Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting

A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q-;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases

Estimation of the proportion of genetically unbalanced spermatozoa in the semen of boars carrying chromosomal rearrangements using FISH on sperm nuclei

Many chromosomal rearrangements are detected each year in France on young boars candidates for reproduction. The possible use of these animals requires a good knowledge of the potential effect of the rearrangements on the prolificacy of their mates. This effect can be estimated by an accurate determination of the rate of unbalanced spermatozoa in the semen of boars which carry the rearrangements. Indeed, these spermatozoa exhibiting normal fertilizing ability are responsible for an early embryonic mortality, and then, for a decrease of the litter sizes. The "spermFISH" technique, i.e. fluorescent in situ hybridization on decondensed sperm heads, has been used on several occasions in Man, in this perspective. In livestock species, this method was formerly used mainly for semen sexing purposes. We used it, for the first time, to estimate the rates of imbalance in the semen of four boars carrying chromosomal rearrangements: two reciprocal translocations, rcp(3;15)(q27;q13) and rcp(12;14)(q13;q21), as well as two independent cases of trisomy 18 mosaicism. The rates of unbalanced gametes were relatively high for the two reciprocal translocations (47.83% and 24.33%, respectively). These values differed from the apparent effects of the rearrangements estimated using a limited number of litters: a decrease in prolificacy of 23% (estimation obtained using the results of 6 litters) and 39% (57 litters), respectively for the 3/15 and 12/14 translocations. The imbalance rates were much lower for the trisomy mosaics (0.58% and 1.13%), suggesting a very moderate effect of this special kind of chromosomal rearrangement

Functional study and regional mapping of 44 hormono-regulated genes isolated from a porcine granulosa cell library

cDNA clones from a pig granulosa cell cDNA library were isolated by differential hybridisation for follicle stimulating hormone (FSH) regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH) and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs

Characterization of 3D genomic interactions in fetal pig muscle

Genome sequence alone is not sufficient to explain the overall coordination of nuclear activity in a particular tissue. The nuclear organisation and genomic long-range intra- and inter-chromosomal interactions play an important role in the regulation of gene expression and the activation of tissue- specific gene networks. Here we present an overview of the pig genome architecture in muscle at two late developmental stages. The muscle maturation process occurs between the 90th day and the end of gestation (114 days), a key period for survival at birth. To characterise this period we profiled chromatin interactions genome-wide with in situ Hi-C (High Throughput Chromosome Conformation Capture) in muscle samples collected at 90 and 110 days of gestation, specific moments where a drastic change in gene expression has been reported. About 200 million read pairs per library were generated (3 replicates per condition). This allowed: (a) the design of an experimental Hi-C protocol optimized for frozen fetal tissues, (b) the first Hi-C contact heatmaps in fetal porcine muscle cells, and (c) to profile Topologically Associated Domains (TADs) defined as genomic domains with high levels of chromatin interactions. Using the new assembly version Sus scrofa v11, we could map 82% of the Hi-C reads on the reference genome. After filtering, 49% of valid read pairs were used to infer the genomic interactions in both developmental stages. In addition, ChIP-seq experiments were performed to map the binding of the structural protein CTCF, known to regulate genome structure by promoting interactions between genes and distal enhancers. The Hi-C and ChIP-seq data were analysed in combination with the results of a previous transcriptome analysis, focusing on the hun-dreds of genes that were reported as differentially expressed during muscle maturation. We will report the observed general differences between both developmental stages in terms of transcription and structure

Analyses of pig genomes provide insight into porcine demography and evolution

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model

Estimation of the proportion of genetically unbalanced spermatozoa in the semen of boars carrying chromosomal rearrangements using FISH on sperm nuclei

Many chromosomal rearrangements are detected each year in France on young boars candidates for reproduction. The possible use of these animals requires a good knowledge of the potential effect of the rearrangements on the prolificacy of their mates. This effect can be estimated by an accurate determination of the rate of unbalanced spermatozoa in the semen of boars which carry the rearrangements. Indeed, these spermatozoa exhibiting normal fertilizing ability are responsible for an early embryonic mortality, and then, for a decrease of the litter sizes. The “spermFISH” technique, i.e. fluorescent in situ hybridization on decondensed sperm heads, has been used on several occasions in Man, in this perspective. In livestock species, this method was formerly used mainly for semen sexing purposes. We used it, for the first time, to estimate the rates of imbalance in the semen of four boars carrying chromosomal rearrangements: two reciprocal translocations, rcp(3;15)(q27;q13) and rcp(12;14)(q13;q21), as well as two independent cases of trisomy 18 mosaicism. The rates of unbalanced gametes were relatively high for the two reciprocal translocations (47.83% and 24.33%, respectively). These values differed from the apparent effects of the rearrangements estimated using a limited number of litters: a decrease in prolificacy of 23% (estimation obtained using the results of 6 litters) and 39% (57 litters), respectively for the 3/15 and 12/14 translocations. The imbalance rates were much lower for the trisomy mosaics (0.58% and 1.13%), suggesting a very moderate effect of this special kind of chromosomal rearrangement

Functional study and regional mapping of 44 hormono-regulated genes isolated from a porcine granulosa cell library

cDNA clones from a pig granulosa cell cDNA library were isolated by differential hybridisation for follicle stimulating hormone (FSH) regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH) and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs

3D organization of telomeres in porcine neutrophils and analysis of LPS-activation effect

