Mechanisms Regulating The Meiotic Spindle Assembly, Chromosome Segregation And Aneuploid In Mouse And Porcine Oocytes

ObjectiveAbnormal germinal cell during genesis and maturation is the fundamental cause of infertility and birth-defect, which lead to failured fertilization and defective embryo development. The high incidence of chromosomally abnormal pregnancies in humans has been an intractable genetic puzzle for decades. Approximately30%of oocytes and9%of sperm may be chromosomally abnormal （aneuploid）. Furthermore, the incidence is strongly affected by age. For women at the end of their reproductive lifespan, the risk of ovulating a chromosomally abnormal egg might be50%or higher. Approximately7-10%of clinically recognized pregnancies are chromosomally abnormal,60%of miscarriaged fetus are chromosomally abnormal;5-10%aborted fetus are chromosomally abnormal. High rate of aneuploid in human oocytes seriously affect human fertility and compromise the population qulity.The quality of oocytes has the greatest influence on results of the monospermic fertilization, early development, and implantation of embryos. Therefore, the quality of oocytes can be a determining factor in the fertilization of oocytes, culture of high quality embryos, and treatment of the infertility. In humans, the oocyte plays a central role in providing RNAs and proteins that maintain genomic integrity of the zygote and cleavage-stage embryos until Day3when embryonic genome activation occurs60%embryos are anueploid, most of which can not be implanted. However, if the anueploid oocytes are implanted, the fetus will be miscarriaged and arrested development. High anueploid is the main reason of birth-defects, the low pregrant rate in ART and recurrent abortion, which were confirmed by preimplantation genetic detect in aged females and implantation failure.The age of the mother is the main reason of the increased aneuploidy in birth defect. Germ cells go aging after fetus were born, only2-3%of the women at age of20years old have the oocytes with abnormal chromosomes, but at40, the risk increases to30-35%. The age increase the rate of anueploid in meiosis or mitosis of MI and MII oocytes. In recent30years, the age of the first birth increasing3.6year caused the high rate of abnormal chromosomes in ageing oocytes, which lead to the dramatic decline of fertility in female at35years old. Along with the mature of human assistant reproduction technology, high aneuploidy in oocytes is the main reason in the implantation failure. The reproduction in aged women is the giant challenge for assistant article technology. Some reports think that young women have show ed increasing recurrent spontaneous abortion, aneubploid oocytes and implantation failure after IVF. The phenomenon attribute to over-doses of FSH injection in control ovarian stimulation and progressively environmental pollution, which affected the recombination, synapsis development of oocytes in follicle pool at fetal period and oocytes susceptibility to aneuploidy.Therefore, clarifing the mechanism of aneuploidy in female oocytes play an important role in reducing the generation of aneuploidy and approving the quality of oocytes. Chromosome segregation error lead to aneuploidy in oocytes, directly resulting in embryo development arrest or spontaneous abortion. The normal spindle assembly is the guarantee of correct chromosome segregation. The spindle pole assembly, the spindle form and length obviously affect the chromosome segregation. ensure correct spindle assembly and chromosome segregation. The proteins at spindle poles such as, protein kinase, dynein and microtuble-associated protein and spindle checkpoint protein interact with each other, which regulate the spindle assemble and chromosome assignment. The mitogen-activated protein kinase （MAPK） superfamily comprises classical MAPK （also called ERK）, c-Jun amino-terminal or stress-activated protein kinase （JNK or SAPK） and p38, all of which are highly conserved in all eukaryotic systems. MAPK （ERK） plays important roles in stabilizing and facilitating pole and chromosome separation. In addition, MEK, the upstream regulator of ERK participates in spindle assembly and chromosome alignment p38mitogen-activated protein kinase （p38MAPK） is one of the three major members of the MAPK family that regulates cellular responses including apoptosis, cell proliferation and immune response as well as cell growth, differentiation, and cytoskeletal rearrangements. A lot of research focus on somatic cell, but the mechanism of aneuploidy which is caused by abnormal chromosome segregation in oocytes. mitogen-activated protein kinase family （MAPK） pathway is the one of the most important signaling