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Abstract |
In somatic cells, chromosomes occupy specific nuclear regions called chromosome territories which are involved in the
maintenance and regulation of the genome. Preliminary data in male germ cells also suggest the importance of chromosome
territoriality in cell functionality. Nevertheless, the specific characteristics of testicular tissue (presence of different
cell types with different morphological characteristics, in different stages of development and with different ploidy)
makes difficult to achieve conclusive results. In this study we have developed a methodology to approach the threedimensional
study of all chromosome territories in male germ cells from C57BL/6J mice (Mus musculus). The method
includes the following steps: i) Optimized cell fixation to obtain an optimal preservation of the three-dimensionality cell
morphology, ii) Chromosome identification by FISH (Chromoprobe Multiprobe® OctoChrome™ Murine System; Cytocell)
and confocal microscopy (TCS-SP5, Leica Microsystems), iii) Cell type identification by immunofluorescence
iv) Image analysis using Matlab scripts, v) Numerical data extraction related to chromosome features, chromosome
radial position and chromosome relative position. This methodology allows the unequivocally identification and the
analysis of the chromosome territories of all spermatogenic stages. Results will provide information about the features
that determine chromosomal position, preferred associations between chromosomes, and the relationship between chromosome
positioning and genome regulation. |
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