Inhibition Of Racl GTPase Activity Affects Porcine Oocyte Maturation And Early Embryo Development

With the rapid development of agriculture and animal husbandry,although livestock and poultry production has been staying stable,the continuous development of society,life quality and increasing demands leaded to a lack of animal production.So how to improve production has become a primary goal.There are many factors that affect the productive process,including environmental factors,disease factors,nutritional factors and their own factors.One of its own factors is the existence of infertility in livestock itself,the defect may be caused by hormone disorders or low quality of livestock sperms or oocytes.Therefore,basic research on the maturation process of maternal oocytes is essentialThe maturation of mammalian oocytes requires two meiosis processes.The first division of mammalian oocytes in the asymmetric meiosis depends on the eccentric localization of the mandrel,leading to the formation and extrusion of polar bodies.The first meiosis and the second meiosis need to ensure the precise separation of the chromosomes in the oocytes,thus ensuring that the large oocytes formed have sufficient genetic material to sustain the development of the offspring.The microfilaments and microtubules in oocytes were reported to regulate the polarization process of oocytes,while the asymmetric division of mammalian oocytes relies on the spindle formation and polar body emission.Mammalian early embryo development is a very complex process,which occurs before the embryo implantation,from zygote development to the blastocyst process.Early embryo development includes three processes:cell division,cell differentiation,and cell morphology changes.Zygote began to occur after fertilization cleavage,and then developed into two-cell,four-cell and eight-cell stage.In the eight-cell stage,the blastomere polarization occurred,embryo continues to cleavage,and then occurred the differentiation into the inner cell mass and trophoblast cells.While the trophoblast cells develop into placenta and fetal membranes,the inner cell mass develops into the various tissues of the fetus in the next process.Small GTPase Racl is a member of the small GTPase and is a member of the Rac subfamily of the Rho family.The family of GTPase is able to regulate a variety of cellular events,including control of cell growth,cytoskeleton recombination and activation of protein kinases.Racl is a multicomponent modulator that includes many cell processes including cell cycle,cell adhesion,and epithelial differentiation.However,how Racl regulates the porcine oocyte maturation and early embryo development is still unknown.In present study we investigated the role of Racl in oocyte maturation and embryo cleavage.We first found that Racl localized at the cortex of the porcine oocytes,and disrupting the Racl activities by treating with NSC 23766 led to the failure of polar body emission.In addition,a majority of treated oocytes exhibited abnormal spindle morphology,indicating that Racl may involve into porcine oocyte spindle formation.This might be due to the regulation of Racl on MAPK,since p-MAPK expression decreased after NSC 23766 treatments.Moreover,we found that the position of most meiotic spindles in treated oocytes were away from the cortex,indicating the roles of Racl on meiotic spindle positioning.Our results also showed that inhibition of Racl activity caused the failure of early embryo development.Therefore,our study showed the critical roles of Racl GTPase on porcine oocyte maturation and early embryo cleavage.Experiment 1 small GTPase Racl affects porcine oocyte maturation by regulating spindle assemblyThe formation and positioning of the spindle plays an important role in the meiosis of oocytes.Studies already showed that Racl regulates the spindle anchoring in mouse oocytes,and further causes the first polar body extrusion failed.However,the dynamics of how Racl regulates the porcine oocytes maturation is still unclear.Therefore,we explore how the small GTPase Racl regulates the meiosis in porcine oocyte.Firstly,immunofluorescence staining showed that Racl localized in the cortical region of porcine oocyte.The activity of Racl in the oocytes was inhibited by NSC 23766,which resulted in the failure of the first polar body extrusion.After subsequent immunofluorescence staining observation and found that the inhibition of Racl leads to the abnormal spindle formation and incorrect migration;In addition,with the inhibitor of Racl,p-MAPK expression of the treatment oocytes was significantly reduced when comparing to the control group.As a result,this experiment shows that Racl plays an important role for the formation and migration of porcine oocyte spindles and polar bodies emission.Experiment 2 Racl affects the early embryo development of porcineAlthough our study shows that Rac1 affects the maturation of porcine oocytes by affecting the formation and migration of porcine oocyte spindles,whether it acts on early embryo development is still unknown.In our study,we utilized immunofluorescence staining to detect localization of Rac1 and we found it located at the cortex of the early embryos.And the localization is similar to that of Rac1 in porcine oocytes,co-located with microfilaments.Then,we treated porcine early embryos with the Rac1 inhibitor and found that a great amount of embryos did not grow to blastocysts stage.The results of these experiments showed that inhibition of Rac1 led to a failure of early embryo development.