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1. Oral exposure to bisphenol S is associated with alterations in the oviduct proteome of an ovine model, with aggravated effects in overfed females

Author

Coline Mahé, Marie-Emilie Lebachelier de la Riviere, Olivier Lasserre, Guillaume Tsikis, Daniel Tomas, Valérie Labas, Sébastien Elis, and Marie Saint-Dizier

Subjects
Bisphenol S, Diet, Mass spectrometry, Oviduct, Secretions, Protein, Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract

Abstract Background Bisphenol S (BPS) is a substitute for bisphenol A in plastic manufacturing and, as a potential endocrine disruptor, may alter the physiology of the oviduct, in which fertilization and early embryo development take place in mammals. The objective of this study was to assess the effect of a daily dietary exposure to BPS combined with a contrasted diet on the oviduct fluid proteome using an ovine model. Results Eighty adult cyclic ewes were allotted to four groups (20/group): overfed (OF) consuming 50 µg/kg/day of BPS in their diet, underfed (UF) consuming 50 µg/kg/day of BPS, and non-exposed controls in each diet group. After three months, the mean body condition score, plasma levels of glucose and non-esterified fatty acids were significantly higher in OF than in UF females. The proteins in collected OF samples (50 µg) were analyzed by nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). Overall, 1563 proteins were identified, among which 848 were quantified. Principal component analysis of the data revealed a clear discrimination of samples according to the diet and a segregation between BPS-exposed and non-exposed females in overfed ewes. Hierarchical clustering of differentially abundant proteins (DAPs) identified two clusters of 101 and 78 DAPs according to the diet. Pairwise comparisons between groups revealed a stronger effect of BPS in OF than in UF females (70 vs. 24 DAPs) and a stronger effect of the diet in BPS-exposed than non-exposed females (56 vs. 36 DAPs). Functional analysis of DAPs showed an enrichment in metabolic processes, immune system, cell response to stress, and reproductive processes. Conclusions This work highlights for the first time the important impact of BPS on the oviduct proteome, with larger effects seen in OF than UF females. These results, together with previous ones, raise health concerns for everyone and call for a greater regulation of BPS in the food industry.

Published
2024
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3. Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens

Author

Thierry Moreau, Emilie Recoules, Marion De Pauw, Valérie Labas, and Sophie Réhault-Godbert

Subjects
chicken, pea, digestion, inhibitor, protease, Animal culture, SF1-1100
Abstract

ABSTRACT: Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies.

Published
2024
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4. The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo

Author

Charles Banliat, Coline Mahé, Régis Lavigne, Emmanuelle Com, Charles Pineau, Valérie Labas, Benoit Guyonnet, Pascal Mermillod, and Marie Saint-Dizier

Subjects
Embryo, Proteomics, Mass spectrometry, Morula, Blastocyst, Cattle, Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract

Abstract Background Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level. Results A total of 38 pools of grade-1 quality bovine embryos at the 4–6 cell, 8–12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development. Conclusions The maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4–6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

Published
2022
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5. Protein expression reveals a molecular sexual identity of avian primordial germ cells at pre-gonadal stages

Author

Laura Soler, Sabine Alves, Aurélien Brionne, Aurore Jacques, Vanessa Guérin, Maeva Cherif-Feildel, Lucie Combes-Soia, Sophie Fouchécourt, Aurore Thélie, Elisabeth Blesbois, Michael J. McGrew, Valérie Labas, and Marina S. Govoroun

Subjects
Medicine, Science
Abstract

Abstract In poultry, in vitro propagated primordial germ cells (PGCs) represent an important tool for the cryopreservation of avian genetic resources. However, several studies have highlighted sexual differences exhibited by PGCs during in vitro propagation, which may compromise their reproductive capacities. To understand this phenomenon, we compared the proteome of pregonadal migratory male (ZZ) and female (ZW) chicken PGCs propagated in vitro by quantitative proteomic analysis using a GeLC-MS/MS strategy. Many proteins were found to be differentially abundant in chicken male and female PGCs indicating their early sexual identity. Many of the proteins more highly expressed in male PGCs were encoded by genes localised to the Z sex chromosome. This suggests that the known lack of dosage compensation of the transcription of Z-linked genes between sexes persists at the protein level in PGCs, and that this may be a key factor of their autonomous sex differentiation. We also found that globally, protein differences do not closely correlate with transcript differences indicating a selective translational mechanism in PGCs. Male and female PGC expressed protein sets were associated with differential biological processes and contained proteins known to be biologically relevant for male and female germ cell development, respectively. We also discovered that female PGCs have a higher capacity to uptake proteins from the cell culture medium than male PGCs. This study presents the first evidence of an early predetermined sex specific cell fate of chicken PGCs and their sexual molecular specificities which will enable the development of more precise sex-specific in vitro culture conditions for the preservation of avian genetic resources.

Published
2021
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6. Three‐dimensional structures of avian beta‐microseminoproteins: insight from the chicken egg‐specific beta‐microseminoprotein 3 paralog

Author

Franck Coste, Thierry Moreau, Valérie Labas, Magali Chessé, Mégane Bregeon, Hervé Meudal, Karine Loth, Bertrand Castaing, Nicolas Guyot, and Sophie Réhault‐Godbert

Subjects
beta‐microseminoproteins, birds, crystal structure, egg, MSMB3, paralogs, Biology (General), QH301-705.5
Abstract

Beta‐microseminoproteins (MSMBs) are small disulfide‐rich proteins that are conserved among vertebrates. These proteins exhibit diverse biological activities and were mainly reported to play a role in male fertility, immunity, and embryogenesis. In this work, we focused on the chicken MSMB3 protein that was previously depicted as an egg antibacterial protein. We report that MSMB3 protein is exclusively expressed in the reproductive tissues of laying hens (in contrast to chicken MSMB1 and MSMB2 paralogs), to be incorporated in the egg white during the process of egg formation. We also showed that chicken MSMB3 possesses highly conserved orthologs in bird species, including Neognathae and Palaeognathae. Chicken MSMB3 was purified from egg white using heparin affinity chromatography and was analyzed by top‐down and bottom‐up proteomics. Several proteoforms could be characterized, and a homodimer was further evidenced by NMR spectroscopy. The X‐ray structure of chicken MSMB3 was solved for the first time, revealing that this protein adopts a novel dimeric arrangement. The highly cationic MSMB3 protein exhibits a distinct electrostatic distribution compared with chicken MSMB1 and MSMB2 structural models, and with published mammalian MSMB structures. The specific incorporation of MSMB3 paralog in the egg, and its phylogenetic conservation in birds together with its peculiar homodimer arrangement and physicochemical properties, suggests that the MSMB3 protein has evolved to play a critical role during the embryonic development of avian species. These new data are likely to stimulate research to elucidate the structure/function relationships of MSMB paralogs and orthologs in the animal kingdom.

Published
2021
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7. Dynamic Changes in the Proteome of Early Bovine Embryos Developed In Vivo

Author

Charles Banliat, Coline Mahé, Régis Lavigne, Emmanuelle Com, Charles Pineau, Valérie Labas, Benoit Guyonnet, Pascal Mermillod, and Marie Saint-Dizier

Subjects
embryo, proteomics, mass spectrometry, morula, blastocyst, cattle, Biology (General), QH301-705.5
Abstract

Early embryo development is a dynamic process involving important molecular and structural changes leading to the embryonic genome activation (EGA) and early cell lineage differentiation. Our aim was to elucidate proteomic changes in bovine embryos developed in vivo. Eleven females were used as embryo donors and pools of embryos at the 4–6 cell, 8–12 cell, morula, compact morula and blastocyst stages were analyzed by nanoliquid chromatography coupled with label free quantitative mass spectrometry. A total of 2,757 proteins were identified, of which 1,950 were quantitatively analyzed. Principal component analysis of data showed a clear separation of embryo pools according to their developmental stage. The hierarchical clustering of differentially abundant proteins evidenced a first cluster of 626 proteins that increased in abundance during development and a second cluster of 400 proteins that decreased in abundance during development, with most significant changes at the time of EGA and blastocyst formation. The main pathways and processes overrepresented among upregulated proteins were RNA metabolism, protein translation and ribosome biogenesis, whereas Golgi vesicle transport and protein processing in endoplasmic reticulum were overrepresented among downregulated proteins. The pairwise comparison between stages allowed us to identify specific protein interaction networks and metabolic pathways at the time of EGA, morula compaction and blastocyst formation. This is the first comprehensive study of proteome dynamics in non-rodent mammalian embryos developed in vivo. These data provide a number of protein candidates that will be useful for further mechanistic and functional studies.

Published
2022
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8. Proteomic Changes Associated With Sperm Fertilizing Ability in Meat-Type Roosters

Author

Anaïs Vitorino Carvalho, Laura Soler, Aurore Thélie, Isabelle Grasseau, Luiz Cordeiro, Daniel Tomas, Ana-Paula Teixeira-Gomes, Valérie Labas, and Elisabeth Blesblois

Subjects
sperm, chicken, proteomic, fertility, semen, Biology (General), QH301-705.5
Abstract

The molecular basis of male fertility remains unclear, especially in chickens, where decades of genetic selection increased male fertility variability as a side effect. As transcription and translation are highly limited in sperm, proteins are key molecules defining their functionality, making proteomic approaches one of the most adequate methods to investigate sperm capacity. In this context, it is interesting to combine complementary proteomic approaches to maximize the identification of proteins related to sperm-fertilizing ability. In the present study, we aimed at identifying proteins related to fertility in meat-type roosters, showing fertility variability. Fertile roosters (fertility rates higher than 70% after artificial insemination) differed from subfertile roosters (fertility rates lower than 40%) in their sperm mass motility. Fertile and subfertile sperm protein contents were compared using two complementary label-free quantitative proteomic methods: Intact Cell MALDI-TOF-Mass Spectrometry and GeLC-MS/MS. Combining the two strategies, 57 proteins were identified as differentially abundant. Most of them were described for the first time as differentially abundant according to fertility in this species. These proteins were involved in various molecular pathways including flagellum integrity and movement, mitochondrial functions, sperm maturation, and storage in female tract as well as oocyte–sperm interaction. Collectively, our data improved our understanding of chicken sperm biology by revealing new actors involved in the complexity of male fertility that depends on multiple cell functions to reach optimal rates. This explains the inability of reductionist in vitro fertility testing in predicting male fertility and suggests that the use of a combination of markers is a promising approach.

Published
2021
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9. Docosahexaenoic acid mechanisms of action on the bovine oocyte-cumulus complex

Author

Sebastien Elis, Mouhamad Oseikria, Anais Vitorino Carvalho, Priscila Silvana Bertevello, Emilie Corbin, Ana-Paula Teixeira-Gomes, Jérôme Lecardonnel, Catherine Archilla, Véronique Duranthon, Valérie Labas, and Svetlana Uzbekova

Subjects
Bovine, Oocyte-cumulus complex, FFAR4, DHA, Oocyte maturation, Gynecology and obstetrics, RG1-991
Abstract

Abstract Background Supplementation of bovine oocyte-cumulus complexes during in vitro maturation (IVM) with 1 μM of docosahexaenoic acid (DHA), C22:6 n-3 polyunsaturated fatty acid, was reported to improve in vitro embryo development. The objective of this paper was to decipher the mechanisms of DHA action. Results Transcriptomic analysis of 1 μM DHA-treated and control cumulus cells after 4 h IVM showed no significant difference in gene expression. MALDI-TOF mass spectrometry analysis of lipid profiles in DHA-treated and control oocytes and cumulus cells after IVM showed variations of only 3 out of 700 molecular species in oocytes and 7 out of 698 species in cumulus cells (p

Published
2017
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10. The Defibrillation Conundrum: New Insights into the Mechanisms of Shock-Related Myocardial Injury Sustained from a Life-Saving Therapy

Author

Nicolas Clementy, Alexandre Bodin, Arnaud Bisson, Ana-Paula Teixeira-Gomes, Sebastien Roger, Denis Angoulvant, Valérie Labas, and Dominique Babuty

Subjects
defibrillation, mechanism, injury, electrical, shock, proteomics, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

Implantable cardiac defibrillators (ICDs) are recommended to prevent the risk of sudden cardiac death. However, shocks are associated with an increased mortality with a dose response effect, and a strategy of reducing electrical therapy burden improves the prognosis of implanted patients. We review the mechanisms of defibrillation and its consequences, including cell damage, metabolic remodeling, calcium metabolism anomalies, and inflammatory and pro-fibrotic remodeling. Electrical shocks do save lives, but also promote myocardial stunning, heart failure, and pro-arrhythmic effects as seen in electrical storms. Limiting unnecessary implantations and therapies and proposing new methods of defibrillation in the future are recommended.

