DNA Physical Properties and Nucleosome Positions Are Major Determinants of HIV-1 Integrase Selectivity

Retroviral integrases (INs) catalyse the integration of the reverse transcribed viral DNA into the host cell genome. This process is selective, and chromatin has been proposed to be a major factor regulating this step in the viral life cycle. However, the precise underlying mechanisms are still under investigation. We have developed a new in vitro integration assay using physiologically-relevant, reconstituted genomic acceptor chromatin and high-throughput determination of nucleosome positions and integration sites, in parallel. A quantitative analysis of the resulting data reveals a chromatin-dependent redistribution of the integration sites and establishes a link between integration sites and nucleosome positions. The co-activator LEDGF/p75 enhanced integration but did not modify the integration sites under these conditions. We also conducted an in cellulo genome-wide comparative study of nucleosome positions and human immunodeficiency virus type-1 (HIV-1) integration sites identified experimentally in vivo. These studies confirm a preferential integration in nucleosome-covered regions. Using a DNA mechanical energy model, we show that the physical properties of DNA probed by IN binding are important in determining IN selectivity. These novel in vitro and in vivo approaches confirm that IN has a preference for integration into a nucleosome, and suggest the existence of two levels of IN selectivity. The first depends on the physical properties of the target DNA and notably, the energy required to fit DNA into the IN catalytic pocket. The second depends on the DNA deformation associated with DNA wrapping around a nucleosome. Taken together, these results indicate that HIV-1 IN is a shape-readout DNA binding protein

Identification of the Inner Cell Mass and the Trophectoderm Responses after an In Vitro Exposure to Glucose and Insulin during the Preimplantation Period in the Rabbit Embryo

International audienceThe prevalence of metabolic diseases is increasing, leading to more women entering pregnancy with alterations in the glucose-insulin axis. The aim of this work was to investigate the effect of a hyperglycemic and/or hyperinsulinemic environment on the development of the preimplantation embryo. In rabbit embryos developed in vitro in the presence of high insulin (HI), high glucose (HG), or both (HGI), we determined the transcriptomes of the inner cell mass (ICM) and the trophectoderm (TE). HI induced 10 differentially expressed genes (DEG) in ICM and 1 in TE. HG ICM exhibited 41 DEGs involved in oxidative phosphorylation (OXPHOS) and cell number regulation. In HG ICM, proliferation was decreased (p < 0.01) and apoptosis increased (p < 0.001). HG TE displayed 132 DEG linked to mTOR signaling and regulation of cell number. In HG TE, proliferation was increased (p < 0.001) and apoptosis decreased (p < 0.001). HGI ICM presented 39 DEG involved in OXPHOS and no differences in proliferation and apoptosis. HGI TE showed 16 DEG linked to OXPHOS and cell number regulation and exhibited increased proliferation (p < 0.001). Exposure to HG and HGI during preimplantation development results in common and specific ICM and TE responses that could compromise the development of the future individual and placenta

Mares’ age affects embryo ICM and trophoblast RNA-seq transcriptome at about D8

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Nulliparity affects the expression of a limited number of genes and pathways in Day 8 equine embryos

Nulliparous mares produce lighter and smaller foals compared to mares having previously foaled, with effects observed at least until 4 months of age. The need for a first gestation priming for the uterus to reach its full capacity has been proposed to explain this observation. Embryo developmental defects could be hypothesized but effects of maternal parity on the embryo have only been described once, in old mares, thus combining effects of parity and old age. The aim of this study was to determine effects of mare parity on embryo gene expression. Day-8 post ovulation blastocysts were collected from young (5/6 years old) nulliparous (YN, N=6) or multiparous (YM, N=4) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner-cell-mass-enriched (ICMandTE) trophoblast were obtained by embryo bisection for RNA sequencing (paired end, non-oriented, Illumina, NextSeq500). Deconvolution was performed on the ICMandTE dataset. Differential expression, with embryo sex and diameter as cofactors and gene set enrichment analysis (GO BP, KEGG, REACTOME databases) were performed using a false discovery rate <0.05 cutoff. Only a few genes were altered (ICM: n=18; TE: n=6) but several gene sets were perturbed (ICM: n=62; TE: n=50) by maternal parity. In YM, only pathways related to transcription, RNA processing and vesicle transport functions were enriched in the ICM whereas only pathways related to RNA localization were enriched in TE. In YN, while only gene sets related to ribosomes and extracellular matrix were enriched in the ICM, functions related to energy and lipid metabolism, lipid transport and interleukin-1 signaling were enriched in the TE. In conclusion, several genes and pathways are affected in embryos collected from nulliparous mares, with different effects on TE and ICM. Embryo development is altered in nulliparous mares, which could partially explain the term phenotype. Whether differences in gene expression result/induce poor embryo-maternal communication remains to be determined

