Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

BACKGROUND: Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. METHODS: Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. RESULTS: We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. CONCLUSION: Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes

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

Profiling the landscape of transcription, chromatin accessibility and chromosome conformation of cattle, pig, chicken and goat genomes [FAANG pilot project]

Functional annotation of livestock genomes is a critical and obvious next step to derive maximum benefit for agriculture, animal science, animal welfare and human health. The aim of the Fr-AgENCODE project is to generate multi-species functional genome annotations by applying high-throughput molecular assays on three target tissues/cells relevant to the study of immune and metabolic traits. An extensive collection of stored samples from other tissues is available for further use (FAANG Biosamples ‘FR-AGENCODE’). From each of two males and two females per species (pig, cattle, goat, chicken), strand-oriented RNA-seq and chromatin accessibility ATAC-seq assays were performed on liver tissue and on two T-cell types (CD3+CD4+&CD3+CD8+) sorted from blood (mammals) or spleen (chicken). Chromosome Conformation Capture (in situ Hi-C) was also carried out on liver. Sequencing reads from the 3 assays were processed using standard processing pipelines. While most (50–70%) RNA-seq reads mapped to annotated exons, thousands of novel transcripts and genes were found, including extensions of annotated protein-coding genes and new lncRNAs (see abstract #69857). Consistency of ATAC-seq results was confirmed by the significant proportion of called peaks in promoter regions (36–66%) and by the specific accumulation pattern of peaks around gene starts (TSS) v. gene ends (TTS). Principal Component Analyses for RNA-seq (based on quantified gene expression) and ATAC-seq (based on quantified chromatin accessibility) highlighted clusters characterised by cell type and sex in all species. From Hi-C data, we generated 40kb-resolution interaction maps, profiled a genome-wide Directionality Index and identified from 4,100 (chicken) to 12,100 (pig) topologically-associating do- mains (TADs). Correlations were reported between RNA-seq and ATAC-seq results (see abstract #71581). In summary, we present here an overview of the first multi-species and -tissue annotations of chromatin accessibility and genome architecture related to gene expression for farm animals

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

3D nuclear organization of theCMHcomplex in control and LPS-activated porcine and human macrophages

3D nuclear organization of theCMHcomplex in control and LPS-activated porcine and human macrophages. 22. International Colloquim on Animal Cytogenetics and Genomics (ICACG

Low persistence of a large-plasmid-cured variant of Salmonella enteritidis in ceca of chicks

International audienceIn order to estimate the contribution of Salmonella in the persistence of this bacterium in chicks, we compared the persistence of a Salmonella enteritidis strain and its plasmid-cured variant in a chicken asymptomatic carrier state model. After oral inoculation, colonization with the plasmid-cured strain was significantly reduced (P < 0.001) in the ceca of chicks from the third week postinoculation and persisted for a shorter period than the wild-type strain. Moreover, numbers of S. enteritidis-infected livers were also significantly lower (P < 0.01) for the plasmid-cured strain compared with the wild-type strain from the third to the seventh week postinoculation. No difference in spleen colonization was observed. These results did not correlate with any in vitro difference in attachment, entry to, or intracellular multiplication of bacteria within intestinal or macrophage avian cell lines.Con el objeto de estimar la persistencia de la Salmonella en los pollos, se comparó la persistencia de una cepa de Salmonella enteritidis y su variante privada de gran plásmido, en un modelo aviar de portador asintomático. Después de la inoculación oral, la colonización con el organismo sin el plásmido fue reducida significantemente (P < 0.001) en el ciego de pollos desde la tercera semana después de la inoculación y persistió por un corto período, comparado con la cepa normal. Además, el número de hígados infectados con S. enteritidis fue también significantemente menor (P < 0.01) para la cepa carente del plásmido comparada con la cepa normal, desde la tercera hasta la séptima semana después de la inoculación. No se observaron diferencias en la colonización del bazo. Estos resultados no mostraron correlación con ninguna diferencia in vitro en la adhesión, entrada o multiplicación intracelular de la bacteria dentro de líneas celulares aviares intestinales o macrófagos

