The Mechanism Research Of D4Mil1e On Refulating Female Gametes Maturation And Early Embryonic Development In Mice

BACKGROUND:Fertility decline of human all over the world has become a serious social problem which concerned by international,accompanied with the environmental degradation and the pace of life changes.Infertility has became a global problem in the medical field.Although Assisted Reproductive Technology(ART)bring benefit to infertility patients,but such as oocytes with the low ratio of in vitro manturation and fertility,high ratio of heteroploidy,as well as low potency of embryonic development,are reason why the abortion of early embryo and the birth defect of offspring.The chromosome ploidy of mature gametes are halved,which is regulated accurately as the one of the critical factors to ensure the normal development of offspring.A mass of researchs showed that lots of molecules to regulate and participate the dynamic progress of spindle and chromosomes.This process can't do without the important cell function is material transportation.Kinesin is a kind of monomer molecular motor proteins,it was discovered in 1985.It consumes energy provide from adenosine triphosphate(ATP)hydrolyzation to drive itself and carries the cargo moved along with microtubule.Although they shares a conserved catalytic core,each kinesin has distinct subcellular localization and function.D4Mil1e also known as kinesin family member 1B(KIF1B),as the a important microtubule-based cis-form motor molecule,is a important member of this family.The main function of D4Mil1e is in charge for transporting membranous organelles including mitochondria and synaptic vesicle in somatic cell,it also involved in some kinds of malignant diseases and tumor development by regulating cell apoptosis as a potential tumor-suppression factor.OBJECTIVE:Mouse oocytes were uesd for in this experiment,in vitro maturation,in vitro fertilization,microinjection,western blotting,immunofluorescence and chromosome spread were employed to further research the accurately expression,localization,function of D4Mil1a and the role of D4Mi11e in zygote first mitosis after fertilization,our thesis all-around and systematic showed the ration and localization of D4Mil1e in 9 successive stages of oocyte meiosis maturation and early embryo development,further explore the interaction and mechanism of D4Mil1e with spindle,chromosome,mitochondria in several important stages of gamete and embryo.Base on these studys,not only we all-sided illuminate the regulating mechanism of D4Mil1e in mammal oocyte meiosis maturation and early embryo development,but also provided estimable theoretical basis to cure infertility and improve ART.METHODS:(1)The oocyte and early embryo of germinal vesicle(GV),germinal vesicle breakdown(GVBD),metaphase I(MI),anaphase I/telophase I(AI-TI),metaphase II(Mil),anaphase II/telophase ?(AII-TII),pronucleus(PN),Late 1-cell,2-cell during 9 successive stage were obtained by sperovulation with pregnant mare serum gonadotropin(PMSG)united human chorionic gonadotrop(hCG)and in vitro fertilization(IVF).Western blotting,immunofluorescence,laser scanning confocal were employed to detect the precise ration and elaborate subcellular localization of D4Mil1e.(2)On the basis of above experiment,the spindle-perturbing drugs,taxol and nocodazole were used to treat MII oocyte.Immunofluorescence and laser scanning confocal microscope were employed to detect the subcellular localization of D4Mil1e and spindle after microtubule destroyed.Further confirm the interrelation between D4Mil1e and microtubule.(3)On the basis of ensuring the interrelation between D4Mil1e and tubulin,the specific mopholino(MO)of D4Mil1e is more efficient and stable was employed to knock down D4Mil1e to inhibit mRNA translation by microinjection into GV stage oocytes of mice.Western blotting,immunofluorescence were employed to detect the effectiveness;statistics the ratio of GVBD and the first polar body(PB1)at 4 hours,8 hours,12 hours,16hours after the oocyte released from milrinone;CellSens Standard software was used to measure size of spindle and thickness of metaphase plate;analyze the spindle-chromosome complex(SCC)dynamics;chromosome spread were employed to detect the aneuploidy.(4)To further confine the role of D4Mil1e in early embryonic development,the specific MO of D4Mil1e was microinjected into PN stage zygote which beginning mitosis.Western blotting was employed to detect the effectiveness;trace statistics condition of embryonic development in each stages;immunofluorescence was employed to analyze embryo quality.