230 Slight alterations of trophoblast gene expression are related to the term placenta morphology and gene expression in horses

The first foal of a mare is typically smaller and lighter than subsequent foals. A poorer uterine potential to support fetal growth during the first pregnancy could be responsible, but modifications already present in the embryo could also be involved. The aim of this study was to evaluate effects of maternal parity on gene expression in blastocyst’s trophoblast, term placenta, and term placenta histology. Young (4–6 years old) nulliparous (YN), or multiparous (YM) mares were inseminated with the semen of one stallion. Embryos were collected eight days after ovulation in YN (n = 6) and YM (n = 5). Trophoblast was obtained by embryo bisection. Placentas from YN (n = 18) and YM (n = 10) were recovered at foaling, measured, and weighed. Stereology was performed using MercartorPro software on HES-stained paraffin-embedded samples and analysed using a linear model with permutations with R software. Paired-end RNA-sequencing was performed on frozen trophoblast and placenta (Illumina, NextSEqn 500). Differential expression was analysed (DESEqn 2) using a false discovery rate (FDR) < 0.05 cutoff. Gene Set Enrichment Analysis was performed using KEGG and GO BP databases. In trophoblast and placenta, 13,178 and 14,696 expressed genes were identified, only 6 and 2 of which were differentially expressed according to mare parity, respectively. Differentially expressed genes were partly related to molecule transport. Gene pathways disturbed in trophoblast were related to energy and lipid metabolism, transport, and interleukin-1 signalling. Only eight pathways were perturbed by maternal parity in term placenta and were involved in innate immune response. Gestation averaged 5.5 days longer (P < 0.05) in YN compared with YM mares. Lighter placentas were observed in YN, but they produced more kg of foal/cm2 of placental macroscopic surface (P < 0.01). No difference was shown in placenta volume or all histological measurements. In conclusion, term placentas appeared to be smaller than for YM, but similar in morphology and potentially more efficient at supporting the fetus. Maternal parity, however, slightly affected gene expression in both trophoblast and term placenta, indicated by changes related to molecule transport and immune regulation. Due to the importance of these functions in placenta establishment, these results suggest that altered exchanges from the embryo stage with the YN dam could be one cause of reduced foal size at birth

142 Nulliparity alters gene expression in inner cell mass and trophoblast of equine blastocysts in old mares

International audienceAn increased incidence in early embryo loss has been observed in aged mares. Moreover, the first foal born to a mare is lighter than her subsequent foals, with reported impaired placental function at term. Because trophoblast function may be affected from the embryo stage, the aim of this project was to determine the effect of parity in aged mares on gene expression in Day-8.5 embryos. Middle-aged (13.5±2.2 years) nulliparous (never foaled) (ON) or multiparous (1.8±1.6 foals) (OM) Saddlebred, non-nursing mares were inseminated with the semen of one unique stallion. At 8 days post-ovulation (10 days post-hCG), embryos were recovered by uterine flushing and bisected to obtain samples of pure (trophectoderm, TE) or inner cell mass enriched (ICM) trophoblast. Paired end, non-oriented RNA sequencing was performed with Illumina (NextSEqn 500) on 5 and 6 TE and ICM collected from ON and OM, respectively. Differential expression was analysed with DESEqn 2. Embryo size was included in the model and a P<0.05 cutoff was used after false discovery rate correction. Gene set enrichment analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. Out of the 13 007 and 12 706 genes expressed in ICM and TE, respectively, only 8 in ICM and 6 in TE were differentially expressed, with 2 genes in common. Nevertheless, 19 gene sets were enriched and 6 depleted in the ICM of ON, whereas 2 gene sets were enriched and 8 depleted in the TE of ON compared with OM. Gene sets involved in ribosomal activity and structure, proteasome, integral component of plasma membrane, and immune response were enriched in ICM from ON embryos, and gene sets linked to sphingolipid metabolism, nucleosome, and constituents of the extracellular matrix (ECM) were depleted. In TE from ON mares, enriched gene sets were involved with ribosomes and depleted gene sets were linked to extracellular matrix, focal adhesion, myosin complex, and sequence-specific DNA binding. Overall, 1 enriched (linked to extracellular matrix) and 1 depleted gene set (involved in ribosomal structure) were common to ICM and TE. Thus, embryos from aged nulliparous mares seem to have higher protein turnover and higher immune response compared with those of OM, whereas the depletion of gene sets associated with extracellular matrix and membrane may indicate differences in cellular organisation into lineages. More work is ongoing to study effects on subsequent development

