprotrain,a specific inhibitor of MKlp2,resulted in polar body extrusion failure.This could be rescued after rescuing oocytes from paprotrain in fresh medium.Cell cycle analysis showed that most oocytes were arrested at metaphase I or telophase I.However,oocyte spindle structure and chromosome alignment were not disrupted after the inhibition of MKlp2 by paprotrain.Taken together,our results demonstrated that MKlp2 is crucial for oocyte maturation by regulating polar body extrusion.Project 2.Profilin 1 plays feedback role on actin-mediated polar body extrusion in mouse oocytesAs a small actin-binding protein,profilin plays a central role in the regulation of actin assembly.However,the functions of profilin in mammalian oocytes are uncertain.To investigate the function of profilin in oocytes,immunofluorescent staining was first used to examine the localization of profilin 1.The results showed that profilin 1 was localized around the meiotic spindles and was eo-localized with cytoplasmic actin.Knockdown(KD)of profilin 1 with specific morpholino(MO)microinjection resulted in polar body extrusion failure.This failure was resulted from an increase of actin expression both at membranes and in the cytoplasm.Furthermore,western blot analysis revealed that the expression of ROCK and the phosphorylation level of myosin light chain(MLC)were significantly decreased after profilin 1 KD.Thus,our results indicated that a feedback mechanism between profilin-actin(PA)and ROCK-MLC2 regulates the actin assembly during mouse oocyte maturation.