Neutrophils are essential components of the innate immune system due to their ability to kill and phagocytose invading microbes. They possess a lobulated nucleus and are capable of extensive and complex changes in response to bacterial stimulation. The aim of our study was to investigate whether the 3D nuclear organization of porcine neutrophils was modified upon stimulation. The organization of centromeres, telomeres, and chromosome territories (chromosomes 2, 3, 7, 8, 12, 13, and 17) was studied on structurally preserved nuclei using 3D fluorescence in situ hybridization, confocal microscopy, and image analysis. By differential labeling of centromeres of acrocentric and metacentric/submetacentric chromosomes, we showed that centromeres associated to form chromocenters but did so preferentially between chromosomes with the same morphology. Upon activation, some of these chromocenters dispersed. Telomeres were also found to form clusters, but their number remained unchanged in lipopolysaccharide-stimulated neutrophils. The analysis of the position of chromocenters and telomere clusters showed a more internal location of the latter compared to the former. The analysis of chromosome territories revealed that homologs were distributed randomly among lobes whatever the cell's status. The volume of these territories was not proportional to chromosome length, and some chromosomes (chr 3, 12, 13, and 17) were more prone to decondensation when neutrophils were stimulated. Thus, our study demonstrated that activation of neutrophils resulted in several modifications of their nuclear architecture: a decrease in the number of non-acrocentric chromocenters and the decondensation of several chromosomes.

Male infertility is an increasing health issue in today's society for both human and livestock populations. In livestock, male infertility slows the improvement of animal selection programs and agricultural productivity. There is increasing evidence that epigenetic marks play an important role in the production of good-quality sperm. We therefore screened for specific or common epigenetic signatures of livestock infertility. To do so, we compared DNA methylation level in sperm DNA from fertile and infertile boars. We evaluated first the global level of sperm DNA methylation and found no difference between the two groups of boars. We then selected 42 loci of interest, most of them known to be imprinted in human or mice, and assessed the imprinting status of five of them not previously described in swine tissues: WT1, CNTN3, IMPACT, QPCT, and GRB10. DNA methylation level was then quantified in fertile and infertile boars at these 42 loci. Results from fertile boars indicated that the methylation level of the selected loci is highly conserved between pig, human, and mice, with a few exceptions, including the POU5F1 (OCT4) promoter and RTL1. Comparison between fertile and infertile boars revealed that one imprinted region, the GNAS locus, shows an increase in sperm DNA methylation in three out of eight infertile boars with low semen quality. This increase in DNA methylation is associated with an altered expression of the genes belonging to the GNAS locus, suggesting a new role for GNAS in the proper formation of functional gametes.

Changes in the nuclear positioning of specific genes, depending on their expression status, have been observed in a large diversity of physiological processes. However, gene position is poorly documented for immune cells which have been subjected to activation following bacterial infection. Using a pig model, we focused our study on monocyte-derived macrophages and neutrophils, as they are the first lines of defence against pathogens. We examined whether changes in gene expression due to LPS activation imply that genes have repositioned in the nuclear space. We first performed a transcriptomic analysis to identify the differentially expressed genes and then analysed the networks involved during lypopolysaccharide/interferon gamma activation in monocyte-derived macrophages. This allowed us to select four up-regulated (IL1β, IL8, CXCL10 and TNFα) and four down-regulated (VIM, LGALS3, TUBA3 and IGF2) genes. Their expression statuses were verified by quantitative real-time RT-PCR before studying their behaviour in the nuclear space during macrophage activation by means of 3D fluorescence in situ hybridization. No global correlation was found between gene activity and radial positioning. Only TNFα belonging to the highly transcribed MHC region on chromosome 7 became more peripherally localized in relation to the less decondensed state of its chromosome territory (CT) in activated macrophages. The analysis of gene positioning towards their CT revealed that IL8 increases significantly its tendency to be outside its CT during the activation process. In addition, the gene to CT edge distances increase for the three up-regulated genes (IL8, CXCL10 and TNFα) among the four analysed. Contrarily, the four down-regulated genes did not change their position. The analysis of gene behaviour towards their CT was extended to include neutrophils for three (TNFα, IL8 and IL1β) up- and two (IGF2 and TUBA3) down-regulated genes, and similar results were obtained. The analysis was completed by studying the four up-regulated genes in fibroblasts, not involved in immune response. Our data suggest that relocation in the nuclear space of genes that are differentially expressed in activated immune cells is gene and cell type specific but also closely linked to the entire up-regulation status of their chromosomal regions.