Mare aging as young as 10 years old alters gene expression and structure of term placenta without affecting foal growth

International audienceMaternal aging, excluding extreme ages, is associated with increased foal weight/size and heavier/more voluminous placenta. Maternal parity is a confounding factor as it is often strongly correlated with maternal age. The aim of this study was to analyze the effect of mare age, while excluding a parity effect, on placental structure and gene expression and foal growth and health.Placentas from 10 young (6-year-old, YM) and 12 older (>10-year-old, OM) multiparous mares of similar parity were recovered, measured and weighted after foaling. Placental structure was analyzed by stereology on HES stained histological slides. Paired-end RNA-sequencing was performed on 6 placentas/group (Illumina, NextSeq500). Differential expression was analyzed (DESeq2) using a false discovery rate <0.05 cutoff. Gene Set Enrichment Analysis was performed using KEGG and GO BP databases. Foal growth was monitored from birth to 18 months of age (m). At 6 and 12 m, carbohydrate metabolism was assessed using frequently sampled intravenous glucose tolerance tests and joint X-rays were performed to detect osteoarticular lesions. Stereology, growth and FSIVGTT parameters were analyzed using a linear model with permutations. Fisher tests were used to analyze the prevalence of osteochondrosis (OCD).Placental weight, volume and surface did not vary according to maternal age. The area of allantoic vessels was reduced (p=0.02) while volume of microcotyledon connective tissue (p=0.05) and total microcotyledon (p=0.08) tended to be increased in OM. Of the 14,716 expressed genes identified in placentas, only 9 were differentially expressed between groups. Pathways enriched in OM were related to immunity whereas those enriched in YM were related to tissue genesis. Neither foal growth nor OCD were affected by maternal age. A decreased insulin pancreas response (p=0.08) with increased basal glycemia (p=0.05) tended to be present in female OM foals at 6m. Basal glycemia (p=0.02) and insulinemia (p=0.03) were increased in YM foal at 12m.Mares’ age did not alter gross morphology but slightly perturbed term placenta histology and function, suggesting long term adaptation. Growth and prevalence of OCD did not differ but carbohydrate metabolism of foals was affected by maternal age

Mare aging as young as 10 years old alters gene expression and structure of term placenta without affecting foal growth

International audienceMaternal aging, excluding extreme ages, is associated with increased foal weight/size and heavier/more voluminous placenta. Maternal parity is a confounding factor as it is often strongly correlated with maternal age. The aim of this study was to analyze the effect of mare age, while excluding a parity effect, on placental structure and gene expression and foal growth and health.Placentas from 10 young (6-year-old, YM) and 12 older (>10-year-old, OM) multiparous mares of similar parity were recovered, measured and weighted after foaling. Placental structure was analyzed by stereology on HES stained histological slides. Paired-end RNA-sequencing was performed on 6 placentas/group (Illumina, NextSeq500). Differential expression was analyzed (DESeq2) using a false discovery rate <0.05 cutoff. Gene Set Enrichment Analysis was performed using KEGG and GO BP databases. Foal growth was monitored from birth to 18 months of age (m). At 6 and 12 m, carbohydrate metabolism was assessed using frequently sampled intravenous glucose tolerance tests and joint X-rays were performed to detect osteoarticular lesions. Stereology, growth and FSIVGTT parameters were analyzed using a linear model with permutations. Fisher tests were used to analyze the prevalence of osteochondrosis (OCD).Placental weight, volume and surface did not vary according to maternal age. The area of allantoic vessels was reduced (p=0.02) while volume of microcotyledon connective tissue (p=0.05) and total microcotyledon (p=0.08) tended to be increased in OM. Of the 14,716 expressed genes identified in placentas, only 9 were differentially expressed between groups. Pathways enriched in OM were related to immunity whereas those enriched in YM were related to tissue genesis. Neither foal growth nor OCD were affected by maternal age. A decreased insulin pancreas response (p=0.08) with increased basal glycemia (p=0.05) tended to be present in female OM foals at 6m. Basal glycemia (p=0.02) and insulinemia (p=0.03) were increased in YM foal at 12m.Mares’ age did not alter gross morphology but slightly perturbed term placenta histology and function, suggesting long term adaptation. Growth and prevalence of OCD did not differ but carbohydrate metabolism of foals was affected by maternal age

Effect of sex on gene expression in equine blastocysts

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