Fig. 5: Spatial location of chromosomal territories in late primary spermatocyte nuclei.

a Schematic model summarizing chromosomal spatial location in the spermatocyte nucleus and probe localization on chromosomes. There are two autosomal territories (A/A), X and 4/4 territory close to the nucleolus and Y chromosome. Experimental 1.688 satellite probe to detect X heterochromatin (red), AATAC satellite to detect Y heterochromatin (green), AATAT to detect 4th heterochromatin (cyan), oligo paints to detect X-euchromatin (yellow) and 2 L (purple) are represented on the fly chromosomes (not to scale). b DAPI (gray) distinguishing three chromosomal territories where X and Y satellite probes clearly detects the chromosomes, respectively. Primary spermatocyte nucleus is outlined (white-dashed line) based on DAPI. Asterless was used to stage individual cells by progression of centriole elongation (insets). c, d Spatial location of X and 4th chromosomes. e, g Representative samples showing spatial location of X and 2 L by oligopaints. f Box plots showing the distributions of mean distances between the X (red), or 4th chromosomes (cyan), to the nucleolus (N = 75 chromosome pairs). To measure chromosome distance to the nucleolus, we averaged the distance from the outer heterochromatin probe edges to the closest nucleolus point (p-value ≤ 0.01 two-sided Wilcoxon signed-rank test with continuity correction (asterisk). Images of X and 2 L spatial localization by oligopaints “masked” in Imaris to obtain 3D images to measure the probe-length corrected h and the probe-length and copy number corrected i volume and j sphericity (N = 23 nuclei). The sphericity measure accounts for differences in volume: ϕ = ((π^(1/3))(6Volume)^(2/3))/(Area). h–j Sample size was 23. Boxplots (box = interquartile range (IQR), notch = 95% confidence interval of median, whiskers = ± 1.5xIQR). Significance (p ≤ 0.01, two-sided Welch’s t-test) (asterisk).