Géraldine Pascal

Structural and functional annotation of the porcine immunome

BackgroundThe domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.ResultsThe Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.ConclusionsThis extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

BackgroundOviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.Methods & resultsIn the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes.In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates.ConclusionOur study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.

Evolution of Genes Involved in Gamete Interaction: Evidence for Positive Selection, Duplications and Losses in Vertebrates

Genes encoding proteins involved in sperm-egg interaction and fertilization exhibit a particularly fast evolution and may participate in prezygotic species isolation [1], [2]. Some of them (ZP3, ADAM1, ADAM2, ACR and CD9) have individually been shown to evolve under positive selection [3], [4], suggesting a role of positive Darwinian selection on sperm-egg interaction. However, the genes involved in this biological function have not been systematically and exhaustively studied with an evolutionary perspective, in particular across vertebrates with internal and external fertilization. Here we show that 33 genes among the 69 that have been experimentally shown to be involved in fertilization in at least one taxon in vertebrates are under positive selection. Moreover, we identified 17 pseudogenes and 39 genes that have at least one duplicate in one species. For 15 genes, we found neither positive selection, nor gene copies or pseudogenes. Genes of teleosts, especially genes involved in sperm-oolemma fusion, appear to be more frequently under positive selection than genes of birds and eutherians. In contrast, pseudogenization, gene loss and gene gain are more frequent in eutherians. Thus, each of the 19 studied vertebrate species exhibits a unique signature characterized by gene gain and loss, as well as position of amino acids under positive selection. Reflecting these clade-specific signatures, teleosts and eutherian mammals are recovered as clades in a parsimony analysis. Interestingly the same analysis places Xenopus apart from teleosts, with which it shares the primitive external fertilization, and locates it along with amniotes (which share internal fertilization), suggesting that external or internal environmental conditions of germ cell interaction may not be the unique factors that drive the evolution of fertilization genes. Our work should improve our understanding of the fertilization process and on the establishment of reproductive barriers, for example by offering new leads for experiments on genes identified as positively selected.

What Makes an Egg Unique? Clues from Evolutionary Scenarios of Egg-Specific Genes

The avian egg, which contains the egg yolk, the egg white, and the eggshell, represents the mostly advanced amniotic egg in oviparous vertebrates. In mammals, this reproductive strategy of laying egg has gradually evolved toward placentation. In order to better understand the unique status of the avian egg in the evolution of the vertebrate reproduction, we investigated the evolution of some Gallus gallus egg-specific protein-coding genes. Based on our finding and other recent research, we have summarized here that gene formation (such as ovalbumin genes, ovocalyxin-36 and apovitellenin-1 encoding genes in the G. gallus), gene divergence between G. gallus and mammals (such as the ovocalyxin-32 gene with its ortholog, the mammalian RARRES1, and the ovocleidin-116 with its ortholog, the mammalian MEPE), and gene loss (egg-expressed genes lost during the evolution of the mammals, such as vitellogenin and RBP encoding genes) play significant roles in the evolution of egg-specific genes.

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12

In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.

Positive selection in bone morphogenetic protein 15 targets a natural mutation associated with primary ovarian insufficiency in human.

Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women.

The evolutionary history of the SAL1 gene family in eutherian mammals

BackgroundSAL1 (salivary lipocalin) is a member of the OBP (Odorant Binding Protein) family and is involved in chemical sexual communication in pig. SAL1 and its relatives may be involved in pheromone and olfactory receptor binding and in pre-mating behaviour. The evolutionary history and the selective pressures acting on SAL1 and its orthologous genes have not yet been exhaustively described. The aim of the present work was to study the evolution of these genes, to elucidate the role of selective pressures in their evolution and the consequences for their functions.ResultsHere, we present the evolutionary history of SAL1 gene and its orthologous genes in mammals. We found that (1) SAL1 and its related genes arose in eutherian mammals with lineage-specific duplications in rodents, horse and cow and are lost in human, mouse lemur, bushbaby and orangutan, (2) the evolution of duplicated genes of horse, rat, mouse and guinea pig is driven by concerted evolution with extensive gene conversion events in mouse and guinea pig and by positive selection mainly acting on paralogous genes in horse and guinea pig, (3) positive selection was detected for amino acids involved in pheromone binding and amino acids putatively involved in olfactory receptor binding, (4) positive selection was also found for lineage, indicating a species-specific strategy for amino acid selection.ConclusionsThis work provides new insights into the evolutionary history of SAL1 and its orthologs. On one hand, some genes are subject to concerted evolution and to an increase in dosage, suggesting the need for homogeneity of sequence and function in certain species. On the other hand, positive selection plays a role in the diversification of the functions of the family and in lineage, suggesting adaptive evolution, with possible consequences for speciation and for the reinforcement of prezygotic barriers.

Fumonisin-Exposure Impairs Age-Related Ecological Succession of Bacterial Species in Weaned Pig Gut Microbiota

Pigs are highly affected by dietary mycotoxin contamination and particularly by fumonisin. The effects of fumonisin on pig intestinal health are well documented, but little is known regarding its impact on gut microbiota. We investigate the effects of the fumonisin (FB1, 12 mg/kg feed) on the fecal microbiota of piglets (n = 6) after 0, 8, 15, 22, and 29 days of exposure. A control group of six piglets received a diet free of FB1. Bacterial community diversity, structure and taxonomic composition were carried out by V3–V4 16S rRNA gene sequencing. Exposure to FB1 decreases the diversity index, and shifts and constrains the structure and the composition of the bacterial community. This takes place as early as after 15 days of exposure and is at a maximum after 22 days of exposure. Compared to control, FB1 alters the ecological succession of fecal microbiota species toward higher levels of Lactobacillus and lower levels of the Lachnospiraceae and Veillonellaceae families, and particularly OTUs (Operational Taxonomic Units) of the genera Mitsuokella, Faecalibacterium and Roseburia. In conclusion, FB1 shifts and constrains age-related evolution of microbiota. The direct or indirect contribution of FB1 microbiota alteration in the global host response to FB1 toxicity remains to be investigated.