Studies of chromosome and genome biology often focus on condensed chromatin in the form of chromosomes and neglect the non-dividing cells. Even when interphase nuclei are considered, they are often then treated as interchangeable round objects. However, different cell types can have very different nuclear shapes, and these shapes have impacts on cellular function; indeed, many pathologies are linked with alterations to nuclear shape. In this review, we describe some of the nuclear morphologies beyond the spherical and ovoid. Many of the leukocytes of the immune system have lobed nuclei, which aid their flexibility and migration; smooth muscle cells have a spindle shaped nucleus, which must deform during muscle contractions; spermatozoa have highly condensed nuclei which adopt varied shapes, potentially associated with swimming efficiency. Nuclei are not passive passengers within the cell. There are clear effects of nuclear shape on the transcriptional activity of the cell. Recent work has shown that regulation of gene expression can be influenced by nuclear morphology, and that cells can drastically remodel their chromatin during differentiation. The link between the nucleoskeleton and the cytoskeleton at the nuclear envelope provides a mechanism for transmission of mechanical forces into the nucleus, directly affecting chromatin compaction and organisation.
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.
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.
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.
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