Volume ratios of painted chromosome territories 5, 7 and X in female human cell nuclei studied with confocal laser microscopy and the Cavalieri estimator

Abstract

Chromosome territories 5, 7 and X were painted in female human amniotic fluid or fibroblast cell nuclei with chromosome specific DNA library probes. For probe detection the fluorochromes FITC or TRITC were applied. Using confocal laser scanning fluorescence microscopy ten to twenty light optical sections of FITC or TRITC images were registered with equal spacing (approximately 400 nm) for each of 131 nuclei showing well separated homologous chromosome territories. Chromosome territory areas were segmented applying a range of gray value thresholds to each section. For each gray value threshold the territory volumes for a given pair of homologs were computed by means of the Cavalieri estimator. The larger volume was divided by the smaller one to obtain a volume ratio. Between 9 and 26 territory volume ratios were calculated for a pair of homologs in a given nucleus. This approach yielded the following results: (i) the range of volume ratios was very similar for both autosome and X‐chromosome territories and (ii) most volume ratios (> 70%) were between 1 and 1.5. The same results were obtained for cells fixed with 4% buffered formaldehyde or methanol/acetic acid indicating that volume ratio measurements are relatively robust against shrinkage effects induced by the latter type of fixation. These similarities of volume ratios argue against the view that the genetic inactivation of one X‐chromosome territory in female cell nuclei should be correlated with a decrease of its volume. Such a decrease was expected in case of a strong overall condensation of this chromosome as compared to other chromosomes.

The “lnc” between 3D chromatin structure and X chromosome inactivation

“…DNA FISH probes that paint chromosomes have generally been employed to estimate chromosomal volume and through application of this technique, multiple studies using RNA/DNA FISH to identify the Xist RNA-coated Xi, have reported that the volume occupied by the Xi is smaller than that of the Xa. Using the total nuclear volume as a normalizer, a standard ratio of between 1.25 +/− 0.05 is consistently reported [55,94,95]. However, to decisively conclude that the nuclear space occupied by the Xi is indeed smaller than that of Xa, the difference between the Xa and Xi should be compared to, and significantly differ from, that between homologous autosomes.…”

“…By conducting this additional normalization step, Rinke and colleagues showed that the average difference in normalized chromosomal volume between X homologs falls within the range observed for autosome pairs. Thus, rather than being caused by increased compaction, the difference in the volume of the Xi compared to the Xa may simply be due to a natural variation in the nuclear space occupied by homologous chromosomes [95]. Since XCI is random, the authors did not distinguish one X-chromosome homologue from another, thus it remains possible that the Xi is indeed consistently smaller than the Xa and that the chance of an autosome occupying a larger volume than its homologue is instead randomly distributed between the two.…”

Morphological Parameters, Medial Lines and Skeletons Computed in the 3D-Voronoi Diagram with Applications to 3D CLSM Images

“…The Voronoi volume estimator is given by equations (3.2) and (3.3). The values obtained by the Voronoi volume estimator were compared with the volume estimates obtained by the Cavalieri estimator after a simple thresholding segmentation of each individual serial section (Rinke et al, 1995). Fig.…”

Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses

“…The spatial intranuclear distribution of chromosome territories appears to depend on cell type and cell cycle (Billia and DeBoni 1991;Vourc'h et al 1993;Cremer et al 1994;Zalensky et al 1995). The different packaging of the DNA is assumed to be associated to different transcription efficiencies of genes (Gerdes et al 1994;Rinke et al 1995).…”