Published
2021
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11. Data on endogenous bovine ovarian follicular cells peptides and small proteins obtained through Top-down High Resolution Mass Spectrometry

Author

Valérie Labas, Ana-Paula Teixeira-Gomes, Laura Bouguereau, Audrey Gargaros, Lucie Spina, Aurélie Marestaing, and Svetlana Uzbekova

Subjects
Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract

The endogenous peptides and small proteins extracted from bovine ovarian follicular cells (oocytes, cumulus and granulosa cells) were identified by Top-down High Resolution Mass Spectrometry (TD-HR-MS/MS) in order to annotate peptido- and proteoforms detected using qualitative and quantitative profiling method based on ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The description and analysis of these Top-down MS data in the context of oocyte quality biomarkers research are available in the original research article of Labas et al. (2017) http://dx.doi.org/10.1016/j.jprot.2017.03.027 [1]. Raw data derived from this peptidomic/proteomic analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD004892). Here, we described the inventory of all identified peptido- and proteoforms including their biochemical and structural features, and functional annotation of correspondent proteins. This peptide/protein inventory revealed that TD-HR-MS/MS was appropriate method for both global and targeted proteomic analysis of ovarian tissues, and it can be further employed as a reference for other studies on follicular cells including single oocytes. Keywords: Top-down proteomics, Bovine, Ovary, Follicular cells

Published
2017
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12. Oviduct Fluid Extracellular Vesicles Change the Phospholipid Composition of Bovine Embryos Developed In Vitro

Author

Charles Banliat, Daniel Le Bourhis, Ophélie Bernardi, Daniel Tomas, Valérie Labas, Pascal Salvetti, Benoît Guyonnet, Pascal Mermillod, and Marie Saint-Dizier

Subjects
extracellular vesicles, exosomes, microvesicles, tubal fluid, fallopian tube, oviduct, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

Oviduct fluid extracellular vesicles (oEVs) have been proposed as bringing key molecules to the early developing embryo. In order to evaluate the changes induced by oEVs on embryo phospholipids, fresh bovine blastocysts developed in vitro in the presence or absence of oEVs were analyzed by intact cell MALDI-TOF (Matrix assisted laser desorption ionization—Time of flight) mass spectrometry (ICM-MS). The development rates, cryotolerance, and total cell number of blastocysts were also evaluated. The exposure to oEVs did not affect blastocyst yield or cryotolerance but modified the phospholipid content of blastocysts with specific changes before and after blastocoel expansion. The annotation of differential peaks due to oEV exposure evidenced a shift of embryo phospholipids toward more abundant phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingomyelins (SM) with long-chain fatty acids. The lipidomic profiling of oEVs showed that 100% and 33% of the overabundant masses in blastocysts and expanded blastocysts, respectively, were also present in oEVs. In conclusion, this study provides the first analysis of the embryo lipidome regulated by oEVs. Exposure to oEVs induced significant changes in the phospholipid composition of resulting embryos, probably mediated by the incorporation of oEV-phospholipids into embryo membranes and by the modulation of the embryonic lipid metabolism by oEV molecular cargos.

Published
2020
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13. Identification of 56 Proteins Involved in Embryo–Maternal Interactions in the Bovine Oviduct

Author

Charles Banliat, Guillaume Tsikis, Valérie Labas, Ana-Paula Teixeira-Gomes, Emmanuelle Com, Régis Lavigne, Charles Pineau, Benoit Guyonnet, Pascal Mermillod, and Marie Saint-Dizier

Subjects
tubal fluid, fallopian tube, oviduct, embryo, bovine, cattle, proteomics, secretions, morula, 4–6 cell, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4−5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4−6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

Published
2020
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14. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification

Author

Pauline Marie, Valérie Labas, Aurélien Brionne, Grégoire Harichaux, Christelle Hennequet-Antier, Alejandro B. Rodriguez-Navarro, Yves Nys, and Joël Gautron

Subjects
Chicken, Eggshell, Biomineralisation, Quantitative proteomics, Eggshell matrix, Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract

Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

Published
2015
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15. Data for chicken semen proteome and label free quantitative analyses displaying sperm quality biomarkers

Author

Valérie Labas, Isabelle Grasseau, Karine Cahier, Audrey Gargaros, Grégoire Harichaux, Ana-Paula Teixeira-Gomes, Sabine Alves, Marie Bourin, Nadine Gérard, and Elisabeth Blesbois

Subjects
Chicken (Gallus gallus), Spermatozoa, Seminal plasma, Proteome, Fertility, Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract

Understanding of biology of the avian male gamete is essential to improve the conservation of genetic resources and performances in farming. In this study, the semen proteome of the main domestic avian species (Gallus gallus) and evaluation of the molecular phenotype related to sperm quality were investigated using GeLC–MS/MS approach and label-free quantitative proteomic based on Spectral Counting (SC) and extracted ion chromatograms (XIC) methods. Here we describe in details the peptide/protein inventory of chicken ejaculated spermatozoa (SPZ) and seminal plasma (SP). We also show differential analyses of chicken semen (SPZ and corresponding SP) from 11 males demonstrating different levels of fertilizing capacity and sperm motility. The interpretation and description of these data can be found in a research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1]. This is a new resource for exploring the molecular mechanisms involved in fertilizing capacity and to reveal new sets of fertility biomarkers.

Published
2014
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16. Data in support of peptidomic analysis of spermatozoa during epididymal maturation

Author

Valérie Labas, Lucie Spina, Clémence Belleannee, Ana-Paula Teixeira-Gomes, Audrey Gargaros, Françoise Dacheux, and Jean-Louis Dacheux

Subjects
Peptidome, Spermatozoa, Maturation, Epididymis, Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract

The final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several epididymal maturation states could be an informative approach to finding sperm modifications related to maturation and fertility. Here we show the data from differential peptidomic/proteomic analyses on spermatozoa isolated from 4 epididymal regions (immature to mature stage) using a profiling approach based on MALDI-TOF mass spectrometry and, combined to top-down MS in order to identify peptidoforms and proteoforms. By this way, 172m/z peaks ranging between 2 and 20 kDa were found to be modified during maturation of sperm. A total of 62m/z were identified corresponding to 32 different molecular species. The interpretation of these data can be found in the research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1].

Published
2014
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17. Data set for the proteomic inventory and quantitative analysis of chicken uterine fluid during eggshell biomineralization

Author

Pauline Marie, Valérie Labas, Aurélien Brionne, Grégoire Harichaux, Christelle Hennequet-Antier, Yves Nys, and Joël Gautron

Subjects
Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract

Chicken eggshell is the protective barrier of the egg. It is a biomineral composed of 95% calcium carbonate on calcitic form and 3.5% organic matrix proteins. Mineralization process occurs in uterus into the uterine fluid. This acellular fluid contains ions and organic matrix proteins precursors which are interacting with the mineral phase and control crystal growth, eggshell structure and mechanical properties. We performed a proteomic approach and identified 308 uterine fluid proteins. Gene Ontology terms enrichments were determined to investigate their potential functions. Mass spectrometry analyses were also combined to label free quantitative analysis to determine the relative abundance of 96 proteins at initiation, rapid growth phase and termination of shell calcification. Sixty four showed differential abundance according to the mineralization stage. Their potential functions have been annotated. The complete proteomic, bioinformatic and functional analyses are reported in Marie et al., J. Proteomics (2015) [1].

Published
2014
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18. Lipid Identification and Transcriptional Analysis of Controlling Enzymes in Bovine Ovarian Follicle

Author

Priscila Silvana Bertevello, Ana-Paula Teixeira-Gomes, Alexandre Seyer, Anaïs Vitorino Carvalho, Valérie Labas, Marie-Claire Blache, Charles Banliat, Luiz Augusto Vieira Cordeiro, Veronique Duranthon, Pascal Papillier, Virginie Maillard, Sebastien Elis, and Svetlana Uzbekova

Subjects
ovarian follicle, lipids, mass spectrometry imaging, MALDI MS profiling, gene expression, oocyte, granulosa, theca, follicular fluid, bovine, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

Ovarian follicle provides a favorable environment for enclosed oocytes, which acquire their competence in supporting embryo development in tight communications with somatic follicular cells and follicular fluid (FF). Although steroidogenesis in theca (TH) and granulosa cells (GC) is largely studied, and the molecular mechanisms of fatty acid (FA) metabolism in cumulus cells (CC) and oocytes are emerging, little data is available regarding lipid metabolism regulation within ovarian follicles. In this study, we investigated lipid composition and the transcriptional regulation of FA metabolism in 3–8 mm ovarian follicles in bovine. Using liquid chromatography and mass spectrometry (MS), 438 and 439 lipids were identified in FF and follicular cells, respectively. From the MALDI-TOF MS lipid fingerprints of FF, TH, GC, CC, and oocytes, and the MS imaging of ovarian sections, we identified 197 peaks and determined more abundant lipids in each compartment. Transcriptomics revealed lipid metabolism-related genes, which were expressed constitutively or more specifically in TH, GC, CC, or oocytes. Coupled with differential lipid composition, these data suggest that the ovarian follicle contains the metabolic machinery that is potentially capable of metabolizing FA from nutrient uptake, degrading and producing lipoproteins, performing de novo lipogenesis, and accumulating lipid reserves, thus assuring oocyte energy supply, membrane synthesis, and lipid-mediated signaling to maintain follicular homeostasis.

Published
2018
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19. The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.

Author

Geoffrey Bailleul, Amanda Kravtzoff, Alix Joulin-Giet, Fabien Lecaille, Valérie Labas, Hervé Meudal, Karine Loth, Ana-Paula Teixeira-Gomes, Florence B Gilbert, Laurent Coquet, Thierry Jouenne, Dieter Brömme, Catherine Schouler, Céline Landon, Gilles Lalmanach, and Anne-Christine Lalmanach

Subjects
Medicine, Science
Abstract

Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion.

Published
2016
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20. Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate.

Author

Kévin Baranger, Marie-Louise Zani, Valérie Labas, Sandrine Dallet-Choisy, and Thierry Moreau

Subjects
Medicine, Science
Abstract

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.