Sperm nuclear architecture is locally modified in presence of a Roberstsonian translocation t(13;17)

In mammals, the non-random organization of the sperm nucleus supports an early function during embryonic development. Altering this organization may interfere with the zygote development and reduce fertility or prolificity. Thus, rare studies on sperm cells from infertile patients described an altered nuclear organization that may be a cause or a consequence of their respective pathologies. Thereby, chromosomal rearrangements and aneuploidy can be studied not only for their adverse effects on production of normal/balanced gametes at meiosis but also for their possible impact on sperm nuclear architecture and the epigenetic consequences of altered chromosome positioning. We decided to compare the global architecture of sperm nuclei from boars, either with a normal chromosome composition or with a Robertsonian translocation involving chromosomes 13 and 17. We hypothesized that the fusion between these chromosomes may change their spatial organization and we examined to what extend it could also modify the global sperm nuclear architecture. Analysis of telomeres, centromeres and gonosomes repartition does not support a global nuclear disorganization. But specific analysis of chromosomes 13 and 17 territories highlights an influence of chromosome 17 for the positioning of the fused chromosomes within the nucleus. We also observed a specific clustering of centromeres depending of the chromosome subtypes. Altogether our results showed that chromosome fusion does not significantly alter sperm nucleus architecture but suggest that centromere remodelling after chromosome fusion locally impacts chromosome positioning

Virulence levels of biofilm-grown Listeria monocytogenes LO28 are lower than those of planktonic cells in an oral inoculation test on mice

International audienceThe aim of this study was to produce Listeria monocytogenes biofilms suitable for virulence assays and to determine whether the released bacteria had the same virulence potential as their planktonic counterparts. Biofilms of Listeria monocytogenes LO28 strain, with or without Sphingomonas paucimobilis CCL10 strain, containing up to 7 log(10) cfu/cm(2) were produced in polypropylene syringes. The virulence of strain LO28 was analyzed in mice after intravenous, subcutaneous and oral inoculation. Its virulence level in binary cultures was not significantly different from that of monocultures. L. monocytogenes LO28 virulence in biofilms was lower than that of their planktonic counterparts after oral inoculation. Our results suggest that biofilms pose no greater health risk to the consumer than planktonic bacteria

Precise excision and secondary transposition of TnphoA in non-motile mutants of a Salmonella enterica serovar Enteritidis clinical isolate.

International audienceMutagenesis with TnphoA has been widely used in many bacteria. Here, we report the excision and secondary transposition of this transposon in three non-motile (fliC, fliF and motB) mutants of Salmonella enterica serovar Enteritidis (S. Enteritidis). Isolation of motile revertants showed that they were kanamycin resistant and conserved a copy of TnphoA in their genome in an insertion site different from the initial one. They also expressed an intact flagella. Characterization of the motile revertant derived from the fliC mutant showed that TnphoA excised precisely from the fliC gene, resulting in an equivalent amount of FliC secreted protein in the revertant compared to that of the wild-type strain. These results show that TnphoA mutants should be used with care and underline the value of using transposon derivatives lacking the transposase gene

Evolutionary breakpoints through a high-resolution comparative map between porcine chromosomes 2 and 16 and human chromosomes

AbstractThis study reports a high-resolution comparative map between human chromosomes and porcine chromosomes 2 (SSC2) and 16 (SSC16), pointing out new homologies and evolutionary breakpoints. SSC2 is of particular interest because of the presence of several important QTLs. Among 226 porcine ESTs selected according to their expected localization, 151 were RH mapped and ordered on SSC2. This study confirmed the extensive conservation between SSC2 and HSA11 and HSA19 and refined the homology with HSA5 (three blocks defined). Furthermore the SSC2q pericentromeric region was shown to be homologous to another human chromosome (HSA1). A complex organization of these syntenies was demonstrated on SSC2q. Our strategy led us to improve also the SSC16 RH map by adding 45 markers. Two-color fluorescence in situ hybridization of markers representative of each synteny confirmed block order. Finally, 29 breakpoints were identified in both species, and porcine BACs containing two breakpoints were isolated