A high-throughput method for unbiased quantitation and categorisation of nuclear morphology

Abstract

2The physical arrangement of chromatin in the nucleus is cell type and species specific. This 3 is particularly evident in sperm, in which most of the cytoplasm has been lost; the shape of 4 the nucleus reflects the shape of the cell. Mice have distinctive falciform ('hook shaped') 5 sperm heads and nuclei. Quantification of the differences in shape variation between mouse 6 species and lines often relies on manual measurement and classification that leads to 7 subjective results, making comparisons within and between samples difficult. 8 9We have developed an analysis program for assessing the morphology of asymmetric 10 nuclei, and characterised the sperm of mice from a range of inbred, outbred and wild-derived 11 mouse lines. We find that laboratory lines have elevated sperm shape variability both within 12 and between samples in comparison to wild-derived inbred lines, and that sperm shape in 13 the F1 offspring of CBA and C57Bl6J lines is subtly affected by the direction of the cross. 14 15Hierarchical clustering can distinguish distinct sperm shapes with greater efficiency and 16 reproducibility than even experienced manual assessors. We quantified the range of 17 morphological defects in the inbred BALB/c line, demonstrating we can identify different 18 morphological subgroups. This approach has applications for studies of sperm development, 19 infertility and toxicology. 20

A conserved requirement forFbxo7during male germ cell cytoplasmic remodelling

“…Heterozygous Fbxo7 LacZ/+ males also showed a slight increase in the frequency of abnormally shaped sperm, with the most severely deformed sperm resembling the homozygous phenotype ( Figure 1G). Using a newly-developed image analysis programme for sperm morphometry (Skinner et al, 2019), we determined that the remaining sperm from Fbxo7…”

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

“…Animals were housed singly or in small groups, sacrificed via CO2 followed by cervical dislocation, and tissues were collected post mortem for analysis. Sperm were collected and fixed in 3:1 methanol-acetic acid as previously described [18].…”

“…Analysis was performed using our image analysis software (Nuclear Morphology Analysis, available from http://bitbucket.org/bmskinner/nuclear_morphology/wiki/Home/, version 1.15.0) for morphometric analysis of mouse sperm shape [18]. Here, we combine nuclear morphometry with FISH signal detection in order to rigorously quantify the distribution of chromosome territories within the asymmetric mouse sperm head.…”

“…The process of warping FISH images. A) Examples of un-FISHed nuclei from the three strains, as described in [18]. B) After FISH, nuclei are automatically identified and landmarks are discovered.…”

“…First, we identify key landmarks around the periphery of the nucleus (i.e. the apical hook, tail attachment site, and other areas of maximal curvature), as described previously [18]. Next, semi-landmarks are constructed by spacing a set number of equidistant points between each landmark (Figure 1Ci).…”