Published
2011
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22. Pluripotent stem cell derived dopaminergic subpopulations model the selective neuron degeneration in Parkinson’s disease

Author

Clement Soleilhavoup, Kieran Patrick, Ulrika Marklund, Emma Moles-Garcia, Lucie Combes-Soia, Peter Hohenstein, Rosa Peltrini, Marco Travaglio, Shahida Sheraz, Lia Panman, Pedro Garção, Tony Oosterveen, and Valérie Labas

Subjects
Pluripotent Stem Cells, Parkinson's disease, derivation of substantia nigra dopaminergic neuronal lineage from pluripotent stem cells, Human Embryonic Stem Cells, Induced Pluripotent Stem Cells, Models, Neurological, Substantia nigra, Biology, Biochemistry, Article, Cell Line, Mice, Genetics, medicine, Animals, Humans, directed differentiation of pluripotent stem cells into distinct dopaminergic subpopulations, Induced pluripotent stem cell, Transcription factor, Otx Transcription Factors, Dopaminergic Neurons, Dopaminergic, Ventral Tegmental Area, Wnt signaling pathway, pluripotent stem cell-based model of Parkinson's disease, Cell Differentiation, Mouse Embryonic Stem Cells, Parkinson Disease, Cell Biology, modeling selective dopaminergic vulnerability in vitro, medicine.disease, Cell biology, Ventral tegmental area, Substantia Nigra, medicine.anatomical_structure, nervous system, Nerve Degeneration, Neuron, SOXD Transcription Factors, Developmental Biology
Abstract

Summary In Parkinson’s disease (PD), substantia nigra (SN) dopaminergic (DA) neurons degenerate, while related ventral tegmental area (VTA) DA neurons remain relatively unaffected. Here, we present a methodology that directs the differentiation of mouse and human pluripotent stem cells toward either SN- or VTA-like DA lineage and models their distinct vulnerabilities. We show that the level of WNT activity is critical for the induction of the SN- and VTA-lineage transcription factors Sox6 and Otx2, respectively. Both WNT signaling modulation and forced expression of these transcription factors can drive DA neurons toward the SN- or VTA-like fate. Importantly, the SN-like lineage enriched DA cultures recapitulate the selective sensitivity to mitochondrial toxins as observed in PD, while VTA-like neuron-enriched cultures are more resistant. Furthermore, a proteomics approach led to the identification of compounds that alter SN neuronal survival, demonstrating the utility of our strategy for disease modeling and drug discovery., Highlights • Derivation of distinct dopaminergic subpopulations from pluripotent stem cells • Wnt signaling inhibitors promote SN dopaminergic neuron specification • Modeling selective vulnerability of SN dopaminergic neurons in vitro • Proteomics reveals pathways that promote SN dopaminergic neuron survival, In this article, Panman and colleagues show that by modulating Wnt signaling levels, distinct subpopulations of dopaminergic neurons can be derived from mouse and human pluripotent stem cells. Cultures enriched for substantia nigra dopaminergic neurons recapitulate the selective sensitivity to mitochondrial toxins as observed in Parkinson’s disease patients. The developed strategy facilitates disease modeling and drug discovery.

Published
2021

23. Three‐dimensional structures of avian beta‐microseminoproteins: insight from the chicken egg‐specific beta‐microseminoprotein 3 paralog

Author

Bertrand Castaing, Mégane Bregeon, Valérie Labas, Magali Chessé, Karine Loth, Nicolas Guyot, Hervé Meudal, Thierry Moreau, Sophie Réhault-Godbert, Franck Coste, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Biologie des Oiseaux et Aviculture (BOA), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plate-forme Phénotypage par imagerie in/ex vivo de l'Animal à la Molécule (Plate-forme PIXANIM), Université de Tours-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université d'Orléans (UO), Région Centre Val de Loire : MUSE Project (Grant No. 2014-00094512) and Grant No. 2013-00082978, European Regional Development Fund (ERDF): SMHART Project, 35069, Conseil Régional du Centre, INRAE, INSERM, ANR-11-BSV5-0020,SPOREPAIR,Caractérisation d'une enzyme radicalaire de réparation de l'ADN : la lyase du photoproduit des spores(2011), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours (UT)-Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), REHAULT-GODBERT, Sophie, BLANC - Caractérisation d'une enzyme radicalaire de réparation de l'ADN : la lyase du photoproduit des spores - - SPOREPAIR2011 - ANR-11-BSV5-0020 - BLANC - VALID, Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Models, Molecular, 0301 basic medicine, crystal structure, animal structures, QH301-705.5, Eggs, [SDV]Life Sciences [q-bio], Crystallography, X-Ray, Proteomics, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 0302 clinical medicine, Affinity chromatography, Animals, MSMB, Amino Acid Sequence, paralogs, Biology (General), Research Articles, biology, Phylogenetic tree, Embryogenesis, Prostatic Secretory Proteins, biology.organism_classification, [SDV] Life Sciences [q-bio], 030104 developmental biology, Biochemistry, 030220 oncology & carcinogenesis, Neognathae, birds, beta‐microseminoproteins, egg, Chickens, Sequence Alignment, MSMB3, Function (biology), Research Article, Egg white
Abstract

Beta‐microseminoproteins (MSMBs) are small disulfide‐rich proteins that are conserved among vertebrates. These proteins exhibit diverse biological activities and were mainly reported to play a role in male fertility, immunity, and embryogenesis. In this work, we focused on the chicken MSMB3 protein that was previously depicted as an egg antibacterial protein. We report that MSMB3 protein is exclusively expressed in the reproductive tissues of laying hens (in contrast to chicken MSMB1 and MSMB2 paralogs), to be incorporated in the egg white during the process of egg formation. We also showed that chicken MSMB3 possesses highly conserved orthologs in bird species, including Neognathae and Palaeognathae. Chicken MSMB3 was purified from egg white using heparin affinity chromatography and was analyzed by top‐down and bottom‐up proteomics. Several proteoforms could be characterized, and a homodimer was further evidenced by NMR spectroscopy. The X‐ray structure of chicken MSMB3 was solved for the first time, revealing that this protein adopts a novel dimeric arrangement. The highly cationic MSMB3 protein exhibits a distinct electrostatic distribution compared with chicken MSMB1 and MSMB2 structural models, and with published mammalian MSMB structures. The specific incorporation of MSMB3 paralog in the egg, and its phylogenetic conservation in birds together with its peculiar homodimer arrangement and physicochemical properties, suggests that the MSMB3 protein has evolved to play a critical role during the embryonic development of avian species. These new data are likely to stimulate research to elucidate the structure/function relationships of MSMB paralogs and orthologs in the animal kingdom., Beta‐microseminoproteins (MSMBs) are small proteins conserved among vertebrates. The chicken genome contains three MSMB paralogs, which exhibit distinct tissue expression profiles, with chicken beta‐MSMB 3 (MSMB3) protein being egg‐specific. Collectively, results from protein sequence analysis, mass spectrometry analysis, and 3D structure data highlight that chicken MSMB3 possesses specific features that may be related to its presumed role in avian reproduction. ​

Published
2021

24. Structure, function, and evolution of Gga -AvBD11, the archetype of the structural avian-double-β-defensin family

Author

Sascha Trapp, Vincent Aucagne, A. Silvestre, P. Reverdiau, Céline Landon, Sophie Iochmann, Sophie Réhault-Godbert, Valérie Labas, Virginie Hervé, Karine Loth, Nicolas Guyot, Hervé Meudal, Guillaume Jousset, Agnès F. Delmas, Biologie des Oiseaux et Aviculture (BOA), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Infectiologie et Santé Publique (UMR ISP), Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pôle d’Analyse et d’Imagerie des Biomolécules, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Frapart, Isabelle, Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
beta-Defensins, animal structures, avian egg, Antiparasitic, medicine.drug_class, [SDV]Life Sciences [q-bio], Embryonic Development, Chicken Cells, avian influenza virus, Chick Embryo, Biology, Genome, Avian Proteins, Evolution, Molecular, 03 medical and health sciences, Influenza A Virus, H1N1 Subtype, Protein Domains, medicine, Animals, Cytotoxic T cell, Amino Acid Sequence, Cytotoxicity, Nuclear Magnetic Resonance, Biomolecular, Defensin, Gene, Phylogeny, 030304 developmental biology, 0303 health sciences, Multidisciplinary, Coccidiosis, 030302 biochemistry & molecular biology, Bacterial Infections, alarmin, Biological Sciences, Immunity, Innate, Cell biology, [SDV] Life Sciences [q-bio], Influenza in Birds, NMR structure, Biological Assay, Antibacterial activity, Chickens, Eimeria tenella
Abstract

Out of the 14 avian β-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian β-defensin 11 ( Gga -AvBD11). Given its specific localization and its established antibacterial activity, Gga -AvBD11 appears to play a protective role in embryonic development. Gga -AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to β-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga- AvBD11 is a compact fold composed of 2 packed β-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-β-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga -AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella , and avian influenza virus. Gga -AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga -AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

Published
2019

25. Protein palmitoylation in bovine ovarian follicle

Author

Ekaterina N. Shedova, Valérie Labas, Pascal Papillier, Peggy Jarrier-Gaillard, Svetlana Uzbekova, Rustem Uzbekov, G. N. Singina, Aurélie Marestaing, Ana-Paula Teixeira-Gomes, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), L.K. Ernst Federal Science Center for Animal Husbandry, Université de Tours, Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Tours (UT)

Subjects
Proteomics, follicular fluid extracellular vesicles, 0302 clinical medicine, Ovarian Follicle, Protein palmitoylation, Biology (General), Cells, Cultured, Spectroscopy, 0303 health sciences, Cumulus Cells, Chemistry, bovine, General Medicine, Protein subcellular localization prediction, Transmembrane protein, Computer Science Applications, Cell biology, medicine.anatomical_structure, Female, lipids (amino acids, peptides, and proteins), Signal transduction, QH301-705.5, Lipoylation, Article, Catalysis, protein palmitoylation, Inorganic Chemistry, 03 medical and health sciences, granulosa, Palmitoylation, medicine, Animals, Physical and Theoretical Chemistry, Ovarian follicle, oocyte, QD1-999, Molecular Biology, 030304 developmental biology, Granulosa Cells, Organic Chemistry, Proteins, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Oocyte, Cytosol, ZDHHC, Oocytes, Cattle, 030217 neurology & neurosurgery, cumulus
Abstract

International audience; Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.

