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NLRPs (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing Proteins) are members of NLR (Nod-like receptors) protein family. Recent researches have shown that NLRP genes play important roles in both mammalian innate immune system and reproductive system. Several of NLRP genes were shown to be specifically expressed in the oocyte in mammals. The aim of the present work was to study how these genes evolved and diverged after their duplication, as well as whether natural selection played a role during their evolution. By using in silico methods, we have evaluated the evolution and functional divergence of NLRP genes, in particular of mouse reproduction-related Nlrp genes. We found that (1) major NLRP genes have been duplicated before the divergence of mammals, with certain lineage-specific duplications in primates (NLRP7 and 11) and in rodents (Nlrp1, 4 and 9 duplicates); (2) tandem duplication events gave rise to a mammalian reproduction-related NLRP cluster including NLRP2, 4, 5, 7, 8, 9, 11, 13 and 14 genes; (3) the function of mammalian oocyte-specific NLRP genes (NLRP4, 5, 9 and 14) might have diverged during gene evolution; (4) recent segmental duplications concerning Nlrp4 copies and vomeronasal 1 receptor encoding genes (V1r) have been undertaken in the mouse; and (5) duplicates of Nlrp4 and 9 in the mouse might have been subjected to adaptive evolution. In conclusion, this study brings us novel information on the evolution of mammalian reproduction-related NLRPs. On the one hand, NLRP genes duplicated and functionally diversified in mammalian reproductive systems (such as NLRP4, 5, 9 and 14). On the other hand, during evolution, different lineages adapted to develop their own NLRP genes, particularly in reproductive function (such as the specific expansion of Nlrp4 and Nlrp9 in the mouse).

Background Mammalian milk oligosaccharides serve as the first natural prebiotics for newborns, promoting the development of a beneficial gut microbiota. The ability of bacteria to use these complex sugars depends on their structure, but data are limited to bacteria isolated from newborn humans. This study aims to investigate in vitro the functional relationship between the structural variability of milk oligosaccharides and the metabolic capacities of newly intestinal commensal bacteria isolated from suckling rabbits and piglets. Results A total of 240 anaerobic intestinal bacterial strains were isolated from suckling piglets and rabbits, and 9 strains were cultivated in the presence of structurally different milk oligosaccharides: lacto-N-tetraose, 2’-fucosyllactose, and 3’-sialyllactose or 6’-sialyllactose. Five strains, belonging to Bacteroides fragilis , Bacteroides thetaiotaomicron , Bacteroides sp. D2, Bacteroides sp. 3_1_33FAA and Phocaeicola vulgatus were able to utilize milk oligosaccharides. Growth curves revealed that glucose supported faster growth, while, leading to a lower final biomass compared to milk oligosaccharides. Both the growth rate and the final bacterial biomass varied depending on the milk oligosaccharide structure, with higher final biomass reached with 2’-fucosyllactose. The consumption rates of milk oligosaccharides exceeded 40% for all oligosaccharides in B. fragilis , Bacteroides sp. 3_1_33FAA and P. vulgatus strains. Conversely, B. thetaiotaomicron with 6’-sialyllactose and Bacteroides sp. D2 strains for each milk oligosaccharide displayed a consumption rate below 40%. Milk oligosaccharide fermentation generated a more diverse metabolome compared to glucose. Utilization of milk oligosaccharides increased the production of propionate, isobutyrate, isovalerate, 2-methylbutyrate and 1,2-propanediol. Remarkably, fermentation of 2’-fucosyllactose resulted in substantial 1,2-propanediol production. Whole genome sequencing of the bacterial strains revealed the presence of diverse glycoside hydrolase in the strains capable of metabolizing milk oligosaccharides. Conclusions This study demonstrates the capacity of diverse intestinal commensal bacteria from suckling rabbits and piglets to ferment diverse milk oligosaccharide structures, revealing species-specific and milk oligosaccharide structure-dependent metabolization profiles. These findings highlight the potential application of milk oligosaccharides as prebiotic supplements to support gut health in farm animals.

Background In dairy cows, the transition period around parturition is a critical period with the highest incidence of infectious and metabolic diseases compared to the rest of the lactation. Over the past few years, several studies have highlighted the central role of the microbiota in health and disease. In mammals, gut microbiota is typically studied by analysing faecal samples. In cattle, most research on the gastrointestinal microbiota has focused on the ruminal microbiota, while the composition and evolution of the faecal microbiota in transitioning dairy cows remain poorly studied. We aimed to describe the composition of the faecal bacterial microbiota in a large number of dairy cows around parturition on commercial farms. Faecal samples were collected three weeks before and one week after calving from a cohort of 411 Holstein dairy cows in their 2nd and 3rd lactations across 25 dairy herds. DNA was extracted from faeces, and the 16S rRNA gene (hypervariable region V3-V4) was sequenced after amplification. Results A loss of microbial diversity was observed after calving, with no significant association with the lactation rank. The analysis identified different genera when comparing pre- and post-calving samples, indicating significant changes in the faecal microbiota of dairy cows after calving compared to the dry period, closer to calving. Among the major changes, Verrucomicrobiota were less abundant in the two unknown genera from the phylum after calving. In contrast, the proportion of Bifidobacterium was higher after than before calving. Conclusion Shifts in faecal microbiota around calving may be attributed to changes in diet composition, feed intake modifications, or physiological changes from the dry period to lactation. However, other factors such as genetic background and health factors may also influence the microbiota composition. This could be further investigated to identify biomarkers for predicting imbalances or identifying maladaptation to the lactation stage.

