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

Abstract

The physical arrangement of chromatin in the nucleus is cell type and species-specific, a fact particularly evident in sperm, in which most of the cytoplasm has been lost. Analysis of the characteristic falciform (“hook shaped”) sperm in mice is important in studies of sperm development, hybrid sterility, infertility, and toxicology. However, quantification of sperm shape differences typically relies on subjective manual assessment, rendering comparisons within and between samples difficult. We have developed an analysis program for morphometric analysis of asymmetric nuclei and characterized the sperm of mice from a range of inbred, outbred, and wild-derived mouse strains. We find that laboratory strains have elevated sperm shape variability both within and between samples in comparison to wild-derived inbred strains, and that sperm shape in F1 offspring from a cross between CBA and C57Bl6J strains is subtly affected by the direction of the cross. We further show that hierarchical clustering can discriminate distinct sperm shapes with greater efficiency and reproducibility than even experienced manual assessors, and is useful both to distinguish between samples and also to identify different morphological classes within a single sample. Our approach allows for the analysis of nuclear shape with unprecedented precision and scale and will be widely applicable to different species and different areas of biology.

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

“…On both genetic backgrounds, our morphological analysis confirmed the known ma of Yq deletion including increased acrosomal curvature, and shortening of the apical hook (Figure 2A,B). Surprisingly, both Yq marked reduction in all linear dimension measures and in overall cross relative to wild type sperm, which has not been previously described ( [44]. The X axis is an index representing percentage of the total perimeter effect size on both genetic backgrounds.…”

“…The insets highlight the increased acrosomal curvature seen in the mutant genotypes, with '*' indicating the point of greatest difference in curvature between genotypes, at index 85. C) Comparison of X-and Y-bearing sperm in standard morphometric parameters (see [44] for definitions), compared to mean wild-type values. Italics indicate size-independent parameters.…”

“…Mice were sacrificed by cervical dislocation and sperm collected from cauda epididymis and vas deferens as previously described [44]. The sperm samples were rinsed in 3 washes of 1xPBS and fixed in 3:1 methanol:acetic acid.…”

“…Morphology analysis was performed using the ImageJ plugin 'Nuclear Morphology Analysis' [44] , version 1.15.1, available at https://bitbucket.org/bmskinner/nuclear_morphology/wiki/Home. In addition to the previouslydescribed measurements, we developed a simple user interface to allow FISH images and pre-FISH images to be displayed side by side.…”

Myosin VI moves on nuclear actin filaments and supports long-range chromatin rearrangements

“…Images were taken using an Olympus BX53 with a 40x oil-immersion objective. Over 60 nuclei per condition were then analysed using the nuclear morphology analysis software, (https://bitbucket.org/bmskinner/nuclear_morphology/wiki /Home) (Skinner et al, 2019).…”