Published
2021

26. Multifaceted roles of the egg perivitelline layer in avian reproduction: Functional insights from the proteomes of chicken egg inner and outer sublayers

Author

Mégane Brégeon, Daniel Tomas, Benoît Bernay, Céline Zatylny-Gaudin, Sonia Georgeault, Valérie Labas, Sophie Réhault-Godbert, Nicolas Guyot, Biologie des Oiseaux et Aviculture (BOA), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plate-forme Phénotypage par imagerie in/ex vivo de l'Animal à la Molécule (Plate-forme PIXANIM), Interactions Cellules Organismes Environnement (ICORE), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), Biologie des Organismes et Ecosystèmes Aquatiques (BOREA), Normandie Université (NU)-Normandie Université (NU)-Muséum national d'Histoire naturelle (MNHN)-Institut de Recherche pour le Développement (IRD)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université des Antilles (UA), Plateforme des Microscopies, Université de Tours (UT), This work was supported by the Région Centre-Val de Loire [grant number 2017-119983] and the Agence Nationale de la Recherche [grant number ANR-19-CE21-0006]., and ANR-19-CE21-0006,EQLIPSE,Amélioration de la qualité et des défenses antimicrobiennes internes de l'oeuf(2019)

Subjects
Proteomics, Sperm-Ovum Interactions, Outer perivitelline layer, Proteome, [SDV]Life Sciences [q-bio], Biophysics, Functional annotation, Gallus gallus, Biochemistry, Chicken, stomatognathic diseases, Inner perivitelline layer, Ultrastructure, Tandem Mass Spectrometry, Egg, Animals, Female, sense organs, Gene expression, Avian reproduction, Chickens, Ovum
Abstract

The avian egg perivitelline layer (PL) is a proteinaceous structure that encloses the egg yolk. It consists of the inner and the outer perivitelline layers (IPL and OPL, respectively) that are assumed to play distinct roles in bird reproduction. To gain insight into their respective function, we analyzed the proteome of IPL and OPL in chicken unfertilized eggs after mechanical separation, using a GeLC-MS/MS strategy. Of the 412 proteins identified, 173 proteins were uniquely recovered in IPL and 98 proteins in OPL, while 141 proteins were identified in both sublayers. Genes coding the most abundant proteins were shown to be expressed either in the liver/ovary (IPL formation) or in the oviduct (OPL formation), but rarely in both. The presence of oviduct-specific proteins (including LYZ, VMO1, AvBD11, PTN, OVAL and LOC10175704) in IPL strongly suggests that they participate in the physical association of IPL to OPL, whose tight attachment was further evidenced by analyses of IPL/OPL interfaces (by scanning electron microscopy). Functional annotation of identified proteins revealed functions associated with fertilization and early development for IPL, while OPL would rather participate in egg defense and embryogenesis. Collectively, our data highlight the complementary functions of IPL and OPL that are major determinants of bird reproductive success. SIGNIFICANCE: The present study unveils for the first time the individual proteomes of the two sublayers composing the chicken egg perivitelline layer (PL), which allowed to assign their respective putative biological roles in avian reproduction. The combination of proteomics with gene expression and ultrastructural analyses provides insightful data on the structure and biochemistry of the avian PL. The functional annotation of PL proteins highlights the multifaceted biological functions of this structure in reproduction including fertilization, embryonic development, and antimicrobial protection. This work will stimulate further research to validate predicted functions and to compare the physiology and the functional specificities of PL in egg-laying species.

Published
2021

27. PPILOW: innovations for improving animal welfare and human well-being in low-input outdoor and organic poultry and pig production systems

Author

Lucia Rocchi, Anne Collin, Giuditta Meloni, Claire Bonnefous, Martina Re, Laura van Vooren, An Jamart, Jarkko Niemi, Minna Väre, Katja Lähtinen, Tuyttens, Frank A. M., Mirjan Thys, Evelien Graat, Charlotte Vanden Hole, Tb (bas) Rodenburg, Saskia Kliphuis, Mona Giersberg, Antoine Roinsard, Olivia Tavares, Brieuc Desaint, Sanna Steenfeldt, Pedersen, Lene J., Engberg, Ricarda M., Isam Almadani Mohamad, Riccardo Carelli, Marlene Sciarretta, Andrea Rosati, Guilloteau, Laurence A., Sophie Réhault-Godbert, Joël Gautron, Elisabeth Le Bihan-Duval, Sandrine Mignon-Grasteau, Cécile Berri, Alexandre Barbin, Elodie Guettier, Elisabeth Baéza, Pascal Chartrin, Thierry Bordeau, Emilie Raynaud, Nathalie Couroussé, Estelle Cailleau-Audouin, Sabine Crochet, Pauline Bodin, Léa Tourneur, Adrien Guichaoua, Henry van den Brand, Bas Kemp, Cesare Castellini, Maxime Reverchon, Daniel Guemene, Michel Sourdioux, Sophie Brard-Fudulea, Roland Akakpo, Frédérique Renard-Dewinter, Ninfa Rangel Pedersen, Brunsgaard Grete, Rene Schepens, Maria Almind, Stefaan Depraetere, Katia Grenier, David Dubuc, Marie-Véronique Le Lann, Raffaella Ponzio, Marina Mainardi, Caterina Accotto, Monica Coletta, Vanessa Guesdon, Hélène Leruste, Bérangère Billiard, Vitor Hugo Bessa Ferreira, Keith Walley, Patricia Parrott, Shelley Allan, Lisa Baldinger, Helen Pluschke, Elsa Delanoue, Florence Macherez, Warin Laura, Jonathan Hercule, Marion Pertusa, Mathilde Stomp, Angelique Travel, Isabelle Bouvarel, Karine Germain, Laure Ravon, Ludovic Calandreau, Valérie Labas, Armelle Prunier, Elodie Merlot, Céline Tallet, Marie-Hélène Perruchot, Isabelle Louveau, Milgen, Jaap J., Frederic Dessauge, Caroline Clouard, Marie-Christine Meunier-Salaün, Bénédicte Lebret, Lucile Montagne, Justine Faure, Anna Zuliani, Pietro Venezia, Canario, Laurianne L., Stéphane Ferchaud, Vasile Cozma, Marina Spinu, Mihai Horia Bǎieş, Valérie Courboulay, Christine Roguet, Didier Gaudré, Patrick Chevillon, Laurent Alibert, Virginie Decruyenaere, Sophie Herremans, José Wavreille, Pia Vanggaard, Jan Bro Vanggaard, Cristina Micheloni, Petra Thobe, Università degli Studi di Perugia = University of Perugia (UNIPG), Biologie des Oiseaux et Aviculture (BOA), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Associazione Italiana per l'Agricoltura Biologica (AIAB), Scuela Santa Anna (SSSA), Scuola Universitaria Superiore Sant'Anna [Pisa] (SSSUP), BioForum Vlaanderen, Natural Resources Institute Finland (LUKE), Research Institute for Agricultural, Fisheries and Food (ILVO), Utrecht University [Utrecht], Institut Technique de l'Agriculture Biologique (ITAB), Aarhus University [Aarhus], Thuenen Institute of Farm Economics, European Association for Animal Production (EAAP), Les instituts techniques agricoles (Acta), Wageningen University and Research [Wageningen] (WUR), Syndicat des Sélectionneurs Avicoles et Aquacoles Français (SYSAAF), Fermentation Experts, Circular Organics, Équipe Micro et nanosystèmes HyperFréquences Fluidiques (LAAS-MH2F), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Équipe DIagnostic, Supervision et COnduite (LAAS-DISCO), Slow Food Biodiversity, JUNIA (JUNIA), Université catholique de Lille (UCL), Harper Adams University, Thuenen-Institute of Organic Farming, ITAVI, Institut de l'élevage (IDELE), Elevage Alternatif et Santé des Monogastriques (UE EASM), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie, Environnement et Génétique pour l'Animal et les Systèmes d'Elevage [Rennes] (PEGASE), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-INSTITUT AGRO Agrocampus Ouest, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), SIVtro VSF Italia, Génétique Physiologie et Systèmes d'Elevage (GenPhySE ), Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-École nationale supérieure agronomique de Toulouse (ENSAT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Unité Expérimentale Elevages Porcins Innovants (GenESI), USAMV Cluj-Napoca, Institut du Porc (IFIP), Centre Wallon de Recherches Agronomiques (CRA-W), Vanggaard Staldmontage, The PPILOW project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement N°816172., One Welfare CIC, PPILOW, and European Project: 816172,H2020-EU.3.2.1.1. - Increasing production efficiency and coping with climate change, while ensuring sustainability and resilience ,PPILOW (2019)

Subjects
[SDV.SA]Life Sciences [q-bio]/Agricultural sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, [SCCO]Cognitive science, [SDV.BA]Life Sciences [q-bio]/Animal biology, [SDV.AEN]Life Sciences [q-bio]/Food and Nutrition, [SDV.BDD]Life Sciences [q-bio]/Development Biology, ComputingMilieux_MISCELLANEOUS, [SHS]Humanities and Social Sciences
Abstract

International audience

Published
2021

28. The Defibrillation Conundrum: New Insights into the Mechanisms of Shock-Related Myocardial Injury Sustained from a Life-Saving Therapy

Author

Valérie Labas, Dominique Babuty, Nicolas Clementy, Ana-Paula Teixeira-Gomes, Denis Angoulvant, Alexandre Bodin, Sébastien Roger, and Arnaud Bisson

Subjects
medicine.medical_specialty, Defibrillation, injury, QH301-705.5, medicine.medical_treatment, mechanism, Review, shock, 030204 cardiovascular system & hematology, electrical, Catalysis, Sudden cardiac death, Inorganic Chemistry, 03 medical and health sciences, 0302 clinical medicine, proteomics, Response effect, Internal medicine, medicine, Animals, Humans, 030212 general & internal medicine, Life saving, Physical and Theoretical Chemistry, Biology (General), Molecular Biology, QD1-999, Spectroscopy, Inflammation, Myocardial stunning, Electroshock, business.industry, Myocardium, Organic Chemistry, General Medicine, Limiting, medicine.disease, defibrillation, Computer Science Applications, Defibrillators, Implantable, Oxidative Stress, Chemistry, Heart failure, Shock (circulatory), Cardiology, medicine.symptom, business
Abstract

Implantable cardiac defibrillators (ICDs) are recommended to prevent the risk of sudden cardiac death. However, shocks are associated with an increased mortality with a dose response effect, and a strategy of reducing electrical therapy burden improves the prognosis of implanted patients. We review the mechanisms of defibrillation and its consequences, including cell damage, metabolic remodeling, calcium metabolism anomalies, and inflammatory and pro-fibrotic remodeling. Electrical shocks do save lives, but also promote myocardial stunning, heart failure, and pro-arrhythmic effects as seen in electrical storms. Limiting unnecessary implantations and therapies and proposing new methods of defibrillation in the future are recommended.

Published
2021

29. Genetic determinants of seed protein plasticity in response to the environment in Medicago truncatula

Author

Julia Buitink, Valérie Labas, Joseph Ly Vu, Lucie Combes-Soia, Christine Le Signor, Kévin Cartelier, Delphine Aimé, Karine Gallardo, Jean‐Marie Prosperi, Agroécologie [Dijon], Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Recherche en Horticulture et Semences (IRHS), Université d'Angers (UA)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur], Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), National Science Foundation (grant NSF-0703285). The PhD fellowship of Kévin Cartelier was funded by the INRAE BAP (Plant Biology and Breeding) department and the Regional Council of Bourgogne-Franche-Comté., ANR-15-CE20-0001,REGULEG,Identification des régulateurs participant à la plasticité d'adaptation des graines de légumineuses aux changements environnementaux(2015), and Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0106 biological sciences, 0301 basic medicine, Candidate gene, Genotype, legumes, Mutant, Vitamin U, Genome-wide association study, Plant Science, Biology, methionine recycling, 01 natural sciences, [SDV.GEN.GPL]Life Sciences [q-bio]/Genetics/Plants genetics, 03 medical and health sciences, chemistry.chemical_compound, Methionine, Stress, Physiological, Medicago truncatula, Genetics, Storage protein, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Genome-wide association studies (GWAS), Gene, Plant Proteins, 2. Zero hunger, chemistry.chemical_classification, Seed Storage Proteins, food and beverages, Globulins, Cell Biology, biology.organism_classification, Metabolic pathway, Phenotype, 030104 developmental biology, chemistry, storage proteins, 13. Climate action, plasticity, Mutation, Seeds, seed, Genome-Wide Association Study, 010606 plant biology & botany
Abstract

As the frequency of extreme environmental events is expected to increase with climate change, identifying candidate genes for stabilizing the protein composition of legume seeds or optimizing this in a given environment is increasingly important. To elucidate the genetic determinants of seed protein plasticity, major seed proteins from 200 ecotypes of Medicago truncatula grown in four contrasting environments were quantified after one-dimensional electrophoresis. The plasticity index of these proteins was recorded for each genotype as the slope of Finlay and Wilkinson's regression and then used for genome-wide association studies (GWASs), enabling the identification of candidate genes for determining this plasticity. This list was enriched in genes related to transcription, DNA repair and signal transduction, with many of them being stress responsive. Other over-represented genes were related to sulfur and aspartate family pathways leading to the synthesis of the nutritionally essential amino acids methionine and lysine. By placing these genes in metabolic pathways, and using a M. truncatula mutant impaired in regenerating methionine from S-methylmethionine, we discovered that methionine recycling pathways are major contributors to globulin composition establishment and plasticity. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed protein composition.

