In common with other mammalian sex chromosomes, the mouse sex chromosomes are enriched for genes with male-specific function such as testis genes. However, in mouse there has been an unprecedented expansion of ampliconic sequence containing spermatid-expressed genes. We show via a phylogenetic analysis of gene amplification on the mouse sex chromosomes that multiple families of sex-linked spermatid-expressed genes are highly amplified in Mus musculus subspecies and in two further species from the Palaearctic clade of mouse species. Ampliconic X-linked genes expressed in other cell types showed a different evolutionary trajectory, without the distinctive simultaneous amplification seen in spermatid-expressed genes. The Palaearctic gene amplification occurred concurrently with the appearance of Sly, a Yq-linked regulator of post-meiotic sex chromatin (PMSC) which acts to repress sex chromosome transcription in spermatids. Despite the gene amplification, there was comparatively little effect on transcript abundance, suggesting that the genes in question became amplified in order to overcome Sly-mediated transcriptional repression and maintain steady expression levels in spermatids. Together with the known sex-ratio effects of Yq/Sly deficiency, our results suggest that Sly is involved in a genomic conflict with one or more X-linked sex-ratio distorter genes. The recent evolution of the novel PMSC regulator Sly in mouse lineages has significant implications for the use of mouse-model systems in investigating sex chromosome dynamics in spermatids.

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.

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

Fbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here, we show that in addition to the previously known Parkinsonian and hematopoietic phenotypes, male mice with reduced Fbxo7 expression are sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the spermatids undergo cytoplasmic remodeling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7 mutant males. Mutation of the Drosophila Fbxo7 ortholog, nutcracker (ntc) also leads to sterility with germ cell death during cytoplasmic remodeling, indicating that the requirement for Fbxo7 at this stage is conserved. The ntc phenotype was attributed to decreased levels of the proteasome regulator, DmPI31 and reduced proteasome activity. Consistent with the fly model, we observe a reduction in PI31 levels in mutant mice; however, there is no alteration in proteasome activity in whole mouse testes. Our results are consistent with findings that Fbxo7 regulates PI31 protein levels, and indicates that a defect at the late stages of spermiogenesis, possibly due to faulty spatial dynamics of proteasomes during cytoplasmic remodeling, may underlie the fertility phenotype in mice.

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

Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the nucleus into zones, and manually scoring which zone a fluorescence in-situ hybridisation (FISH) signal lies in. This is time consuming, limiting the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and two autosomes in three mouse lineages (Mus musculus domesticus, Mus musculus musculus and Mus spretus). We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse evolution.

statementFbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here we show that in addition to the previously-known Parkinsonian and haematopoietic phenotypes, Fbxo7deficient male mice are completely sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the cells undergo cytoplasmic remodelling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7-deficient males. Mutation of the Drosophila Fbxo7 orthologue, nutcracker (ntc) was previously shown to cause sterility at a similar stage of germ cell development, indicating that the requirement for Fbxo7 is conserved.The ntc phenotype was attributed to proteasome mis-regulation via an interaction with the proteasome regulator, DmPI31. Our data suggest rather that in mice, the requirement for Fbxo7 is either independent of its interaction with PI31, or relates specifically to cytoplasmic proteasome activity during spermiogenesis.

This paper reports on our experience with providing Information Flow Control (IFC) as a library. Our aim was to support the use of an unmodified Platform as a Service (PaaS) cloud infrastructure by IFC-aware web applications. We discuss how Aspect Oriented Programming (AOP) overcomes the limitations of RubyTrack, our first approach. Although use of AOP has been mentioned as a possibility in past IFC literature we believe this paper to be the first illustration of how such an implementation can be attempted.We discuss how we built FlowR (Information Flow Control for Ruby), a library extending Ruby to provide IFC primitives using AOP via the Aquarium open source library. Previous attempts at providing IFC as a language extension required either modification of an interpreter or significant code rewriting. FlowR provides a strong separation between functional implementation and security constraints which supports easier development and maintenance; we illustrate with practical examples. In addition, we provide new primitives to describe IFC constraints on objects, classes and methods that, to our knowledge, are not present in related work and take full advantage of an object oriented language (OO language).The experience reported here makes us confident that the techniques we use for Ruby can be applied to provide IFC for any Object Oriented Program (OOP) whose implementation language has an AOP library.