| BackgroundThe quality of oocyte is fundamental to fertility.Oocytes with good potential are essential for the early development of embryos,and proper meiosis is the premise of producing high-quality oocytes.The meiosis process of mammalian oocytes prior to fertilization can be morphologically divided into four stages:GV stage,GVBD stage,the first meiosis stage and the second meiosis stage.Finally,the mature oocytes stay in the MⅡ stage and wait for fertilization.Unlike mitosis,the meiosis of oocytes is asymmetric,with the oocyte dividing in two consecutive asymmetric divisions to form two small polar bodies and a large polar oocyte to ensure that most of the genetic material remains in the oocyte.In recent years,the Hippo signaling pathway has become a research hotspot because of its important functions in controlling organ size,tissue proliferation,tumor occurrence and so on.When the Hippo signaling pathway is activated,its core effector YAP is phosphorylated and anchored in the cytoplasm.When Hippo signaling pathway is inactivated,non-phosphorylated YAP enters the nucleus to play a transcriptional regulatory role.YAP plays an indispensable role in the reproductive system.In the ovary,oocytes activate YAP in granulosa cells to promote their proliferation,and activation of YAP by drugs or mechanical stimulation can promote the growth of follicles.During preimplantation embryo development,YAP is involved in the establishment of trophoblast ectoderm and the subsequent correct differentiation of the inner cell mass.Knockout of the gene encoding YAP mRNA in mouse zygotes will lead to the inability of embryos to establish normal structures.The role of YAP in oocytes is still unclear.Meiosis is an important process of oocyte development.In this study,we cultured oocytes in vitro and used drugs to affect the activity of YAP to observe its effect on the meiosis process of oocytes.ObjectiveTo detect the expression and localization of YAP,the key component of Hippo pathway,in each stage of meiosis of mouse oocytes.To investigate how YAP regulate the meiosis of mouse oocytes and its mechanism.MethodsGV oocytes of 5-week-old female KM mice were collected and cultured in vitro.Use the RT-qPCR to detect to detect the expression of YAP1,LATS1 and TEAD1 mRNA in each stage of meiosis.Use the immunofluorescence technique to analyze the location of YAP in each stage of meiosis.The collected GV oocytes were randomly divided into three groups:the control group(same volume of solvent DMSO),the inhibitor group(1 μmol/L VP)and the activator group(6 μmol/L XMU-MP-1).After 4 h and 12 h in vitro culture,the GVBD rate and PBE rate of each group were observed and counted,and the distribution of YAP in oocytes and its effect on the structure of spindle and actin cap were analyzed by immunofluorescence technique.ResultsYAP1 mRNA was expressed in all stages of oocyte meiosis,and the expression level increased gradually.YAP was mainly located in the cytoplasm of oocytes in GV phase,and less distributed in the nucleus.Activation of YAP could make it migrate to the nucleus.Compared with the control group,the meiosis of oocytes in the inhibitor group were not affected,while the PBE rate of oocytes in the activator group decreased significantly(14.52± 2.99%,P<0.05).Immunofluorescence assay showed that after 9 h,the oocytes in the activator group had abnormal chromosome arrangements and shorter spindle structures compared with the control group,and the normal actin caps were not formed on the oocyte membranes,resulting in the inhibition of spindle migrations.After 12 h in vitro culture,oocytes in the activator could not excrete the first polar body.ConclusionYAP,the key component of Hippo signaling pathway,is expressed in all stages of mouse oocyte meiosis.YAP plays an important role in the meiosis of mouse oocytes.Interference the activity of YAP can destroy the spindle structures of mouse oocytes and block the formation of actin caps,resulting in the arrest of oocytes at the MI stage. |
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