pathway, it is pivotal in many important biological progresses, such as cell proliferation, cell differentiation and cell cycle regulation. The p38protein is known to be phosphorylated by MKK3and MKK6for activation in response to cell stress and in turn it phosphorylates a number of substrates, including MAPKAP kinase2（MK2）. P38a forms a stable heterodimer with MK2and mediates the phosphorylation and functions of MK2in vitro and in vivo. In the present study, we in the first explore the function of P38aMAPK on meiotic spindle organization and chromosome assignment in mouse oocytes, and understand the mechanism of aneuplord in the mammal oocyte.Part one:p38a MAPK Is a MTOC-Associated Protein Regulating Spindle Assembly, Spindle Length and Accurate Chromosome Segregation during Mouse Oocyte Meiotic MaturationThe assembly of a functional bipolar spindle is critical for accurate chromosome segregation in mammalian oocytes. The size, shape and length of the spindle is relatively constant in one cell during spindle, which is regulated by motor proteins, protein kinase, microtubel-associated protein and pole-associated protein. The mitogen-activated protein kinase （MAPK） superfamily comprises classical MAPK （also called ERK）, c-Jun amino-terminal or stress-activated protein kinase （JNK or SAPK） and p38, all of which are highly conserved in all eukaryotic systems. p38mitogen-activated protein kinase （p38MAPK） is one of the three major members of the MAPK family that regulates cellular responses including apoptosis, cell proliferation and immune response as well as cell growth, differentiation, and cytoskeletal rearrangements. In the present study, we used the mouse oocytes as the model, investigating the roles of p38MAPK in the spindle assembly and spindle checkpoint activation, which enriches our knowledge about this cellular event in meiosis and provides the theoretical basis for the mechanisms forming the aneuploidy in mammalian oocytes.Methods Immature oocytes were collected from ovaries of6-week-old female ICR mice. Firstly, we examined the expression and subcellular localization of this protein by immunoblotting analysis and immunofluorescence; next, we investigate the space localzation bwtween p38MAPK and MTOC in oocytes treated with Taxol。 Furthermore, we examined the phnotype in oocytes by special P38a inhibitor treatment and P38mophornilo injection. In addtion, we tested a possible interaction between endogenous p-p38a and p-MK2by co-immunoprecipitation with p-p38a antibody in mouse MI oocytes extracts and then performed immunoblot analysis with the anti-p-MK2antibody. Chromosome spreading was done for checking aneuploid in MII oocytes. At last, BubRl signal was detected in p38aMO-injected oocytes.Results1. Expression and subcellular localization of phospho-p38aMAPK （p-p38a） during mouse oocyte meiotic maturation The expression level of p-p38a remained stable from GV to MI stage, and then become reduced at the ATI and MII stages by Westernbloting. At the GV stage, p-p38a was localized in the germinal vesicle; after GVBD, multiple bright foci of p-p38a were detected near the individual chromosomes. By Pro-MI when chromosomes began to migrate to the equator of the spindle, p-p38a was gradually translocated to the spindle poles. Notably, p-p38a were specifically concentrated at the spindle poles at the MI stage. No evident signals of p-p38a were labeled in anaphase/telophase, however, p-p38a reappeared at the spindle poles in oocytes at the MII stage.2.p-p38a colocalizes with y-tubulin at cytoplasmic MTOCs and spindle poles in mouse oocytes. p-p38a was colocalized with y-tubulin at the spindle poles and cytoplasmic MTOCs in oocytes at MI and MII stages. Taxol was employed for oocyte treatment. In this case, p-p38a was specifically colocalized with y-tubulin at the poles of the abnormal spindles, the center of MTOCs, and at the center of the cytoplasmic asters in oocytes at Pro-MI, MI and MII stages. Specifically, p-p38a was strictly colocalized with a broad "C"-shaped y-tubulin configuration unattached at the poles.3.Depletion of p38α resulted in abnormal spindles and misaligned chromosomes To explore the roles of p38α, we employed a morpholino-based gene-silencing approach to deplete p38a in oocytes. Using a specific p38a-targeting morpholino （termed p38a-MO）, we successful depleted most of the endogenous p-p38a protein in oocytes. P-p38a depletion did not affect meiotic cell cycle progression and rates of polar body extrusion. Down-regulation of p-p38a resulted in significant defects in spindle formation and chromosomes alignment. Aberrant spindle organization included elongated spindles and various defects in poles including multipolar spindles and disintegrated spindle poles. The rate of abnormal spindle formation in the p38aMO-injected group was74.4%（128/172）, which was considerably higher than that of the control MO-injected group21.3%（40/188）, x2=101.919, P<0.001. P38α-depleted oocytes displayed severe defects in chromosome alignment, showing lagging chromosomes and irregularly scattered chromosomes. The incidence of misaligned chromosomes in the p38aMO-injected group was up to75%（129/172）, much higher than that in the control group (10.10%（19/188）,（x2=156.241, P<0.001）. 