双光子成像量化观测小鼠卵母细胞染色体三维构象
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摘要
卵母细胞成熟要经历两次减数分裂过程，染色质逐渐卷曲凝聚成染色体。此过程中染色体构象(指染色体空间位置、形态、分布等)经历了许多复杂变化。如何可视化并原位定量分析染色体构象的“时-空”四维发育特点，对认识复杂的遗传与变异过程，及探讨重大疾病的机理有重要作用。由于卵母细胞体积大、光散射强、对发育环境敏感，因此要在微干扰条件下，原位观测并量化分析卵母细胞染色体三维构象随发育的变化特点，存在较大困难，目前此类研究较少。
本文利用穿透能力强、光损伤性小、三维分辨率高的双光子荧光成像系统和图像重构技术，提出量化分析卵母细胞染色体构象的方法，尝试通过染色体体积、表面积、类球因子（RF）、平均距离、粘连程度、包裹染色体圆柱的高宽比（H/D）等指标定量描述染色体的形态、形貌、空间分布等构象特点。并将该方法应用于诱发有机体生理环境改变的内因“衰老”和外因“氨甲喋呤（MTX）”对小鼠卵母细胞染色体构象影响的研究中。
我们的研究结果表明：衰老导致卵母细胞质量下降。在第一次减数分裂前中期和中期，衰老干扰染色体的正常凝聚，引起染色体平均体积和表面积增大，平均距离缩短，粘连加重，形成团状体。在第二次减数分裂中期，衰老增加了小鼠体内成熟卵母细胞染色体错误排列的几率，并造成染色体的总体积、总表面积、H/D值增大。衰老还影响了卵胞质内DNA甲基转移酶蛋白的空间定位及表达水平。
MTX是常用的抗肿瘤药物，但其具有干扰叶酸代谢功能，可能引起生殖异常。实验结果表明，腹腔注射MTX可造成小鼠体内成熟卵母细胞染色体异常空间排布比率升高、纺锤体形态改变。亚叶酸钙和自身代谢两种回救方式都可缓解MTX对体内成熟卵母细胞的毒副影响，恢复染色体空间排布及纺锤体形态至正常状态。
本文还提出一种实时观测减数分裂纺锤体四维运动的便捷方法，并通过空间位置、速度、加速度等指标量化分析其运动特点。该方法基于我们观察到的一种重要实验现象：无损染料Fura-2标记小鼠活卵母细胞内钙离子，由于Fura-2与钙离子结合后双光子激发截面减小，钙离子的聚集使第一次减数分裂中期染色体赤道板两侧的纺锤体区域，形成两个对称荧光弱区。此现象即荧光弱区与纺锤体共定位，通过实时追踪活卵母细胞内荧光弱区的运动，可获知纺锤体的动态行为。
The oocyte maturation undergoes two meiotic divisions, and the chromatintransforms into the chromosome. In this process, the chromosomal conformation, suchas the chromosomal spatial position, morphology, arrangement, etc., experiences thecomplex changes. How to visualize and quantitatively analyze the ‘spatio-temporal’four-dimensional developmental characteristics of the chromosomal conformation, isvery important for learning the complicated process of the heredity and variation, andresearching the mechanism of the emergent diseases. The oocytes have the features oflarge size, high light scattering and sensitivity to the developmental environment whichmake their chromosomal conformation difficult to be observed and qualitativelyassessed nearly without interference. At present, there are few studies on quantitativeanalyses of oocyte chromosomes.
This thesis provides a method for quantitative analysis of the chromosomalconformation in the oocytes by the image reconstruction technique and two-photonfluorescence imaging system which provides high penetration capability, minimalphotodamage, and high three-dimensional spatial resolution. We attemptted to usechromosomal volume, surface area, roundness factor, mean distance, degree ofadherence, and the ratio of height to diameter of a circular cylinder enclosing thechromosomes (H/D), etc., to quantitatively depict the morphology, shape, and spatialarrangement of the chromosomes. This methodology was applied to study the changesof the chromosomal conformation caused by aging and methotrexate which were theinternal and external inducements to the changes in physical environments of anindividual organism respectively.
Our results showed that aging leaded a decline on the oocyte quality. At theprometaphase and metaphase of meiosis I, aging interfered with the condensed foldedform of the chromosomes, increasing both the mean volume and the mean surface areaof chromosomes. The mean distance was shorter, the adhesion among chromosomesbecame more severe, and the chromosomes huddled together. At the metaphase ofmeiosis II, aging caused increased errors in the chromosomal arrangement in in-vivomatured mouse oocytes, and leaded the increased total volume and surface area of chromosomes and the high H/D value. Also, aging had effects on the localizations andexpression levels of the DNA methyltransferases in the oocyte cytoplasm.
Methotrexate is a common antineoplastic agent with the function of interfering themetabolism of folic acid and leading the reproduction abnormality possibly. Our resultsshowed that the peritoneal injection of the methotrexate leaded an increase in the rate ofthe in-vivo matured oocytes having abnormal chromosomal arrangement, and thechanges in the spindle morphology. Both calcium folinate and body metabolism canrescue the in-vivo matured oocytes from the toxic and adverse effects of themethotrexate, and can help the chromosomal arrangement and the spindle morphologyreturn to normal.
Additionally, this thesis provides a convenient method for real-time visualizationof meiotic spindle movements, and quantifies its movements by the spatial position,speed, and acceleration, etc. The method was based on an important phenomenonobserved in our experiment. The calcium ions in living mouse oocytes were labeledwith the non-damaging dye Fura-2. Since the two-photon excitation cross section ofFura-2was much lower after binding to calcium ions, two symmetrical weaklyfluorescent areas, caused by calcium ions accumulation, appeared on both sides of theequatorial plate of the chromosomes where the spindle located at the metaphase ofmeiosis I. This indicated co-localization of calcium ions and the spindles. By real-timetracking of the weakly fluorescent areas in living mouse oocytes, the movements of themeiotic spindle were recorded.
