| 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. |
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