(5)To further confirm the effect of D4Mil1e around the zygotic genome activation(ZGA)on embryonic development,we used the 2-cell stage embryo which obtained by embryo flushing.We first employed the "the same embryo with different blastomere knock down in difference technology" to further ensure effect of D4Mil1e in mitosis during 2-cell embryo of ZGA.The specific MO of D4Mil1e was microinjected into two blastomere by Ctrl MO+Ctrl MO,Ctrl MO+D4mil1e MO and D4mil1e MO+D4mil1e MO,respectively.Immunofluorescence were employed to detect the spindle assembly and chromosome arrangement,trace statistics condition of embryonic development and analyze embryo quality in each stages.(6)On the basis of obtaining the data of MO knock down,to further ensuring the function of D4Mil1e,the specific D4Mil1e antibody was uesd to inhibit D4Mil1e function in protein level,and it was microinjected into GV or MII stage oocyte,respectively.Statistics the ratio of GVBD and the PB1 at 4 hours,8 hours,12 hours,16hours after the oocyte released from milrinone;CellSens Standard software was used to measure size of spindle and calculate ratio of length-width;chromosome spread were employed to detect the aneuploidy;tracking statistics condition of embryonic development in each stages.(7)On the basis of ensuring loss of D4Mil1e lead to abnormal oocyte and embryo development in mice,further explore the downstream mechanism of D4Mil 1 e potential function.The specific MO of D4Mil1e was microinjected into GV stage oocyte to mediate D4Mil1e loss its function,immunofluorescence and NIS Elements Viewer software were employed to detect the changes of mitochondrial distribution and ATP level in mature oocyte.RESULTS:(1)This research systematic displayed the highly correlation between ration,localization of D4Mil1e and oocyte maturation,early embryonic development for the first time.The results showed that expression level,tendency as well as subcellular localization of D4Mil1e during successive 9 stages of oocyte(GV,GVBD,MI,AI-TI,MII)and embryo(AII-TII,PN,Late 1-cell,2-cell).The result of western blotting showed that from GV to 2-cell stage,D4Mil1e all existed,the peak of expression focus on M and AII-TII stage,other stages were low.The result of immunofluorescence showed that D4Mil1e displayed the accumulation of D4Mil1e exhibit a dynamic localization pattern during the progress of meiosis and first mitosis.(2)This research further expounds the correlation between D4Mil1e and microtubule dynamics.The results showed that the oocyte treated with taxol which inhibited microtubule depolymerization,most of D4Mil1e normally colocalized with the abnormal spindle and others dispersed in the cytoplasm.The oocyte treated with nocodazole which promoted microtubule depolymerization,the microtubules completely disassembled and D4Mil1e was also dispersed.(3)This research revealed that the affect and mechanism of D4Mil1e's biological function in oocyte maturation.The results showed that after knock down D4Mil1e by MO in GV stage oocytes,western blotting showed the expression of D4Mil 1 e declined 81.8%(P<0.01),immunofluorescence showed the localization of D4Mil1e abnormal,proved this method with the highly effectiveness;although D4mil1e MO injection groups showed that no effect for percentage of GVBD,but ratio of PB1 declined significantly during oocyte maturation;thickness metaphase plate increased 2.2 times;SCCs displayed arrested in the ball stage,altered spindle pole morphology,chromosome misalignment;compared to the control MO group(arrested in the ball stage:3.5±1.2%,altered spindle pole morphology:9.8± 1.0%,chromosome misalignment:6.3± 1.7%),these ratio of abnormity were increased to 15.5±2.6%,37.1±2.0%and 26.7±2.6%(P<0.01),respectively.Chromosome aneuploidy of D4mil1e MO group also increased 14.4 times than the control MO group after the oocytes were cultured to mature(control MO:2.9±1.8%,D4mil1e MO:44.6±5.2%,P<10-5).(4)This research revealed that the important affect of D4Mil1e on zygotes development.The results showed that after knock down D4Mil1e by MO in PN stage zygotes,western blotting showed the expression of D4Mil1e declined 74.1%(P<0.001);compared to the control MO group(2-cell:94.8I 1.2%,4-cell:81.3±7.1%,Morula:73.5±5.9%,Blastocyst:57.0±7.2%),D4mil1e MO injection groups showed that the ratio of embryo development in 2-cell,4-cell,morula,blastocyst stages declined 85.6±3.6%,27.7±3.8%,19.5±2.5%and 5.7±4.4%-(P<0.05),respectively.Immunofluorescence showed the abnormal spindle assembly and chromosome arrangement when the zygotes beginning first mitosis;cell number declined 56.5%in morula stages compared to control MO group;blastocysts displayed markedly lower quality such as blastocoele form failure,fragmentation and 2-cell stage arrest.