Functional Study Of Protein Regulating Cytokinesis 1 In Mouse Oocyte Meiosis Maturation And Preimplantation Embryo Development

Protein Regulating Cytokinesis 1(PRC1)is an important member of the microtubule binding protein family,and is a substrate of cyclin-dependent kinases.Previous studies reported that PRC1 plays important roles in mitotic spindle organization,cell proliferation,and cytokinesis.However,there are few reports about PRC1 in mammalian oocyte meiosis maturation and preimplantation embryo development.During oocyte meiosis maturation,precise control of chromosome pairing and segregation as well as asymmetric division is the prerequisite for embryo development.Spindle is a temporary dynamic structure during oocyte meiosis.Spindle's proper organization and migration ensures the accurate chromosome separation and asymmetric cytokinesis.Haploid oocyte became a diploid embryo after fertilization,and then developed to blastocyst for implantation after successive and multiple cleavages.Correct cleavage and development of preimplantation embryo is the prerequisite for healthy birth.Therefore,in the present study,we for the first time explored the function and potential mechanism of PRC1 in mouse oocyte meiosis maturation and preimplantation embryo development.OBJECTIVE:To study the function and mechanism of PRC1 in mouse oocyte meiotic maturation and preimplantation embryo development,which providing foundation for clinical screening of aneuploidy oocyte and prevention of birth defects.METHODS:(1)Stages of oocytes at GV,GVBD,MI,AI-TI and MII were collected.The expression and localization of PRC1 were analyzed by Western blot and immunofluorescence with laser confocal scanning.Stages of preimplantation embryos at AII-TII,PN,late 1-cell,2-cell,4-cell,morula,and blastocyst were collected for PRC1 localization analysis by immunofluorescence and confocal laser scanning.(2)MII oocytes were treated with the spindle-perturbing drugs(Taxol and Nocodazole).Immunofluorescence and confocal laser scanning were employed to detect the localization of PRC1 and spindle after microtubule was destroyed.The spatial position interrelation between PRC1 and microtubule were further confirm.(3)In order to explore the function of PRC1 in oocyte meiosis,the technique of functional knockdown was employed.Specific Prc1 Morpholino(MO)was used to microinject into oocyte cytoplasm.The rate of GVBD and first polar body(PB1)extrusion were counted.The volume of PB1 was measured;spindle-chromosome complex(SCC)dynamics were detected by immunofluorescence staining and confocal laser scanning technology.Chromosome spread technique was employed to detect the aneuploidy.MT-kinetochore attachment was detected by immunofluorescence staining.(4)Prc1 MO,GFP-Tubulin and mCherry-H2 B mRNA were microinjected into oocyte cytoplasm.Time-lapse imaging was employed to observe spindle migration process and the migration distance.Bridging microtubule organization was examined by immunofluorescence staining and confocal laser scanning technology.Phalloidin probe was utilized as the microfilament protein(F-Actin)marker for detecting its distribution in cortical.(5)Exogenous transcription Prc1 mRNA was microinjected into oocyte cytoplasm for PRC1 overexpression.The rate of GVBD and PB1 extrusion were counted.The volume of PB1 was measured.SCC dynamics were detected by Timelapse imaging after GFP-Tubulin and mCherry-H2 B mRNA injection.Chromosome spread technique was empoyed to detect the aneuploidy.(6)In order to explore the function of PRC1 in preimplantation embryo,Prc1 MO was microinjected into PN stage embryo,before the first mitosis in zygote.The percentages of embryo developed to 2-cell,4-cell,morula,and blastocyst were counted at 24 h,48 h,72 h and 96 h,respectively.The blastomere numbers of embryos at 72 h were counted by confocal laser scanning technology.(7)To further explore the roles of PRC1 in embryo development after zygotic genome activation,embryo at 2-cell stage with one blastomere injected Ctrl MO-Lissa with a red fluorescent group and the other injected Prc1 MO-Carboxy with a green fluorescent group was utilized to observe blastomeres division.Immunofluorescence staining and confocal laser scanning technology was employed to detect spindle organization and chromosome arrangement in two different blastomeres.Fluorescent protein mRNA of GFP-Tubulin and mCherry-H2 B were microinjected into cytoplasm to detect the dynamic changes of SCC in different blastomeres with PRC1 knockdown.RESULTS:(1)Western blot results showed that the expression of PRC1 in meiotic GV and GVBD was relatively low,but significantly increased from entering metaphase.Immunofluorescence results showed that the localization of PRC1 in oocyte and embryo were divided into three modes: when oocyte or embryo nuclear membrane was intact,PRC1 was mainly distributed in nucleus;when the nuclear membrane broke down,PRC1 was mainly distributed in cytoplasm;when entering metaphase and anaphase,PRC1 was mainly localized in the spindle midzone,co-localized with the central spindle.After treatment with Taxol that inhibiting the depolymerization of microtubule,abnormal aggregation of PRC1 was found.After treatment with Nocodazole that accelerating the depolymerization of microtubule,the location of PRC1 was disappear.The co-localization between PRC1 and the central spindle microtubule was further confirmed.(3)PRC1 knockdown did not affect the process of GVBD,suggesting that PRC1 do not participate in the meiosis resumption.But the rate of PB1 extrusion was significantly decreased(51.4 ± 3.0%).The volum of PB1 was increased after PRC1 was knockdown.Percentage of abnormal spindle organization was increased(spindle arrest in ball stage 24.4 ± 1.6%;cyclindric spindle 36.1 ± 2.5%).The stability of the spindle poles was affected,and the rate of two abnormal poles increased to 87.2 ± 2.5%.The percentage of aneuploidy was increased(78.7 ± 4.4%).The kinetochore microtubule could not accurately capture the kinetochore,and the abnormal rate was increased to a high level of 78.6 ± 4.5%.(4)Spindle migration was failed after bridging microtubule was depolymerized.And the distribution of F-Actin in cortical area became abnormal compared with PRC1 knockdown,which finally led to the approximately symmetrical oocyte cytokinesis.(5)Function of PRC1 in oocyte was further confirmed by overexpression experiment.The results showed that PRC1 overexpression did not affect the process of GVBD,but significantly decreased the percentage of PB1 extrusion(42.4 ± 3.0%).The spindle organization and chromosome arrangement became defective,and the rate of aneuploidy was significantly increased(67.6 ± 4.2%).(6)PRC1 knockdown at PN stage embryo did not affect 2-cell rate(90.6 ± 1.7%),but significantly decreased 4-cell(52.0 ± 8.8%),morula(31.5 ± 4.7%)and blastocyst(13.7 ± 3.2%)development rate.The absence of PRC1 impacted blastomere cleavage and reduced the cell number in embryos.(7)PRC1 knockdown in double blastomere at 2-cell stage embryo exhibited blastomere cytokinesis failures due to absence of PRC1.And this absence also impacted spindle organization and chromosome arrangement.CONCLUSIONS:(1)The protein expression of PRC1 and its subcellular localization are highly related to the progression of oocyte meiosis and preimplantation embryo development.(2)In oocyte,PRC1 maintain spindle stability and integrity via regulating bridge microtubule organization,and thus facilitate the completion of precise chromosome separation through promoting accurately capture between kinetochore microtubule and kinetochore.(3)In oocyte,PRC1 promotes oocyte asymmetric cytokinesis through regulating spindle migration.And this procedure was completed by the PRC1 controlling bridge microtubule organization and cortical F-actin distribution.(4)In preimplantation embryo,PRC1 promote embryo blastomere cleavage and development through regulating SCC dynamic change.