Published
2021

30. Metabolic exchanges between the oocyte and its environment: focus on lipids

Author

Sébastien Elis, Ana-Paula Teixeira-Gomes, Priscila Silvana Bertevello, Valérie Labas, Philippe Monget, Svetlana Uzbekova, Rozenn Dalbies-Tran, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), L.K. Ernst Federal Science Center for Animal Husbandry, Plate-forme Phénotypage par imagerie in/ex vivo de l'Animal à la Molécule (Plate-forme PIXANIM), Université de Tours (UT)-Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Tours-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
endocrine system, Membrane lipids, Reproductive technology, Biology, protein palmitoylation, lipids, 03 medical and health sciences, 0302 clinical medicine, Endocrinology, Ovarian Follicle, Follicular phase, Genetics, medicine, Animals, Protein palmitoylation, Ovarian follicle, oocyte, Molecular Biology, 030304 developmental biology, 0303 health sciences, Cumulus Cells, Granulosa Cells, 030219 obstetrics & reproductive medicine, follicular cells, Lipid metabolism, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Oocyte, Follicular fluid, follicular fluid, Cell biology, medicine.anatomical_structure, Reproductive Medicine, fatty acid metabolism, Oocytes, Female, Animal Science and Zoology, lipids (amino acids, peptides, and proteins), ovary, extracellular vesicles, Developmental Biology, Biotechnology
Abstract

International audience; Finely regulated fatty acid (FA) metabolism within ovarian follicles is crucial to follicular development and influences the quality of the enclosed oocyte, which relies on the surrounding intra-follicular environment for its growth and maturation. A growing number of studies have examined the association between the lipid composition of follicular compartments and oocyte quality. In this review, we focus on lipids, their possible exchanges between compartments within the ovarian follicle and their involvement in different pathways during oocyte final growth and maturation. Lipidomics provides a detailed snapshot of the global lipid profiles and identified lipids, clearly discriminating the cells or fluid from follicles at distinct physiological stages. Follicular fluid appears as a main mediator of lipid exchanges between follicular somatic cells and the oocyte, through vesicle-mediated and non-vesicular transport of esterified and free FA. A variety of expression data allowed the identification of common and cell-type-specific actors of lipid metabolism in theca cells, granulosa cells, cumulus cells and oocytes, including key regulators of FA uptake, FA transport, lipid transformation, lipoprotein synthesis and protein palmitoylation. They act in harmony to accompany follicular development, and maintain intra-follicular homeostasis to allow the oocyte to accumulate energy and membrane lipids for subsequentmeiotic divisions and first embryo cleavages.

Published
2021

31. MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary

Author

Galina Singina, Valérie Labas, Ana-Paula Teixeira-Gomes, Virginie Maillard, Luiz Cordeiro, Pascal Papillier, Svetlana Uzbekova, Marie-Claire Blache, Rustem Uzbekov, Priscila Silvana Bertevello, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Chirurgie et Imagerie pour la Recherche et l'Enseignement [Nouzilly] (Plate-forme CIRE), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), L.K. Ernst Institute of Animal Husbandry, Faculté de Médecine, Laboratoire Biologie Cellulaire et Microscopie Electronique, Université Francois Rabelais [Tours], Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], lcsh:Chemistry, 0302 clinical medicine, Ovarian Follicle, Follicular phase, lcsh:QH301-705.5, Spectroscopy, media_common, 030219 obstetrics & reproductive medicine, Cumulus Cells, Chemistry, General Medicine, Lipids, Computer Science Applications, lipid fingerprints, medicine.anatomical_structure, Theca, Theca Cells, lipids (amino acids, peptides, and proteins), Female, extracellular vesicles, Corpus luteum, Ovulation, endocrine system, media_common.quotation_subject, mass spectrometry imaging, Catalysis, Article, ovarian follicles, Inorganic Chemistry, Andrology, 03 medical and health sciences, Follicle, medicine, Animals, Physical and Theoretical Chemistry, oocyte, Molecular Biology, Granulosa Cells, Organic Chemistry, Ovary, follicular cells, Lipid metabolism, Oocyte, Follicular fluid, follicular fluid, 030104 developmental biology, lcsh:Biology (General), lcsh:QD1-999, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Oocytes, Cattle
Abstract

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p &lt, 0.05, fold change &gt, 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Published
2020

32. Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular amorphous calcium carbonate

Author

Valérie Labas, Joël Gautron, Lucie Combes-Soia, Jacky Ezagal, Lilian Stapane, Nathalie Le Roy, Maxwell T. Hincke, Alejandro B. Rodríguez-Navarro, Biologie des Oiseaux et Aviculture (BOA), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Universidad de Granada (UGR), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Ottawa [Ottawa], Université François Rabelais de Tours, Région Centre-Val de Loire, Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2016-04410, Le STUDIUM, Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, Glycobiology and Extracellular Matrices, [SDV.SA.ZOO]Life Sciences [q-bio]/Agricultural sciences/Zootechny, Eggshell formation, Biochemistry, Mineralization (biology), Calcium Carbonate, Avian Proteins, 03 medical and health sciences, chemistry.chemical_compound, Egg Shell, Extracellular Vesicles, Calcification, Physiologic, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Animals, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Eggshell, Molecular Biology, Extracellular Matrix Proteins, 030102 biochemistry & molecular biology, Vesicle, Cell Biology, Extracellular vesicle, Amorphous calcium carbonate, Vesicular transport protein, 030104 developmental biology, chemistry, [SDV.SA.SPA]Life Sciences [q-bio]/Agricultural sciences/Animal production studies, Biophysics, Female, Chickens, Biomineralization
Abstract

Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also demonstrate that EVs contain ACC in uterine fluid using spectroscopic analysis. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of bicarbonate ions (HCO23 ), for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first-time experimental evidence for the components of vesicular transport to supply ACC in a vertebrate model of biomineralization., Universite Francois Rabelais de Tours, Region Centre-Val de Loire, Natural Sciences and Engineering Research Council of Canada RGPIN-2016-04410, Le STUDIUM

Published
2020

33. Intact cell MALDI-TOF mass spectrometry, a promising proteomic profiling method in farm animal clinical and reproduction research

Author

Elisabeth Blesbois, Laura Soler, Svetlana Uzbekova, Xavier Druart, Valérie Labas, Biosynthèse & Toxicité des Mycotoxines (ToxAlim-BioToMyc), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur], This work was supported by the French National Infrastructure of Research CRB anim funded by 'Investissements d'avenir', ANR-11-INBS-0003 and from the French National Institute of Agronomic Research. The high-resolution mass spectrometer was financed (SMHART project) by the European Regional Development Fund (ERDF), the Conseil Regional du Centre, the French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (Inserm). Laura Soler has received the support of the EU in the framework of the Marie-Curie FP7 COFUND People Programme, through the award of an AgreenSkills fellowship (under grant agreement no 267196)., ANR-11-INBS-0003,CRB-Anim,Réseau de Centres de Ressources Biologiques pour les animaux domestiques(2011), European Project: 267196,EC:FP7:PEOPLE,FP7-PEOPLE-2010-COFUND,AGREENSKILLS(2012), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Ecole d'Ingénieurs de Purpan (INP - PURPAN), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Proteomics, MALDI-TOF, Oocyte, Livestock, media_common.quotation_subject, [SDV]Life Sciences [q-bio], Vitelline membrane, Computational biology, Biology, 03 medical and health sciences, 0302 clinical medicine, Food Animals, medicine, Animals, Small Animals, reproductive and urinary physiology, media_common, 030219 obstetrics & reproductive medicine, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, Mass spectrometry, Equine, Proteomic Profiling, Reproduction, 0402 animal and dairy science, Intact cell, 04 agricultural and veterinary sciences, MALDI-TOF Mass Spectrometry, 040201 dairy & animal science, Sperm, medicine.anatomical_structure, Gene Expression Regulation, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, ICM-MS, [SDV.SA.SPA]Life Sciences [q-bio]/Agricultural sciences/Animal production studies, Proteome, embryonic structures, Animal Science and Zoology, Single-Cell Analysis
Abstract

The objective of this review is to provide new insights into the possible use of a proteomic method known as Intact Cell Matrix-Assisted Laser Desorption-ionization Time-Of-Flight Mass Spectrometry (ICM-MS) in animal clinical research. Here, we give an overview of the basics of this technique, its advantages and disadvantages compared with other proteomic approaches, past applications and future perspectives. A special emphasis on its implementation in animal reproduction science is given, including examples of the reliable use of ICM-MS on fertility screening. In mammals, the ICM-MS profiles from pig epididymal spermatozoa reflect the proteome changes that they undergo during epididymal maturation and could be associated with the acquisition of fertilizing ability. In chicken, using adequate pre-processing and bioinformatics analysis tools, sperm ICM-MS profiles showed characteristic spectral features that allowed their classification according to their actual fertilizing ability. The association of ICM-MS and Top-down proteomic strategies allowed the identification of chicken fertility biomarkers candidates such as protein vitelline membrane outer layer protein 1 (VMO-1) and avian beta-defensin 10 (AvBD10). In female reproduction, a similar approach on ovarian follicular cells allowed the identification of specific markers of oocyte maturation in the oocyte and surrounding cumulus cells. Altogether, these results indicate that ICM-MS profiling could be a suitable approach for molecular phenotyping of male and female gametes.

Published
2020

34. Beta-nerve growth factor stimulates spontaneous electrical activity of in vitro embryonic mouse GnRH neurons through a P75 mediated-mechanism

Author

Catherine Taragnat, Caroline Pinet-Charvet, Renaud Fleurot, Thierry Moreau, Guillaume Tsikis, Xavier Druart, Valérie Labas, Ana-Paula Teixeira-Gomes, Anne Duittoz, Flavie Derouin-Tochon, Xavier Cayla, Simon P. de Graaf, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), School of Life and Environmental Sciences A08 [Australia) (University of Sydney), The University of Sydney, Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Biologie des Oiseaux et Aviculture (BOA), INRA PHASE department (IMPAIC project), IFCE (NOVO project), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Male, INVOLVEMENT, 0301 basic medicine, Physiology, [SDV]Life Sciences [q-bio], Beta-Nerve Growth Factor, lcsh:Medicine, Gonadotropin-Releasing Hormone, Mice, GABA, Endocrinology, 0302 clinical medicine, Induced ovulation, Nerve Growth Factor, OVULATION-INDUCING FACTOR, lcsh:Science, Receptor, media_common, Neurons, GONADOTROPIN-RELEASING-HORMONE, Multidisciplinary, Reproduction, Neurotrophic factors, ENSHEATHING CELLS, Recombinant Proteins, Cell biology, SECRETION, Female, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Ovulation, media_common.quotation_subject, Reproductive biology, STATISTICAL-MODEL, Biology, Article, 03 medical and health sciences, Ovulation Induction, Corpus Luteum, Semen, In vivo, Animals, Humans, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, SEMINAL PLASMA, lcsh:R, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Embryonic stem cell, In vitro, 030104 developmental biology, nervous system, Central Nervous System Stimulants, lcsh:Q, HYPOTHALAMUS, Olfactory ensheathing glia, BIOLOGICAL-ACTIVITY, 030217 neurology & neurosurgery
Abstract

The control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (β-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca β-NGF (aβ-NGF) and its 3D structure and compared it with human recombinant β-NGF (hβ-NGF). We showed that the β-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that β-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism.