Background: While the essential role of 3D nuclear architecture on nuclear functions has been demonstrated for various cell types, information available for neutrophils, essential components of the immune system, remains limited. In this study, we analysed the spatial arrangements of telomeres which play a central role in cell fate. Our studies were carried out in swine, which is an excellent model organism for both biomedical research and agronomic applications. We isolated bacterial artificial chromosome (BAC)-containing subtelomeric p and q sequences specific to each porcine chromosome. This allowed us to study the behaviour of p and q telomeres of homologous chromosomes for seven pairs chosen for their difference in length and morphology. This was performed using 3D-FISH on structurally preserved neutrophils, and confocal microscopy. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen aggression modifies this organization.[br/] Results: The positions of the p and q telomeres relative to the nuclear outer border were determined in the two states. All p telomeres changed their position significantly during the activation process, although the effect was less pronounced for the q telomeres. The patterns of telomeric associations between homologs and their frequencies were analysed for 7 pairs of chromosomes. This analysis revealed that the distribution of pp, qq and pq associations differs significantly among the 7 chromosomes. This distribution does not fit with the theoretical distribution for each chromosome, suggesting that preferential associations occur between subtelomeres.[br/] Conclusions: The percentage of nuclei harbouring at least one telomeric association between homologs varies significantly among the chromosomes, the smallest metacentric chromosome SSC12, which is also the richest in gene-density, harbouring the highest value. The distribution of types of telomeric associations is highly dependent on the chromosomes and is not affected by the activation process. The frequencies of telomeric associations are also highly dependent on the type of association and the type of chromosome. Overall, the LPS-activation process induces only minor changes in these patterns of associations. When telomeric associations occur, the associations of p and q arms from the same chromosome are the most frequent, suggesting that “chromosome bending” occurs in neutrophils as previously observed in gametes

Dynamiques d'expressions et architecture nucléaire des gènes IGF2 et de DLK1 pour une meilleure compréhension de la balance myogenèse/adipogenèse dans l'espèce porcine

National audienceNotre objectif est de caractériser le rôle de l’architecture nucléaire dans la régulation de l'expression de gènes d’intérêt pendant l’adipogénèse et la myogenèse. Il a été choisi d’étudier à la fois la dynamique d’expression des gènes IGF2 et DLK1 (gènes clés dans le développement des tissus adipeux et musculaires) et l’organisation nucléaire de ces gènes au cours de la prolifération puis de la différenciation des populations de cellules stromales/souches adultes issues du tissu adipeux sous-cutané ou du muscle longissimus de porcelets, pour mieux comprendre les mécanismes impliqués dans la balance muscle/gras chez le porc. En effet, il a été montré que l’architecture nucléaire est impliquée dans la régulation de l'expression de nombreux gènes pendant l’adipogénèse (Charo et al., 2016) et la myogenèse y compris chez le porc (Lahbib-Mansais et al., 2016). Cependant, il n’y a pas d’études qui caractérisent la régulation d’IGF2 et DLK1 dans des populations de cellules souches des tissus musculaires et adipeux, triées en fonction de leurs capacités myogéniques ou adipogéniques, et selon le tissu où elles résident. A PEGASE, nous avons testé dans une précédente étude un panel de marqueurs permettant de phénotyper les cellules musculaires et adipeuses et visant à aller plus loin dans la discrimination des cellules satellites (CD56+/CD45-) et des cellules fibroadipogéniques (CD56-/CD45-). Au stade précoce auquel nous récupérons les cellules de porcelets (5-7 jours), les marqueurs CD56 (marqueur myogénique, présent à la fois dans le muscle et le tissu adipeux) et CD45 (marqueur hématopoïétique) paraissent les plus pertinents. Pour cette étude, les variations 1) d’expression génique (partenaire 1, PEGASE) et 2) d’association génique lors d’étude d’architecture nucléaire (partenaire 2, GenPhySE) sont étudiées sur les cellules isolées des tissus prélevés sur des porcelets (Landrace x Large White x Piétrain, femelles) de 5 jours. Cette approche a nécessité de développer des systèmes de culture sur lame de verre qui préservent la structure 3D (sur Matrigel). La culture sur verre nous a confronté à des difficultés d’adhérence des cellules, surtout lors de leur différenciation, certainement en lien avec la migration cellulaire et la réorganisation intracellulaire inhérente à ces modèles de culture. L’étude sur cellules triées n’a pas été poursuivie. En effet, les cellules CD56- n’adhérant pas sur lame de verre, nous avons décidé de travailler sur l’ensemble des cellules stromales sans tri préalable en réalisant des mesures sur un point au cours de la prolifération et sur deux points au cours de la différenciation. A GenPhySE, les premiers résultats d’association génique entre IGF2-DLK1 ont été montrés dans des noyaux de cellules souches non triées issues de tissu adipeux, au stade de prolifération (Jour 3). L’observation en microscopie confocale des noyaux marqués à l’aide de deux molécules fluorescentes distinctes pour chacun des gènes, a permis de mettre en évidence une association ADN-ADN entre un allèle de chacun des gènes. Pour conclure, il a été difficile de transposer les systèmes de culture cellulaire utilisés précédemment à des systèmes de culture sur lame de verre permettant de préserver la structure 3D des cellules et de réaliser les analyses FISH 3D et la microscopie confocale. Des analyses d’association génique et d’expression de gènes cibles dont IGF2 et DLK1 par qPCR sont toujours en cours et les dernières analyses sont prévues pour le printemps 2022