4. P38a depletion may compromise meiotic spindle organization and chromosome alignment via the p38α/MK2signaling pathway. Firstly, we tested a possible interaction between endogenous p-p38a and p-MK2by coimmunoprecipitation. p-MK2was clearly decreased in the p38a-depleted oocytes compared with the control group, indicating that p38a could phosphorylate and activate MK2. Subsequently, we examined the p-MK2localization after p38a depletion and demonstrated that p-MK2was dissociated from the spindle poles and chromosomes, and were scattered in the cytoplasm in the p38a-depleted oocytes.5. p38a is required for recruitment of y-tubulin to MTOCs and stabilization of spindle bipolarity. In p38a-depleted MI oocytes, spindle organization was disrupted in over70%of oocytes （n=114）. The typical defects in spindle assembly included aberrant poles （58.2%, n=114）, with striking additional small poles around spindles or near the main poles. Significant γ-tubulin signals were observed in the cytoplasm in p38a-depleted MI oocytes. Moreover, MTOC asters labeled with y-tubulin loosely dispersed within the abnormal spindles or in the cytoplasm in MI oocytes, which indicates that activation of p38a is required for recruitment of cytoplasmic MTOCs to spindle poles. The oocytes with abnormal spindles displayed multiple bright foci loosely dispersed at the defective spindle poles. When treated with cold, microtubule-nucleating components became dissociated from the spindle ends in the p38a-depleted oocytes. Additionally, the minus ends of kinetochore microtubules were not tethered at the spindle poles. p38a was colocalized with Plkl at MTOCs and spindle poles in oocytes treated with taxol and in control oocytes. In p38a-depleted oocytes, Plkl was either completely dissociated from the spindle poles and chromosomes or Plkl signals became faint.6. P38a and Eg5play opposite roles in determining spindle tension and length P38a-depleted oocytes displayed elongated spindles and increased tension between homologous kinetochores in pole-defocused spindles. The distance between homologous kinetochores in pole-defocused MI spindles of p38a-depleted oocytes （2.88μm±0.05, n=10spindles,64kinetochore pairs） was significantly increased compared to that of control oocytes （0.99μm,±0.04, n=8spindles,52kinetochore pairs） F=21.567, P<0.001, indicating that the tension between homologous kinetochores in pole-defocused spindles was notably increased. To quantify spindle length, we measured the spindle length as shown in the schematic. The spindles were50%longer in p38aMO-injected oocytes than those of control MO-injected oocytes （23.3±3.02μm）. Interesting, spindle elongation was observed in Eg5-overexpressed oocytes （37.80±2.09μm）. In contrast, monopolar spindles were observed in Eg5knocked-down oocytes. We further injected oocytes with both p38αMO and Eg5MO. As expected, in p38αMO and Eg5MO co-injected oocytes （25.3±2.72μm）, bipolar spindle morphology and length were rescued.7. P38a depletion affected the distribution of Eg5along microtubules in abnormal spindles. The results indicated that Eg5or dynein depletion did not affect the localization of p38a, and p38a was still localized at spindle poles. However, p38a depletion significantly affected the distribution of Eg5along the spindles. Eg5was localized at microtubules and concentrated at spindle poles in control MO injected oocytes. In Eg5-overexpressed oocytes, Eg5was localized to microtubules but not concentrated at the poles. However, in p38α-depleted oocytes, the expression of Eg5was decreased at poles, but increased on the spindle microtubules, notably at the midzone of the spindle. In some spindles, Eg5signal was only detected in half of the whole spindle.8. P38a depletion inactivated SAC and increased frequencies of aneuploidy. We observed that the ratio of polar body extrusion （PBE） in p38a-depleted oocytes （78.2%, n=234） was not different from that of the control group （79.8%, n=196）. However, p38a-delepted MI and MII oocytes showed obviously misaligned chromosomes. Thus, chromosome spreads were performed in p38a-depleted MII oocytes. Our results showed that p38a-depleted MII oocytes typically displayed