Published
2020

35. A new model for vertebrate mineralization via stabilized amorphous calcium carbonate for avian eggshell formation

Author

Alejandro B. Rodríguez-Navarro, Lilian Stapane, Valérie Labas, Joël Gautron, Jacky Ezagal, Maxwell T. Hincke, Nathalie Le Roy, and Lucie Combes-Soia

Subjects
0303 health sciences, Chemistry, 030302 biochemistry & molecular biology, chemistry.chemical_element, Eggshell formation, Calcium, Mineralization (biology), Amorphous calcium carbonate, Vesicular transport protein, 03 medical and health sciences, chemistry.chemical_compound, Calcium carbonate, Biophysics, Eggshell, 030304 developmental biology, Biomineralization
Abstract

Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where mineralization takes place. Herein, we report further experimental evidence for this model. We confirmed the presence of extracellular vesicles (EVs) that contain ACC in uterine fluid using transmission electron microscopy and elemental analysis. We also demonstrate high levels of expression of vesicular markers in the oviduct segments where eggshell is formed. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of HCO3−, for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first time experimental evidence for the components of vesicular transport to supply ACC in vertebrate biomineralization. These results could give insight to understand the mineralization of otoconia, which are calcium carbonate biomineralized structures present in all vertebrates and necessary for balance and sensing linear acceleration.

Published
2020

36. Progesterone induces sperm release from oviductal epithelial cells by modifying sperm proteomics, lipidomics and membrane fluidity

Author

Pascal Mermillod, Marina Ramal-Sanchez, Nicola Bernabò, Xavier Druart, Guillaume Tsikis, Marie Saint-Dizier, Valérie Labas, Marie-Claire Blache, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Facoltà di Bioscienze e tecnologie agro-alimentari e ambientali - Faculty of Bioscience and Agro-Food and Environmental Technology [Teramo], Universita degli studi di Teramo - University of Teramo [Italie] (UNITE), Chirurgie et Imagerie pour la Recherche et l'Enseignement [Nouzilly] (Plate-forme CIRE), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), European Project: 675526,H2020,H2020-MSCA-ITN-2015,REP-BIOTECH(2015), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Università degli Studi di Teramo (UniTE)

Subjects
0301 basic medicine, Male, Proteomics, endocrine system, Proteome, Membrane Fluidity, Membrane lipids, 030209 endocrinology & metabolism, Insemination, Biochemistry, law.invention, 03 medical and health sciences, 0302 clinical medicine, Endocrinology, Confocal microscopy, law, Binder of sperm proteins, Lipidomics, Membrane fluidity, Animals, Molecular Biology, Cells, Cultured, Fallopian Tubes, reproductive and urinary physiology, Progesterone, Sperm-Ovum Interactions, Chemistry, urogenital system, Epithelial Cells, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Sperm capacitation, Oviduct, Sperm, Spermatozoa, In vitro, Cell biology, 030104 developmental biology, Cattle, Female
Abstract

International audience; The sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) - 3 and - 5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls.

Published
2020

37. Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets

Author

Mustapha Berri, Isabelle Fleurot, Sandrine Melo, Laura Soler, Ignacio S. Caballero, Ana-Paula Teixeira-Gomes, Valérie Labas, Nicolas Cenac, Mustapha Si-Tahar, Julien Pujo, Andrea Bähr, Déborah Bréa, Claire Chevaleyre, Nikolai Klymiuk, Antoine Guillon, SEGUIN, Nathalie, Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biosynthèse & Toxicité des Mycotoxines (ToxAlim-BioToMyc), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire d'Analyses des Interactions Moléculaires, Plate-forme d'Analyses intégrative des Biomarqueurs cellulaires et moléculaires (PAIB), UMR6175, Institut National de la Recherche Agronomique (INRA), Institut de Recherche en Santé Digestive (IRSD ), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Gene Center and Center for Innovation Medical Models (CiMM), Ludwig-Maximilians University [Munich] (LMU), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Vaincre la Mucoviscidose [grant numbers RF20130500925 and RF2015050357, Region Centre (France) [grant number 320 0 0604 ]., Metatoul- Lipidomique Core Facility-MetaboHub, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Ludwig Maximilians University of Munich, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), and Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, Pulmonary and Respiratory Medicine, Male, Proteomics, Swine, Neutrophils, [SDV.MHEP.PHY] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], [SDV]Life Sciences [q-bio], Inflammation, medicine.disease_cause, Cystic fibrosis, Flow cytometry, 03 medical and health sciences, 0302 clinical medicine, Immunophenotyping, medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Animals, CFTR, chemistry.chemical_classification, Reactive oxygen species, Proteomic Profile, Lung, medicine.diagnostic_test, Pseudomonas aeruginosa, business.industry, Models, Theoretical, medicine.disease, Pig model, Molecular biology, 3. Good health, [SDV] Life Sciences [q-bio], Disease Models, Animal, 030104 developmental biology, medicine.anatomical_structure, 030228 respiratory system, chemistry, Animals, Newborn, ICM-MS, Pediatrics, Perinatology and Child Health, Fatty Acids, Unsaturated, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, medicine.symptom, business, Reactive Oxygen Species
Abstract

International audience; Background: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process.Methods: Peripheral blood neutrophils were isolated from newborn CFTR+/+ and CFTR-/- piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated.Results: Polyunsaturated fatty acid metabolites analysis did not show any difference between CFTR+/+ and CFTR-/- neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR-/- neutrophils to kill pseudomonas aeruginosa in vitro was observed.Conclusions: ICM-MS demonstrated that CFTR-/- neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.

Published
2020

38. Identification of 56 Proteins Involved in Embryo–Maternal Interactions in the Bovine Oviduct

Author

Emmanuelle Com, Charles Banliat, Ana-Paula Teixeira-Gomes, Marie Saint-Dizier, Régis Lavigne, Charles Pineau, Pascal Mermillod, Valérie Labas, Benoit Guyonnet, Guillaume Tsikis, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Chirurgie et Imagerie pour la Recherche et l'Enseignement [Nouzilly] (Plate-forme CIRE), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Infectiologie et Santé Publique (UMR ISP), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de recherche en santé, environnement et travail (Irset), Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Proteomics Core Facility (Protim), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Plateforme Génomique Santé Biogenouest®, Biologie de la Reproduction, Environnement, Epigénétique & Développement (BREED), Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-École nationale vétérinaire d'Alfort (ENVA)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), No grant number, Allan Wilson Centre, 2017/0684, Association Nationale de la Recherche et de la Technologie, Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Biologie du Développement et Reproduction (BDR), Institut National de la Recherche Agronomique (INRA), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Rennes (UR)-Plateforme Génomique Santé Biogenouest®, École nationale vétérinaire - Alfort (ENVA)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Santé et de la Recherche Médicale (INSERM)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Université d'Angers (UA), École nationale vétérinaire d'Alfort (ENVA)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Jonchère, Laurent

Subjects
0301 basic medicine, Blastomeres, Proteome, oviduct, [SDV]Life Sciences [q-bio], Oviducts, morula, Proteomics, lcsh:Chemistry, 0302 clinical medicine, tubal fluid, OVGP1, Zona pellucida, lcsh:QH301-705.5, Spectroscopy, ComputingMilieux_MISCELLANEOUS, fallopian tube, 030219 obstetrics & reproductive medicine, bovine, Serine Endopeptidases, Embryo, General Medicine, Computer Science Applications, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, embryonic structures, 4–6 cell, Oviduct, Female, Vitelline Membrane, endocrine system, animal structures, Annexins, embryo, Perivitelline space, Biology, Catalysis, Article, Protein–protein interaction, Inorganic Chemistry, 03 medical and health sciences, proteomics, medicine, Animals, Physical and Theoretical Chemistry, Molecular Biology, urogenital system, secretions, Organic Chemistry, Blastomere, 030104 developmental biology, lcsh:Biology (General), lcsh:QD1-999, cattle
Abstract

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4&ndash, 5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4&ndash, 6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

Published
2020

39. Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage

Author

Xavier Druart, Valérie Labas, Simon P. de Graaf, Kiri Farmer, Ana Paula Teixeira-Gomes, Guillaume Tsikis, Taylor Pini, T. Leahy, University of Sydney, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA), Australian Wool Innovation [ON_00252], NSW Stud Merino Breeders Association, European Regional Development Fund [3069], Conseil Regional du Centre, French National Institute for Agricultural Research, French National Institute of Health and Medical Research (INSERM), and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, endocrine system, Cryoprotectant, Seminal Plasma Proteins, [SDV]Life Sciences [q-bio], Motility, General Biochemistry, Genetics and Molecular Biology, Cryopreservation, Andrology, 03 medical and health sciences, chemistry.chemical_compound, Cryoprotective Agents, 0302 clinical medicine, Semen, Binder of sperm proteins, Freezing, Animals, [INFO]Computer Science [cs], Phosphatidylethanolamine, Sheep, 030219 obstetrics & reproductive medicine, urogenital system, Phosphatidylethanolamines, 0402 animal and dairy science, Tyrosine phosphorylation, 04 agricultural and veterinary sciences, General Medicine, Spermatozoa, 040201 dairy & animal science, Mucus, Sperm, Ovine, Fertility, chemistry, Sperm Motility, Cattle, General Agricultural and Biological Sciences, Semen Preservation
Abstract

International audience; Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

Published
2018

40. Use of MALDI-TOF mass spectrometry to explore the peptidome and proteome of in-vitro produced bovine embryos pre-exposed to oviduct fluid

Author

Benoit Guyonnet, Charles Banliat, Valérie Labas, Daniel Tomas, Pascal Mermillod, Ana-Paula Teixeira-Gomes, Marie Saint-Dizier, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Union Evolution, Partenaires INRAE, Plate-forme Phénotypage par imagerie in/ex vivo de l'Animal à la Molécule (Plate-forme PIXANIM), Université de Tours (UT)-Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours (UT), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Tours

Subjects
Proteomics, animal structures, Proteome, [SDV]Life Sciences [q-bio], Peptide, Fertilization in Vitro, Oviducts, Fallopian tube, Mass spectrometry, 03 medical and health sciences, 0302 clinical medicine, Endocrinology, Animals, Peptidomics, 030304 developmental biology, Transitional Endoplasmic Reticulum ATPase, chemistry.chemical_classification, 0303 health sciences, 030219 obstetrics & reproductive medicine, Proteins, Embryo, Oviduct, Embryo, Mammalian, In vitro, Biochemistry, chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, embryonic structures, Cattle, Female, Animal Science and Zoology, Developmental Biology
Abstract

International audience; Our objective was to investigate the regulation of the proteome in the bovine oviductal fluid according to the stage of the oestrous cycle, to the side relative to ovulation and to local concentrations of steroid hormones. Luminal fluid samples from both oviducts were collected at four stages of the oestrous cycle: pre-ovulatory (Pre-ov), post-ovulatory (Post-ov), and mid- and late luteal phases from adult cyclic cows (18–25 cows/stage). The proteomes were assessed by nanoLC–MS/MS and quantified by label-free method. Totally, 482 proteins were identified including a limited number of proteins specific to one stage or one side. Proportions of differentially abundant proteins fluctuated from 10 to 24% between sides at one stage and from 4 to 20% among stages in a given side of ovulation. In oviductal fluids ipsilateral to ovulation, Annexin A1 was the most abundant protein at Pre-ov compared with Post-ov while numerous heat shock proteins were more abundant at Post-ov compared with Pre-ov. Among differentially abundant proteins, seven tended to be correlated with intra-oviductal concentrations of progesterone. A wide range of biological processes was evidenced for differentially abundant proteins, of which metabolic and cellular processes were predominant. This work identifies numerous new candidate proteins potentially interacting with the oocyte, spermatozoa and embryo to modulate fertilization and early embryo development.

