The Location And Function Of KIF2A In In Vitro Maturation Of Human Oocyte

Background:Meiosis maturation of mammalian oocytes is a complex and multi-stage process of precise regulation.Before ovulation,oocytes are arrested at the first meiosis.The first meiotic division continues only after the appearance of the luteinizing hormone(LH)peak.Germinal vesicle breakdown(GVBD)marks the beginning of a mature process.In the oocytes of the first meiotic metaphase(MⅠ),the microtubules assembled near the chromosomes formed a bipolar spindle,providing favorable conditions for the accurate separation of chromosomes.Subsequently,the spindle moves to the cortex under the action of filaments,discharges the first polar body(PB1),and the oocytes enter the second meiosis(MⅡ)awaiting fertilization.At this point,the oocyte has reached meiosis,and after fertilization,it will resume meiosis until the split ends.The kinesin Super family is a group of drive proteins that can be subdivided into 14 subtypes,which plays a key role in macromolecular transport,mitosis and even meiosis.kif2a has the activity of polymerase catalysis,and it belongs to 13 families of kif2b and Kif2c/MCAK.It was first discovered in the brain of mice and can be used to cut the axon of mammalian brain neurons by the action of the micro-tube,thus inhibiting the transition growth of the axons.In mitosis,kif2a as a micro-tube negative end of the protein is located at the spindle poles,MACK as the positive end of the protein is located in the chromosomes of the silk grains,they are respectively directed to the spindle of the two poles of the power and point to the silk grain at the power and mutual coordination,thus maintaining the bipolar spindle.The reduction of kif2a leads to the excessive micro-tube assembly during mitosis,which may seriously affect the stability of the micro-dynamics,leading to the formation of unipolar or multipolar spindles.Recent study of our team suggests that kif2a is also critical in the meiosis of mouse oocytes.The knockout kif2a resulted in a significant decrease in the ability of the mouse oocytes to complete their first meiotic division.In these oocytes,the abnormal rate of spindle increased,and could not be transferred to the oocyte cortex accurately.In addition,the researchers found that kif2a was also involved in the regulation of micro-tube dynamics by raising the SAC’s component BubRl and its binding gametes BUB3 to block oocyte in the MI period.Even if the oocytes are able to complete the first meiosis,the polar bodies they expel are abnormally fat,and this erroneous cell asymmetry may have been caused by the failure of the MI spindle migration.Human KIF2A is the homologous gene of mouse kif2a.To date,whether KIF2A participates in meiosis maturation of human oocytes remains unknown.Purpose:In the study,we investigated the expression,localization and potential roles of KIF2A in human oocyte in vitro maturation,especially in meiosis maturation.It provided the novel evidence for diagnosis and treatment of human oocyte maturation deficiency.Method:We used the transcriptome sequencing information of human female FGCs in GEO datasets and analyzed the mRNA expression of KIF2A in four different developmental phases of FGCs.Human oocytes and embryos at different stages were collected by embryologist,and those that were considered to be of poor quality and not in accord with the criteria set for fertilization or embryo transfer were donated with the informed consent from the patients undergoing ICSI cycles at the Nanfang Hospital.The indication for ICSI treatment was types of male infertility.Firstly,we examined the expression of KIF2A mRNA by single cell-qPCR,the expression and subcellular localization of KIF2A protein by immunofluorescence.Then,we investigated the correlation between KIF2A localization and microtubule dynamics by employing taxol and nocodazole through immunofluorescence.Finally,we knockdown the expression of KIF2A by injecting specific siRNA in to human GV oocytes.After in vitro maturation,we investigated the potential functions in human oocyte maturation.Results:1.The expression of KIF2A in meiosis prophase of human female FGCs is significant.Human female FGCs undergo four distinct sequential phases characterized by mitosis,retinoic acid signaling,meiotic prophase,and oogenesis.We analyzed the mRNA expression of KIF2A in these four phases and found that the expression of KIF2A in meiosis prophase is more significant than in mitosis and oogenesis.This indicated that KIF2A may play an important role in oocyte meiosis maturation.2.The expression of KIF2A in different stages of human oocytes and embryos.KIF2A mRNA was expressed from GV oocyte to the blastocyst stage and expression level was highest at GV stage,verifying that the expression of KIF2A in meiosis prophase is more significant than in mitosis and oogenesis.KIF2A mainly distributed in the nucleus of GV oocyte.At MⅠ and MⅡ stage,it localized to the spindle microtubules,being concentrated at two poles.We also found that KIF2A protein was localized in the whole cytoplasm of oocytes and pre-implantation embryos and continuously expressed from oocytes to blastocyst stage.The localization pattern for KIF2 A implicate that it may play an important role in human oocyte maturation and pre-implantation embryo development.In cell division,Taxol has the function of promoting the coalescence of micro-tube subunit and inhibiting the polymerization of micro-tube.After being treated with taxol,the microtubule fibers excessively polymerized,leading to notably enlarged spindles and multiple asters in the cytoplasm of MⅠ oocyte and MⅡoocyte.Also,KIF2A was co-localized with a-tubulin and accumulated at abnormal spindle poles as well as asters in the cytoplasm,overlapping with y-tubulin.In cell division,nocodazole plays an important role in tumor inhibition by means of the coalescence of micro-tube subunit.In nocodazole-treated oocytes,microtubule fibers were disassembled and Kif2a partly dispersed into the cytoplasm.3.The effects of knockout kif2a on the in vitro maturation of human oocytesFirst,we verified the efficiency of oocyte maturation in vitro.We put the GV oocytes in the maturation medium,and after 24 hours,8 of the 10 oocytes were expelled from the first polar body,with a maturation rate of 80%.Then we ordered four KIF2A siRNA labeled with CY3 red fluorescent marker and negative control siRNA labeled with FAM green fluorescent marker.After injection of these siRNA into human GV oocytes,we only detected the fluorescent signals in oocytes that injected siRNA by using a fluorescent microscope.Therefore,siRNA could enter oocytes and disturb the expression of KIF2A by use microinjection.Then we investigated the efficiency of siRNA.Both mRNA and protein expression level of KIF2A decreased after injection KIF2A siRNA.In this study,knockdown of KIF2A results in abnormal spindles and decrease of first polar body exclusion rate,leading to deficiency of oocyte meiosis maturation.Conclusion:This study indicates that KIF2A as a microtubule polymerase,may regulate spindle assembly,chromosome congression and exclusion of first polar body.It reveals a role of KIF2A in human oocyte and embryonic development.It provides a molecular marker for the clincal diagnosis of infertility caused by deficient oocyte or embryonic development and certain reference value for improving the quality of oocytes and embryos.