(5)This research used "the same embryo with different blastomere knock down in difference technology" to further revealed that the regulating effect of D4Mil1e on early embryo development.The results showed that after knock down D4Mil1e by MO in 2-cell stage embryos,western blotting showed the expression of D4Mil1e declined 61.8%in Ctrl MO+D4mil1e MO injection group(P<0.001),the expression of D4Mil1e declined 90.8%in D4mil1e MO+D4mil1e MO injection group(P<10-4),proved this method with the highly effectiveness;the ratio of embryo development at 4-cell,morula,blastocyst stages in Ctrl MO+D4mil1e MO injection group declined to 44.5±2.5%,35.0±2.9%and 23.6±1.2%(P<10 4),respectively.The ratio of embryo development at 4-cell,morula,blastocyst stages in D4mil1e MO+D4mil1e MO injection group declined to 30.9±3.2%,14.3±2.7%and 8.7±0.4%(P<10-5),respectively,they were all declined significantly compared to Ctrl MO+Ctrl MO injection group(4-cell:85.6±2.3%,Morula:77.1 ± 1.8%,Blastocyst:70.8±2.8%).Immunofluorescence showed the spindle and chromosome in blastomere which injected with D4mil1e MO in Ctrl MO+D4mil1e MO injection groups;cell number of morula stage in Ctrl MO+D4mil1e MO or D4mil1e MO+D4mil1e MO injection groups declined 40.0%or 57.2%;blastocysts quality were all deteriorated accompany with aggravation of D4Mil1e depletion.(6)This research via antibody block technology to further revealed that the affect of D4Mil1e 's biological function on oocyte maturation and embryo development.The results showed that after inhibit D4Mil1e by specific antibody on protein level in GV or MII stages oocytes,no effect for percentage of GVBD;but ratio of PB1 declined significantly during oocyte maturation;the ratio of length-width of MI or MII stages oocytes declined significantly.SCCs displayed arrested in the ball stage,altered spindle pole morphology,chromosome misalignment in MI or MII stages,compared to the IgG group(MI stage:arrested in the ball stage,4.0±0.8%,altered spindle pole morphology,10.0± 1.2%,chromosome misalignment,7.7±0.9%;MII stage:arrested in the ball stage,2.9±1.0%,altered spindle pole morphology,8.7±2.0%,chromosome misalignment,5.8± 1.5%),these ratio of abnormity in MI stage were increased to 13.4±1.4%,30.5± 1.3%and 25.6±1.3%(P<0.01),respectively.These ratio of abnormity in MII stage were increased to 11.0±1.3%,25.2±2.8%and 21.4±2.2%(P<0.01),respectively.Chromosome aneuploidy of D4Mil1e antibody group also increased 6.0 times than the IgG group after the oocytes were cultured to mature(IgG group:3.8±2.4%,D4Mil1e antibody group:26.6±1.3%,P<0.01).Antibody block technology was used in MII oocytes,compared with IgG group(2-cell:56.3±4.1%,4-cell:34.6±5.1%,morula:27.9±4.2%,blastocyst:18.9±4.5%),the ratio of embryo development in D4Mil1e antibody group after fertilization at 2-cell,4-cell,morula and blastocyst stages declined to 47.4±2.4%,19.5± 1.8%,13.7± 1.1%and 7.3±2.5%(P<0.05),respectively.(7)This research further revealed that the affect and mechanism of D4Mil1e 's biological function on oocyte maturation from energy metabolism perspective.The results showed that after knock down D4Mil1e by MO in GV stage oocytes.In MI and MII oocytes stages,the mitochondrial aggregations around the metaphase plate disappeared,agglutinate phenomenon of mitochondria in cytoplasm also disappeared;mitochondrial signal which around the nucleus declined significantly,mitochondrial distribution abnormal;the level of ATP at MI stage declined to 0.3±0.1,compared to control MO group(1.0±0.1)declined 72.0%(P<10 3);the level of ATP at MII stage declined to 0.7±0.02,compared to control MO group(1.0±0.1)declined 34.0%(P<0.01).CONCLUSIONS:In short,these results for the first time to indicate(1)the expression level and subcellular localization of D4Mil1e were highly related to oocyte meiotic maturation and early embryo development;(2)Knock down D4Mil1e in oocytes or early embryos lead to meiosis failure and subsequent embryo development damaged;(3)Knock down D4Mil1e in oocytes result in mitochondrial distribution anomalies and ATP level declined;(4)This research all-sided proved D4Mil1e play a important role in spindle assembly and chromosome congression of oocyte via microtubule-based mechanism for the first time.We provided the theoretical foundation of mechanism on gametogenesis and occurrence on chromosome aneuploidy disease.