Published
2021

41. Effect of a long-chain n-3 polyunsaturated fatty acid–enriched diet on adipose tissue lipid profiles and gene expression in Holstein dairy cows

Author

Virginie Maillard, Joëlle Dupont, Sandrine Freret, Sébastien Elis, Alice Desmarchais, Juliette Cognie, Valérie Labas, Céline Hivelin, Eric Briant, Svetlana Uzbekova, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Plateforme d'Analyse Intégrative des Biomolécules, Institut National de la Recherche Agronomique (INRA), Unité Expérimentale de Physiologie Animale de l‘Orfrasiére (UE PAO), Région Centre Val de Loire, INRA, Apis-Gene, Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Unité Expérimentale de Physiologie Animale de l‘Orfrasiére (Unité Expérimentale de Physiologie Animale de l‘Orfrasiére - UE PAO), and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, bovin, mass spectrum analysis, tissu adipeux, Adipose tissue, chemistry.chemical_compound, spectrométrie de masse, dairy cows, 2. Zero hunger, chemistry.chemical_classification, Fatty Acids, food and beverages, 04 agricultural and veterinary sciences, Fish oil, adipose tissue, Milk, Docosahexaenoic acid, vache laitière, Female, Polyunsaturated fatty acid, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], medicine.medical_specialty, expression génique, Biology, acide gras polyinsaturé n 3, Fatty acid-binding protein, 03 medical and health sciences, Perilipin-1, Internal medicine, Fatty Acids, Omega-3, Genetics, medicine, Animals, Lactation, Lipolysis, maldi tof, Fatty acid metabolism, 0402 animal and dairy science, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, 040201 dairy & animal science, Diet, 030104 developmental biology, Endocrinology, chemistry, Dietary Supplements, gene expression, Cattle, Animal Science and Zoology, paramètre zootechnique, Food Science
Abstract

The objective of this study was to determine whether fish oil supplement has an effect on adipose tissue lipid profiles and gene expression in postpartum dairy cows. Holstein cows were supplemented with either long-chain n-3 polyunsaturated fatty acid (PUFA; protected fish oil) or control PUFA (n-6; toasted soybeans) for 2mo after calving (n=23 per diet). These cows showed no difference in milk production or metabolic parameters, but exhibited a tendency toward a decrease in early embryo mortality rate after artificial insemination. We hypothesized that, in addition to this effect, modifications in adipose tissue (AT) gene expression and lipid profiles would occur in response to diet. Subcutaneous AT samples were thus collected from the dewlaps of n-3 and n-6 dairy cows at 1mo antepartum, and 1wk, 2mo, and 5mo postpartum for the analysis of lipids and gene expression. Lipid profiles were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in both positive and negative modes. We found 37 lipid species in the 200 to 1,200 m/z range, which differed between the n-3 and control groups, suggesting that the n-3 supplement affected the lipid composition through the enrichment of lipids integrating long-chain PUFA from fish oil sources: eicosapentaenoic and docosahexaenoic acid. Moreover, a decrease in triacylglycerolipids was observed in AT of n-3 supplemented cows. The expression of 44 genes involved in fatty acid metabolism and the adipokine system was assessed by real-time reverse-transcription PCR. Hierarchical clustering, according to either postpartum stage or diet, enabled us to group genes exhibiting similar kinetic properties during lactation or by those that varied in similar ways after n-3 supplementation, respectively. Among the genes exhibiting a dietary effect, FABP4, LIPE, CD36, and PLIN1 were overexpressed in n-3 AT samples compared with the control, suggesting an increase in lipolysis due to n-3 supplementation, which was reflected on lipolytic activity at the protein level (i.e., protein expression of fatty acid binding protein 4, phosphorylated perilipin 1, and phosphorylated hormone-sensitive lipase). This increase in lipolysis is relevant to the decrease in triglycerides observed in these samples. Gene expression analyses between n-3 and control AT samples also suggested that the n-3 diet could modulate the secretory functions of AT, possibly by affecting adipokine expression; however, this has to be confirmed at the protein level.

Published
2016

42. Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival

Author

Grégoire Harichaux, Cindy Riou, Joël Gautron, Audrey Gargaros, Valérie Labas, Luiz Cordeiro, Aurélien Brionne, Nadine Gérard, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Biologie des Oiseaux et Aviculture (BOA), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme d'Analyse Intégrative des Biomolécules, Institut National de la Recherche Agronomique (INRA), Plateforme Intégrative des Biomolécules, Grant Sponsor: Conseil régional du Centre Val de Loire. Grant name : OVISPERM, Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), UMR0083, Biologie des Oiseaux et Aviculture (BOA), Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Allice, Station de Phénotypage, Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Federal Institute of Rio Grande do Norte [Mossoró] (IFRN), and Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, [SDV.SA]Life Sciences [q-bio]/Agricultural sciences, Male, Proteomics, Proteome, Protein Deglycase DJ-1, Exosomes, 0302 clinical medicine, Valosin Containing Protein, Biologie de la reproduction, mineralization, Annexin A4, protéome, Biological Phenomena, Reproductive Biology, 030219 obstetrics & reproductive medicine, composition protéinique, Spermatozoa, Cell biology, Body Fluids, fluide utérin, medicine.anatomical_structure, survie des spermatozoïdes, Immunohistochemistry, Female, faune aviaire, Autre (Sciences du Vivant), Female circumcision, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], coquille d'oeuf, Biology, sperm, 03 medical and health sciences, Semen, Genetics, medicine, exosome, Animals, HSPA8, avian, Uterus, PARK7, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Cell Biology, Sperm, Microvesicles, Epithelium, 030104 developmental biology, uterine fluid, exosomes, proteome, minéralisation, Chickens, Biomarkers, Developmental Biology
Abstract

International audience; Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom-up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

Published
2019

43. Guinea fowl eggshell quantitative proteomics yield new findings related to its unique structural characteristics and superior mechanical properties

Author

Aurélien Brionne, Nathalie Le Roy, Alejandro B. Rodríguez-Navarro, Lucie Combes-Soia, Valérie Labas, Maxwell T. Hincke, Yves Nys, Joël Gautron, Biologie des Oiseaux et Aviculture (BOA), Institut National de la Recherche Agronomique (INRA)-Université de Tours, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Universidad de Granada (UGR), University of Ottawa [Ottawa], Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Biomineralization, Yield (engineering), Ovalbumin, Quantitative proteomics, Biophysics, Biochemistry, Texture (geology), Mineralization (biology), Calcium Carbonate, 03 medical and health sciences, chemistry.chemical_compound, Egg Shell, Animals, Eggshell, Calcite, 030102 biochemistry & molecular biology, [SDV.BA]Life Sciences [q-bio]/Animal biology, Egg Proteins, Proteins, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, 15. Life on land, 030104 developmental biology, chemistry, Guinea fowl, Organic matrix, Muramidase, Lysozyme, Chickens
Abstract

International audience; The Guinea fowl eggshell is a bioceramic material with the remarkable mechanical property of being twice as strong as the chicken eggshell. Both eggshells are composed of 95% calcite and 3.5% organic matrix, which control its structural organization. Chicken eggshell is made of columnar calcite crystals arranged vertically. In the Guinea fowl, the same structure is observed in its inner half, followed by a dramatic change in crystal size and orientation in the outer region. Guinea fowl eggshell is thicker than chicken eggshell. Both structure and shell thickness confer a superior resistance to breakage compared to eggshells of other bird species. To understand the underlying mechanisms controlling the structural organization of this highly resistant material, we used quantitative proteomics to analyze the protein composition of the Guinea fowl eggshell organic matrix at key stages of the biomineralization process. We identified 149 proteins, which were compared to other bird eggshell proteomes and analyzed their potential functions. Among the 149 proteins, 9 are unique to Guinea fowl, some are involved in the control of the calcite precipitation (Lysozyme, Ovocleidin-17-like, Ovocleidin-116 and Ovalbumin), 61 are only found in the zone of microstructure shift and 17 are more abundant in this zone. Significance: The avian eggshell is a critical physical barrier to protect the contents of this autonomous reproductive enclosure from physical and microbial assault. The Guinea fowl (Numida meleagris) eggshell exhibits a unique microstructure (texture), which confers exceptional mechanical properties compared to eggshells of other species. In order to understand the mechanisms that regulate formation of this texture in the Guinea fowl eggshell, we performed comparative quantitative proteomics at key stages of shell mineralization and particularly during the dramatic shift in shell microstructure. We demonstrate that the Guinea fowl eggshell proteome comprises 149 proteins, of which 61 were specifically associated with the change in size and orientation of calcite crystals. Comparative proteomics analysis with eggshell of other bird species leads to new insights into the biomineralization process. Moreover, our data represents a list of organic compounds as potential additives to regulate material design for industrial fabrication of ceramics. This information also provides molecular markers for efficient genomic selection of chicken strains to lay eggs with improved shell mechanical properties for enhanced food safety.

Published
2019

44. Polymer 'ruthenium-cyclopentadienyl' conjugates - New emerging anti-cancer drugs

Author

Pierre Falson, Philippe Zinck, Rita Francisco, Elisabeta Comsa, Fernanda Marques, A. P. Alves de Matos, Valérie Labas, Andreia Valente, Lucie Combes-Soia, Cyril Rousseau, M. Helena Garcia, Ana Preto, Tiago Moreira, Audrey Favrelle, Ana-Paula Teixeira-Gomes, Sophie Duban-Deweer, School of Applied Social Sciences, Durham University, Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiopathologie de la Barrière Hémato-Encéphalique (LBHE), Université d'Artois (UA), Pôle d'Analyse et d’Imagerie des Biomolécules (PAIB), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Departamento de Química, Instituto Tecnológico e Nuclear, Unité de Catalyse et de Chimie du Solide - UMR 8181 (UCCS), Université d'Artois (UA)-Ecole Centrale de Lille-Ecole Nationale Supérieure de Chimie de Lille (ENSCL)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Informatique pour la Mécanique et les Sciences de l'Ingénieur (LIMSI), Université Paris-Sud - Paris 11 (UP11)-Université Paris-Saclay-Sorbonne Université - UFR d'Ingénierie (UFR 919), Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Saclay (COmUE), Universidade do Minho, Universidade de Lisboa = University of Lisbon (ULISBOA), Universidade do Minho = University of Minho [Braga], Laboratoire de la Barrière Hémato-Encéphalique (LBHE), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Instituto Superior de Ciências da Saúde Egas Moniz [Portugal], Unité de Catalyse et Chimie du Solide - UMR 8181 (UCCS), Université d'Artois (UA)-Centrale Lille-Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Universidade de Lisboa (ULISBOA), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Centrale Lille Institut (CLIL)-Université d'Artois (UA)-Centrale Lille-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Lille, ENSCL, CNRS, Centrale Lille, Univ. Artois, Université de Lille, Faculdade de Ciências [Lisboa], Centre of Molecular and Environmental Biology - CBMA (Braga, Portugal), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry [MMSB], Laboratoire de la Barrière Hémato-Encéphalique [LBHE], Université de Tours [UT], University of Lisboa, Instituto de Engenharia de Sistemas e Computadores Investigação e Desenvolvimento em Lisboa [INESC-ID], Unité de Catalyse et Chimie du Solide - UMR 8181 [UCCS], and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)