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.

Our study was designed to gain a better understanding of how different feeding patterns affect the dynamics of gut microbiomes and microbe–host interactions. This research showed that the timing of solid food introduction is a key component of the gut microbiota shaping in early developmental stages, though with lower impact on settled gut microbiota profiles in older individuals. In mammals, the introduction of solid food is pivotal for the establishment of the gut microbiota. However, the effects of the first food consumed on long-term microbiota trajectory and host response are still largely unknown. This study aimed to investigate the influences of (i) the timing of first solid food ingestion and (ii) the consumption of plant polysaccharides on bacterial community dynamics and host physiology using a rabbit model. To modulate the first exposure to solid nutrients, solid food was provided to suckling rabbits from two different time points (3 or 15 days of age). In parallel, food type was modulated with the provision of diets differing in carbohydrate content throughout life: the food either was formulated with a high proportion of rapidly fermentable fibers (RFF) or was starch-enriched. We found that access to solid food as of 3 days of age accelerated the gut microbiota maturation. Our data revealed differential effects according to the digestive segment: precocious solid food ingestion influenced to a greater extent the development of bacterial communities of the appendix vermiformis , whereas life course polysaccharides ingestion had marked effects on the cecal microbiota. Greater ingestion of RFF was assumed to promote pectin degradation as revealed by metabolomics analysis. However, transcriptomic and phenotypic host responses remained moderately affected by experimental treatments, suggesting little outcomes of the observed microbiome modulations on healthy subjects. In conclusion, our work highlighted the timing of solid food introduction and plant polysaccharides ingestion as two different tools to modulate microbiota implantation and functionality. IMPORTANCE Our study was designed to gain a better understanding of how different feeding patterns affect the dynamics of gut microbiomes and microbe–host interactions. This research showed that the timing of solid food introduction is a key component of the gut microbiota shaping in early developmental stages, though with lower impact on settled gut microbiota profiles in older individuals. This study also provided in-depth analysis of dietary polysaccharide effects on intestinal microbiota. The type of plant polysaccharides reaching the gut through the lifetime was described as an important modulator of the cecal microbiome and its activity. These findings will contribute to better define the interventions that can be employed for modulating the ecological succession of young mammal gut microbiota.

DNA barcoding and metabarcoding are now powerful tools for studying biodiversity and especially the accurate identification of large sample collections belonging to diverse taxonomic groups. Their success depends largely on the taxonomic resolution of the DNA sequences used as barcodes and on the reliability of the reference databases. For wild bees, the barcode sequences coverage is consistently growing in volume, but some incorrect species annotations need to be cared for. The COI (Cytochrome Oxydase subunit 1) gene, the most used in barcoding/metabarcoding of arthropods, suffers from primer bias and difficulties for covering all wild bee species using the classical Folmer primers. We present here a curated database for a 250 bp mini-barcode region of the 16S rRNA gene, suitable for low-cost metabarcoding wild bees in applications, such as eDNA analysis or for sequencing ancient or degraded DNA. Sequenced specimens were captured in Occitania (south-west of France) and morphologically identified by entomologists, with a total of 530 individuals belonging to 171 species and 19 genera. A customised workflow including distance-tree inferences and a second round of entomologist observations, when necessary, was used for the validation of 348 mini-barcodes covering 148 species. Amongst them, 93 species did not have any 16S reference barcode available before our contribution. This high-quality reference library data are freely available to the scientific community, with the aim of facilitating future large-scale characterisation of wild bee communities in a context of pollinators' decline.

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.

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.

Oviduct 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. In 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. Our 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.

Background Relationships between microbial composition and steatosis are being extensively studied in mammals, and causal relations have been evidenced. In migratory birds the liver can transiently store lipids during pre-migratory and migratory phases, but little is known about the implications of the digestive microbiota in those mechanisms. The Landaise greylag goose ( Anser anser ) is a good model to study steatosis in migratory birds as it is domesticated, but is still, from a genetic point of view, close to its wild migratory ancestor. It also has a great ingestion capacity and a good predisposition for hepatic steatosis, whether spontaneous or induced by conventional overfeeding. The conventional (overfeeding) and alternative (spontaneous steatosis induction) systems differ considerably in duration and feed intake level and previous studies have shown that aptitudes to spontaneous steatosis are very variable. The present study thus aimed to address two issues: (i) evaluate whether microbial composition differs with steatosis-inducing mode; (ii) elucidate whether a digestive microbial signature could be associated with variable aptitudes to spontaneous liver steatosis. Results Performances, biochemical composition of the livers and microbiota differed considerably in response to steatosis stimulation. We namely identified the genus Romboutsia to be overrepresented in birds developing a spontaneous steatosis in comparison to those submitted to conventional overfeeding while the genera Ralstonia , Variovorax and Sphingomonas were underrepresented only in birds that did not develop a spontaneous steatosis compared to conventionally overfed ones, birds developing a spontaneous steatosis having intermediate values. Secondly, no overall differences in microbial composition were evidenced in association with variable aptitudes to spontaneous steatosis, although one OTU, belonging to the Lactobacillus genus, was overrepresented in birds having developed a spontaneous steatosis compared to those that had not. Conclusions Our study is the first to evaluate the intestinal microbial composition in association with steatosis, whether spontaneous or induced by overfeeding, in geese. Steatosis induction modes were associated with distinct digestive microbial compositions. However, unlike what can be observed in mammals, no clear microbial signature associated with spontaneous steatosis level was identified.