incorrect numbers （more or less than20） of univalents. The frequency of aneuploidy of p38a-depleted MII oocytes42.4%（14/33） was significantly higher than that in control oocytes （4.8%（1/21））x2=9.07, P=0.003, P<0.05. We observed that BubRl was enriched at numerous kinetochores in control MO injected oocytes at the prometaphase stage. However, no BubRl signal was detected in p38aMO-injected oocytes.Summary1. P38a is an important component of MTOC, which regulates spindle assembly. P38a specifically colocalized with y-tubulin and Plkl at the center of MTOCs and spindle poles. Knockdown of p38a lead to the disperse of y-tubulin and Plkl at spindle pole, which support that p38a regulate spindle and microtuble nuclear and recruit y-tubulin to MTOC. Depletion of p38a by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2dephosphorylation.2. P38a stablized the spindle and spindle pole in mouse oocyte. Depletion of p38a led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased y-tubulin and Plkl at MTOCs, which indicate that P38a connected the pole microtuble and MTOC with the negative of microtuble. The unclustered pole microtuble and kinetochore microtuble can not be assembled into normal spindle pole after P38a, which indicate that P38a is an important component of pole-associated proteins.3. p38a antagonize the Eg5by indirectly producing a counteracting force, which regulate the tension and length of the spindle. Depleted-P38a led to inccreased spindle length and microtubel tension. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation, and its morpholino injection almost completely rescued spindle elongation caused by p38a depletion, the presence of an antagonistic relationship between Eg5and p38a in mouse oocytes. Given that Eg5generates an outward force by sliding antiparallel microtubules, we considered the possibility that p38a may affect other minus proteins, which generate an inward force and partially antagonized Eg5. Thus, Eg5could be responsible for spindle elongation with increased tension in the absence of p38a. P38a depletion led to imbalance in force between spindle poles, which may be explained the abnormal poles in the absence of p38a.4. p38a play an important pole in the formation of anueploid oocytes by regulating the spindle assemble checkpoint protein on kinetochores in mouse oocytes. Impairment of p38a disturbed the localization of BubRl at the kinetochores of chromosomes and compromised the SAC, which caused misaligned chromosome and subsequent aneuploidy in mouse oocyte meiosis.Part two MK2Regulates Spindle Assembly and Accurate Chromosome Segregation during porcine Oocyte Meiotic MaturationMethodsPorcine COCs were collected for culture in vitro. Firstly, the cumulus cell expansion and meiotic maturation of COCs were checked after the specific MK2inhibitor CMPD1treatment. Next, the meiotic maturation of DOs were investigated after CMPD1treatment. MK2were accurately located in spindle and chromosome、of porcine oocytes by immunofluorescence. To further clarify the correlation among MK2, MTOC, Crest and Plkl, taxol was employed for oocyte treatment. Furthermore, the spindle formation and chromosome alignment were explored after depletion of MK2by CMPD1treatment and MK2antibody injection.Results1. The effect of CMPD1on the cumulus expansion of porcine COCs After porcine COCs were cultured for24h in TMP-199with FSH,85.7%（281/328） of COCs in control were examined as cumulus expansion. High density CMPD1（30μmol/L） significantly inhibited the cumulus expansion （3.3%（8/242））;12.5%（30/240）&24.39%（80/328） were expanded in the presence of20μmol/L and10μmol/L（χ2=546.22, P<0.001）, which indicate that CMPD1inhibits porcine cumulus expansion dependly on the concentration of CMPD1. 2. Evaluating the effect of CMPD1on porcine COCs meiotic maturation. CMPD1dose not affect the meiotic recover of porcine oocyte, however, CMPD1affects the extrusion of the first polar body（pbl）. After porcine COCs were cultured for42h in TMP-199with FSH, the rate of pbl extrusion in porcine oocytes in30,20,10μmol/L showed as0%;6.25%（11/176）&37.9%（50/132）, which significantly decreased in comparison with the control （63.16%（60/95）,χ2=178.17, P<0.001）. Thus, CMPD1inhibit the pbl extrusion in porcine oocytes dependly on the concentrtion of CMPD1.3. Evaluating the effect of CMPD1on meiotic maturation of porcine denude oocytes. CMPD1dose not affect the the meiotic recover of porcine oocyte but the pb1extrusion. The rate of pb1extrusion of porcine oocytes in30,20,10μmol/L showed as0%（0/136）;5.41%（10/185）&19.48%（30/154）, which significantly decreased in comparison with the control54.46%（55/101）,χ2=150.055, P<0.001. Moreover, the rate of pbl extrusion in DO treated with10μmol/L CMPD1siginificantly decreased compared with the control （54.46%（55/101））.4. The subcellular location of p-MK2during porcine oocyte meiotic maturation. After germinal vesicle breakdown （GVBD）, p-MK2was localized along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions. When oocytes progressed to MI, homologous chromosomes aligned at the equatorial plate, and p-MK2localized at the spindle plus ends and chromosomal axes. At anaphase I, the homologous chromosomes were segregated, and the p-MK2signals disappeared from chromosomes and localized to the equatorial region between the separating chromosomes. At MII, the p-MK2signals again translocated to the spindle plus ends and was located between Crest and sister chromatids. P-MK2was partly colocalized with Plkl on chromosome and microtubles or was located between Crest and sister chromatids. P-MK2was partly colocalized was y-tubulin on spindle.5. MK2inhibitor CMPD1affect the spindle assembly and chromosome assignment in porcine oocytes. Down-regulation of MK2resulted in significant defects in spindle formation and chromosomes alignment. Aberrant spindle organization included collapsed spindle surrounded by dispersed chromsomes and irregularly scattered spindle. The rate of abnormal spindle formation in CMPD1treatment group was67.78%（61/90）, which was considerably higher than that of the control group18.35%（20/109）;χ2=49.90, P<0.001. P38a-depleted oocytes displayed severe defects in chromosome alignment, showing lagging chromosomes and irregularly scattered chromosomes. The incidence of misaligned chromosomes in CMPD1treatment group was up to63.33%（57/90）, much higher than that in the control group21.10%（23/109）,χ2=36.560, P<0.001.6. MK2antibody injection disrupted spindle assemble and chromosome assignment in porcine oocytes. The rate of abnormal spindle formation in antibody-injected group was66.9%（79/118）, which was considerably higher than that of the control group21.0%（42/200）;（χ2=66.47, P<0.001）; the incidence of misaligned chromosomes in antibody-injected group was up to70.34%（83/118）, much higher than that in the control group₍23.5%（47/200χ2=67.37, P<0.001）.Summary1. MK2play an important role in cumulus expansion of porcine oocytes induced by FSH through the MK2on the cumulus. MK2inhibitor CMPD1significantly inhibited the cumulus expansion of COCs.2. MK2regulates spindle assemble and chromosome assignment in porcine oocytes. collapse spindles surrounded by the chromosomes is a specific phenotype, which indicates that MK2can connected microtuble with crest. The results from CMPD1treatment and antibody injection support the above views.3. MK2regulates the procession of porcine oocytes during meiotic maturation. However, MK2dose not affect the spontaneous meiotic recovery in GV oocytes.Conclusion1. p38aMAPK regulate spindle, spindle pole organization and chromosome assignment. p-p38α was located at spindle pole and was colocalized with γ-tubulin at MTOC and cytoplasm aster; p38aMAPK can recuit Plk1and γ-tubulin to spindle pole and MTOC and promote the microtuble nucleation. p38aMAPK regulates kintochore microtuble polymerized and stablized the spindles and spindle poles. Thus, p38aMAPK is one component of MTOCs in mouse oocytes and also is a negative microtuble-associated protein.2. p38a and Eg5are an antagonism force, which regulate spindle tension and length. p38a can regulate spindle tension and length through changing Eg5distribution at microtubule, but not affect the location of Dynein. p38a is an important kinase. It needs to be further investigated whether p38aMAPK regulates spindle tension through phosphrylating motor proteins.3. p38a affects the spindle checkpoints BubR1in mouse oocytes and regulates chromosome assignment, which is an important mechanism about mouse aneuploid.4. MK2promotes porcine comulus expandation through FSH induction on the cumulus. MK2also regulates spindle assemble, chromosome separation and connected the Crest with microtubles in porcine oocytes. MK2interupts the pbl extrusion, but not affect the spontanous meiotic recovery in porcine oocytes.5. The location of p38a is charaterized with species distinction. p38a is localized at the cytoplasm of porcine oocytes, but at the spindle poles in mouse oocytes. However, the location of MK2in mouse is the same as in the porcine oocytes. In mouse oocytes, p38a is formed a compound with MK2and MK2is the downstream of p38aMAPK, which phosphrylates Mk2. p38a regulates spindles assemble and chromosome assignments through p38aMAPK-MK2pathway. However, the relatation between p38aMAPK and MK2in the porcine oocytes is different from the mouse oocytes. Thus the mechanism about MK2in porcine oocytes need to further be explored.