Subjects
Polymers, Ciências Biológicas [Ciências Naturais], [CHIM.THER]Chemical Sciences/Medicinal Chemistry, 01 natural sciences, Coordination Complexes, Drug Discovery, Cytoskeleton, 0303 health sciences, Molecular Structure, Chemistry, Ruthenium organometallic compounds, Polymer-metal conjugates, Proteomic analysis, Biological activity, General Medicine, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], medicine.symptom, medicine.drug, Cell Survival, Antineoplastic Agents, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cyclopentanes, [CHIM.INOR]Chemical Sciences/Inorganic chemistry, Ruthenium, Structure-Activity Relationship, 03 medical and health sciences, Microscopy, Electron, Transmission, Cell Line, Tumor, medicine, Humans, [CHIM.COOR]Chemical Sciences/Coordination chemistry, Clonogenic assay, Cell Proliferation, 030304 developmental biology, Pharmacology, Cisplatin, Ciências Naturais::Ciências Biológicas, Science & Technology, Dose-Response Relationship, Drug, 010405 organic chemistry, Organic Chemistry, 0104 chemical sciences, Mechanism of action, Apoptosis, Cell culture, Cancer cell, Cancer research, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Drug Screening Assays, Antitumor
Abstract

In this work, we aimed to understand the biological activity and the mechanism of action of three polymer-'ruthenium-cyclopentadienyl' conjugates (RuPMC) and a low molecular weight parental compound (Ru1) in cancer cells. Several biological assays were performed in ovarian (A2780) and breast (MCF7, MDA-MB-231) human cancer derived cell lines as well as in A2780cis, a cisplatin resistant cancer cell line. Our results show that all compounds have high activity towards cancer cells with low IC50 values in the micromolar range. We observed that all Ru-PMC compounds are mainly found inside the cells, in contrast with the parental low molecular weight compound Ru1 that was mainly found at the membrane. All compounds induced mitochondrial alterations. PMC3 and Ru1 caused F-actin cytoskeleton morphology changes and reduced the clonogenic ability of the cells. The conjugate PMC3 induced apoptosis at low concentrations comparing to cisplatin and could overcame the platinum resistance of A2780cis cancer cells. A proteomic analysis showed that these compounds induce alterations in several cellular proteins which are related to the phenotypic disorders induced by them.Our results suggest that PMC3 is foreseen as a lead candidate to future studies and acting through a different mechanism of action than cisplatin. Here we established the potential of these Ru compounds as new metallodrugs for cancer chemotherapy., This work was financed by the Portuguese Foundation for Science and Technology (Fundacao para a Ciencia e a Tecnologia, FCT) within the scope of projects UID/QUI/00100/2013 and PTDC/QUI-QIN/28662/2017. This work was supported by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). Andreia Valente acknowledges the COST action CM1302 (SIPs), the Investigator FCT2013 Initiative for the project IF/01302/2013 (acknowledging FCT, as well as POPH and FSE - European Social Fund) and the Royal Society of Chemistry's Research Fund. Pierre Falson and Elisabeta Comsa warmly acknowledge Thibault Andrieu from the cytometry plateau of SFR bioscience -UMS 3444- at Lyon-Gerland, France for assistance on CytoF. This work was also supported by the Marie Curie Initial Training Network: FP7-PEOPLE-2012-ITN proposal no 317297 - acronym GLYCOPHARM and PITN-GA-2012-317297. The high resolution mass spectrometer at CIRE-PAIB was financed (SMHART project no3069) by the European Regional Development Fund (ERDF), the Conseil Regional du Centre, the French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (Inserm).

Published
2019

45. Graphene oxide: A glimmer of hope for Assisted Reproductive Technology

Author

Valérie Labas, Barbara Barboni, Luca Valbonetti, Xavier Druart, Marie-Claire Blache, Marie Saint-Dizier, Antonella Fontana, Pascal Mermillod, Marina Ramal-Sanchez, Guillaume Tsikis, Florine Dubuisson, Nicola Bernabò, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Faculty of BioScience and Technology for Food, Agriculture and Environment, University of Teramo (UT), Università degli studi 'G. d'Annunzio' Chieti-Pescara [Chieti-Pescara] (Ud'A), and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Infertility, medicine.medical_treatment, 02 engineering and technology, Biology, 010402 general chemistry, 01 natural sciences, Andrology, Human fertilization, Animal model, Capacitation, medicine, General Materials Science, Blastocyst, Assisted reproductive technology, Male factor, urogenital system, [SDV.BA]Life Sciences [q-bio]/Animal biology, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, General Chemistry, oxyde de graphène, 021001 nanoscience & nanotechnology, medicine.disease, Sperm, 3. Good health, 0104 chemical sciences, medicine.anatomical_structure, graphene oxide, 0210 nano-technology
Abstract

International audience; Infertility is a worldwide problem affecting around 48.5 million couples in the word, the male factor being responsible for approximately the 50% of the cases, with a high percentage of unknown causes. For that reason, improving the success of In Vitro Fertilization (IVF) techniques is a primordial aim for researchers working in the reproductive field. Here, by using a mammalian animal model, the bovine, we present an innovative in vitro fertilization system that combines the use of a somatic component, the epithelial oviductal cells, and a carbon-based material, the graphene oxide, with the aim to open new ways in IVF systems design and application. Our results show an increase in the IVF outcomes without harming the blastocyst developmental rate, as well as high modified proteomic and lipidomic profiles of capacitating spermatozoa. Furthermore, we compared the modifications produced by GO with those exerted by the hormone progesterone, finding similar functional effects on sperm capacitation. In conclusion, our results stand out the use of a non-physiological material as graphene oxide in a new and innovative strategy that improves sperm capacitation, conferring them a higher fertilizing competence and thus increasing the in vitro fertilization outcomes. (C) 2019 Published by Elsevier Ltd.

Published
2019

46. Stage-dependent changes in oviductal phospholipid profiles throughout the estrous cycle in cattle

Author

Marie Saint-Dizier, Svetlana Uzbekova, Ana-Paula Teixeira-Gomes, Charles Banliat, Benoit Guyonnet, Daniel Tomas, Valérie Labas, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Union Evolution, Partenaires INRAE, Institut National de la Recherche Agronomique (INRA), Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), Université de Tours (UT), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Université de Tours, and Université de Tours

Subjects
endocrine system, media_common.quotation_subject, Estrous Cycle, Luteal phase, Andrology, 03 medical and health sciences, 0302 clinical medicine, Human fertilization, Food Animals, Capacitation, Animals, Small Animals, Ovulation, Fallopian Tubes, Phospholipids, media_common, Estrous cycle, Sphingolipids, 030219 obstetrics & reproductive medicine, Oviduct fluid, Equine, Chemistry, urogenital system, [SDV.BA]Life Sciences [q-bio]/Animal biology, Embryogenesis, 0402 animal and dairy science, MALDI-TOF mass spectrometry, 04 agricultural and veterinary sciences, Bovine, Lipids, 040201 dairy & animal science, Sperm, Gene Expression Regulation, Estrus cycle, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Oviduct, Cattle, Female, Animal Science and Zoology
Abstract

Appendix A. Supplementary data; International audience; Sperm capacitation, fertilization and embryo development take place in the oviduct during the periovulatory period of the estrous cycle. Phospholipids are crucial metabolites for sperm capacitation and early embryo development. The aim of this study was to monitor the abundance of phospholipids in the bovine oviductal fluid (OF) according to the stage of the estrous cycle and the side relative to ovulation. Pairs of bovine oviducts were collected in a slaughterhouse and classified into four stages of the estrous cycle: post-ovulatory (Post-ov), mid-luteal (Mid-lut), late-luteal (Late-lut) and pre-ovulatory (Pre-ov) phases (n = 17 cows/stage). Cell-free OF from oviducts ipsilateral and contralateral to the site of ovulation were analyzed using MALDI-TOF mass spectrometry. Lipid identification was achieved by high resolution mass spectrometry. A total of 274 lipid masses were detected in the mass range of 400-1000 Da, corresponding mostly to phosphatidylcholines (PC), lysoPC, phosphatidylethanolamine (PE), lysoPE and sphingomyelins (SM). Ipsilateral and contralateral OF did not differ in their lipid profiles at any stage of the cycle. However, 127 and 96 masses were differentially abundant between stages in ipsilateral and contralateral OF, respectively. Highest differences in lipid profiles were observed in the Pre-ov vs. Mid-lut and Pre-ov vs. Late-lut comparisons in both sides relative to ovulation. Differential abundance of specific molecules of PC, PE, SM and l-carnitine were observed at Pre-ov and Post-ov compared with the luteal phase. This work proposes new candidates potentially able to regulate sperm capacitation and early embryo development.

Published
2019

47. Digestion dynamics in broilers fed rapeseed meal

Author

Michel J. Duclos, Agnès Narcy, Sophie Réhault-Godbert, Alain Quinsac, Lucie Combes-Soia, M. Lessire, C. Peyronnet, E. Recoules, Valérie Labas, Lionel Lardic, Biologie des Oiseaux et Aviculture (BOA), Institut National de la Recherche Agronomique (INRA)-Université de Tours, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Terres Univia, Terres Inovia, Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Proteomics, Rapeseed, lcsh:Medicine, Ileum, digestion, Plant Proteins, Dietary, Article, Jejunum, Avian Proteins, 03 medical and health sciences, 0302 clinical medicine, Pepsin, Biologie animale, medicine, Animals, protéine digestible dans l'intestin, Food science, protéomique, lcsh:Science, Gizzard, Animal biology, 2. Zero hunger, Multidisciplinary, biology, [SDV.BA]Life Sciences [q-bio]/Animal biology, lcsh:R, Brassica napus, Proteolytic enzymes, Proventriculus, Animal Feed, Gastrointestinal Contents, Gastrointestinal Tract, 030104 developmental biology, medicine.anatomical_structure, colza graine, appareil digestif, biology.protein, lcsh:Q, Animal Nutritional Physiological Phenomena, Digestion, Chickens, poulet de chair, 030217 neurology & neurosurgery
Abstract

Rapeseed proteins are described to be poorly digestible in chickens. To further identify some molecular locks that may limit their use in poultry nutrition, we conducted a proteomic study on the various chicken digestive contents and proposed an integrative view of the proteins recruited in the crop, proventriculus/gizzard, duodenum, jejunum, and ileum for digestion of rapeseed by-products. Twenty-seven distinct rapeseed proteins were identified in the hydrosoluble fraction of the feed prior ingestion. The number of rapeseed proteins identified in digestive contents decreases throughout the digestion process while some are progressively solubilized in the most distal digestive segment, likely due to a combined effect of pH and activity of specific hydrolytic enzymes. Fifteen chicken proteins were identified in the hydrosoluble proventriculus/gizzard content, including chymotrypsin-like elastase and pepsin. Interestingly, on the 69 distinct proteins identified in duodenum, only 9 were proteolytic enzymes, whereas the others were associated with homeostasis, and carbohydrate, lipid, vitamin and hormone metabolisms. In contrast, chicken proteins identified in jejunal and ileal contents were mostly proteases and peptidases. The present work highlights the relevance of using integrative proteomics applied to the entire digestive tract to better appreciate the protein profile and functions of each digestive segment.

Published
2019

48. Effects of a n-3 polyunsaturated fatty acid-enriched diet on embryo production in dairy cows

Author

Olivier Desnoës, Priscila Silvana Bertevello, Svetlana Uzbekova, M. