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.

Avian genome organisation is characterised, in part, by a set of microchromosomes that are unusually small in size and unusually large in number. Although containing about a quarter of the genome, they contain around half the genes and three quarters of the total chromosome number. Nonetheless, they continue to belie analysis by cytogenetic means. Chromosomal rearrangements play a key role in genome evolution, fertility and genetic disease and thus tools for analysis of the microchromosomes are essential to analyse such phenomena in birds. Here, we report the development of chicken microchromosomal paint pools, generation of pairs of specific microchromosome BAC clones in chicken, and computational tools for in silico comparison of the genomes of microchromosomes. We demonstrate the use of these molecular and computational tools across species, suggesting their use to generate a clear picture of microchromosomal rearrangements between avian species. With increasing numbers of avian genome sequences that are emerging, tools such as these will find great utility in assembling genomes de novo and for asking fundamental questions about genome evolution from a chromosomal perspective.

SummaryThe mouse sex chromosomes exhibit an extraordinary level of copy number amplification of postmeiotically expressed genes [1, 2], driven by an “arms race” (genomic conflict) between the X and Y chromosomes over the control of offspring sex ratio. The sex-linked ampliconic transcriptional regulators Slx and Sly [3, 4, 5, 6, 7] have opposing effects on global transcription levels of the sex chromosomes in haploid spermatids via regulation of postmeiotic sex chromatin (PMSC) [8, 9, 10, 11] and opposing effects on offspring sex ratio. Partial deletions of the Y chromosome (Yq) that reduce Sly copy number lead to global overexpression of sex-linked genes in spermatids and either a distorted sex ratio in favor of females (smaller deletions) or sterility (larger deletions) [12, 13, 14, 15, 16]. Despite a large body of work studying the role of the sex chromosomes in regulating spermatogenesis (recent reviews [17, 18, 19, 20]), most studies do not address differential fertility effects on X- and Y-bearing cells. Hence, in this study, we concentrate on identifying physiological differences between X- and Y-bearing sperm from Yq-deleted males that affect their relative fertilizing ability and consequently lead to sex ratio skewing. We show that X- and Y-bearing sperm in these males have differential motility and morphology but are equally able to penetrate the cumulus and fertilize the egg once at the site of fertilization. The altered motility is thus deduced to be the proximate cause of the skew. This represents the first demonstration of a specific difference in sperm function associated with sex ratio skewing.

The separation of germ cell populations from the soma is part of the evolutionary transition to multicellularity. Only genetic information present in the germ cells will be inherited by future generations, and any molecular processes affecting the germline genome are therefore likely to be passed on. Despite its prevalence across taxonomic kingdoms, we are only starting to understand details of the underlying micro-evolutionary processes occurring at the germline genome level. These include segregation, recombination, mutation and selection and can occur at any stage during germline differentiation and mitotic germline proliferation to meiosis and post-meiotic gamete maturation. Selection acting on germ cells at any stage from the diploid germ cell to the haploid gametes may cause significant deviations from Mendelian inheritance and may be more widespread than previously assumed. The mechanisms that affect and potentially alter the genomic sequence and allele frequencies in the germline are pivotal to our understanding of heritability. With the rise of new sequencing technologies, we are now able to address some of these unanswered questions. In this review, we comment on the most recent developments in this field and identify current gaps in our knowledge.

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.

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

The search for morphological or physiological differences between X-and Y-bearing mammalian sperm has provoked controversy for decades. Many potential differences have been proposed, but none validated, while accumulating understanding of syncytial sperm development has cast doubt on whether such differences are possible even in principle. We present the first ever mammalian experimental model to trace a direct link from a measurable physiological difference between X-and Y-bearing sperm to the resulting skewed sex ratio. We show that in mice with deletions on chromosome Yq, birth sex ratio distortion is due to a relatively greater motility of X-bearing sperm, and not to any aspect of sperm/egg interaction. Moreover, the morphological distortion caused by Yq deletion is more severe in Y-bearing sperm, providing a potential hydrodynamic basis for the altered motility.This reinforces a growing body of work indicating that sperm haploid selection is an important and underappreciated evolutionary force.We thank the animal handling staff at