Automated nuclear cartography reveals conserved sperm chromosome territory localization across 2 million years of mouse evolution

Abstract

16Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. 17 This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the 18 nucleus into zones, and manually scoring which zone a FISH signal lies in. This is time consuming, limiting 19 the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for 20 automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for 21 nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then 22 dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal 23 within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and 24 two autosomes in three mouse lineages (Mus musculus domesticus, Mus musculus musculus and Mus spretus). 25We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from 26 interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse 27 evolution. 62Mus musculus domesticus and Mus spretus. Of these, M. spretus has a notably different nuclear shape [18] to 63 the others, being shorter and wider, allowing us to test whether chromosome position is conserved across 64 structurally equivalent regions. 65 Materials and Methods 66Sample collection 67 68We collected sperm from wild-derived inbred mouse strains Mus musculus musculus (PWK/PhJ), M. m. 69 domesticus (LEWES/EiJ) and Mus spretus (STF). All animal procedures were in accordance with the University 70 of Montana Institute for Animal Care and Use Committee (protocol 002-13) and were subject to local ethical 71 review. Animals were bred at the University of Montana from mice purchased from Jackson Laboratories (Bar 72Harbor, ME) or were acquired from Francois Bonhomme (University of Montpellier). Animals were housed 73 singly or in small groups, sacrificed via CO2 followed by cervical dislocation, and tissues were collected post 74 mortem for analysis. Sperm were collected and fixed in 3:1 methanol-acetic acid as previously described [18]. 75

Differential Sperm Motility Mediates the Sex Ratio Drive Shaping Mouse Sex Chromosome Evolution

“…This in turn would enrich for X-bearing sperm among the population reaching the site of fertilization in the oviduct. We therefore systematically tested for morphological differences between X- and Y-bearing sperm, using a novel image analysis tool for quantitative sperm morphometry [29, 30], and a repeat-imaging protocol (Figure S1) that allowed us to capture pre-fluorescence in situ hybridization (FISH) morphology data and correlate this with post-FISH identification of X- versus Y-bearing status.…”

Differential sperm motility mediates the sex ratio drive shaping mouse sex chromosome evolution

“…Therefore, despite the confounding effects of unrelated background sperm morphology distortions, the cluster analysis overall supports the circularity and regularity measurements showing that on both genetic backgrounds, Yq deletion and shSLY knockdown lead to more severe morphological defects in Y-bearing sperm. On the C57Bl6 background, we also analysed chromosome localisation within the nucleus using dynamic warping [45] (Figure S2). This showed no change in chromosome territory localisation, indicating that the morphological changes are likely driven by changes in cytoskeletal dynamics during spermiogenesis rather than differences in chromatin organisation.…”

“…Slides were washed in 2xSSC for 2x10 minutes to remove mounting medium, and dehydrated through an ethanol series (70%, 80%, 100%, 2mins at RT). FISH was performed as previously described [45] using X and Y chromosome paints (Cytocell, Cambridge, UK), and images were captured as described above. The saved slide positions were used to perform automated batch capture of the previously imaged nuclei.…”

“…Dendrograms were cut, with cluster number determined following visual inspection, and cell cluster membership was imported back into Nuclear Morphology Analysis to allow consensus nucleus building. FISH images from males on the C57BL6 background were further used to assess nuclear organisation via dynamic signal warping, as previously described [45].…”

Differential Sperm Motility Mediates the Sex Ratio Drive Shaping Mouse Sex Chromosome Evolution

“…Therefore, despite the confounding effects of unrelated background sperm morphology distortions, the cluster analysis overall supports the circularity and regularity measurements showing that, on both genetic backgrounds, Yq deletion and shSLY knockdown lead to more severe morphological defects in Y-bearing sperm. On the C57Bl6 background, we also analyzed sex chromosome localization within the nucleus using dynamic warping [30] ( Figure S3). This showed no change [25].…”