本文利用穿透能力强、光损伤性小、三维分辨率高的双光子荧光成像系统和图像重构技术，提出量化分析卵母细胞染色体构象的方法，尝试通过染色体体积、表面积、类球因子（RF）、平均距离、粘连程度、包裹染色体圆柱的高宽比（H/D）等指标定量描述染色体的形态、形貌、空间分布等构象特点。并将该方法应用于诱发有机体生理环境改变的内因“衰老”和外因“氨甲喋呤（MTX）”对小鼠卵母细胞染色体构象影响的研究中。
我们的研究结果表明：衰老导致卵母细胞质量下降。在第一次减数分裂前中期和中期，衰老干扰染色体的正常凝聚，引起染色体平均体积和表面积增大，平均距离缩短，粘连加重，形成团状体。在第二次减数分裂中期，衰老增加了小鼠体内成熟卵母细胞染色体错误排列的几率，并造成染色体的总体积、总表面积、H/D值增大。衰老还影响了卵胞质内DNA甲基转移酶蛋白的空间定位及表达水平。
MTX是常用的抗肿瘤药物，但其具有干扰叶酸代谢功能，可能引起生殖异常。实验结果表明，腹腔注射MTX可造成小鼠体内成熟卵母细胞染色体异常空间排布比率升高、纺锤体形态改变。亚叶酸钙和自身代谢两种回救方式都可缓解MTX对体内成熟卵母细胞的毒副影响，恢复染色体空间排布及纺锤体形态至正常状态。
本文还提出一种实时观测减数分裂纺锤体四维运动的便捷方法，并通过空间位置、速度、加速度等指标量化分析其运动特点。该方法基于我们观察到的一种重要实验现象：无损染料Fura-2标记小鼠活卵母细胞内钙离子，由于Fura-2与钙离子结合后双光子激发截面减小，钙离子的聚集使第一次减数分裂中期染色体赤道板两侧的纺锤体区域，形成两个对称荧光弱区。此现象即荧光弱区与纺锤体共定位，通过实时追踪活卵母细胞内荧光弱区的运动，可获知纺锤体的动态行为。
The oocyte maturation undergoes two meiotic divisions, and the chromatintransforms into the chromosome. In this process, the chromosomal conformation, suchas the chromosomal spatial position, morphology, arrangement, etc., experiences thecomplex changes. How to visualize and quantitatively analyze the ‘spatio-temporal’four-dimensional developmental characteristics of the chromosomal conformation, isvery important for learning the complicated process of the heredity and variation, andresearching the mechanism of the emergent diseases. The oocytes have the features oflarge size, high light scattering and sensitivity to the developmental environment whichmake their chromosomal conformation difficult to be observed and qualitativelyassessed nearly without interference. At present, there are few studies on quantitativeanalyses of oocyte chromosomes.
This thesis provides a method for quantitative analysis of the chromosomalconformation in the oocytes by the image reconstruction technique and two-photonfluorescence imaging system which provides high penetration capability, minimalphotodamage, and high three-dimensional spatial resolution. We attemptted to usechromosomal volume, surface area, roundness factor, mean distance, degree ofadherence, and the ratio of height to diameter of a circular cylinder enclosing thechromosomes (H/D), etc., to quantitatively depict the morphology, shape, and spatialarrangement of the chromosomes. This methodology was applied to study the changesof the chromosomal conformation caused by aging and methotrexate which were theinternal and external inducements to the changes in physical environments of anindividual organism respectively.
Our results showed that aging leaded a decline on the oocyte quality. At theprometaphase and metaphase of meiosis I, aging interfered with the condensed foldedform of the chromosomes, increasing both the mean volume and the mean surface areaof chromosomes. The mean distance was shorter, the adhesion among chromosomesbecame more severe, and the chromosomes huddled together. At the metaphase ofmeiosis II, aging caused increased errors in the chromosomal arrangement in in-vivomatured mouse oocytes, and leaded the increased total volume and surface area of chromosomes and the high H/D value. Also, aging had effects on the localizations andexpression levels of the DNA methyltransferases in the oocyte cytoplasm.
Methotrexate is a common antineoplastic agent with the function of interfering themetabolism of folic acid and leading the reproduction abnormality possibly. Our resultsshowed that the peritoneal injection of the methotrexate leaded an increase in the rate ofthe in-vivo matured oocytes having abnormal chromosomal arrangement, and thechanges in the spindle morphology. Both calcium folinate and body metabolism canrescue the in-vivo matured oocytes from the toxic and adverse effects of themethotrexate, and can help the chromosomal arrangement and the spindle morphologyreturn to normal.
Additionally, this thesis provides a convenient method for real-time visualizationof meiotic spindle movements, and quantifies its movements by the spatial position,speed, and acceleration, etc. The method was based on an important phenomenonobserved in our experiment. The calcium ions in living mouse oocytes were labeledwith the non-damaging dye Fura-2. Since the two-photon excitation cross section ofFura-2was much lower after binding to calcium ions, two symmetrical weaklyfluorescent areas, caused by calcium ions accumulation, appeared on both sides of theequatorial plate of the chromosomes where the spindle located at the metaphase ofmeiosis I. This indicated co-localization of calcium ions and the spindles. By real-timetracking of the weakly fluorescent areas in living mouse oocytes, the movements of themeiotic spindle were recorded.
引文
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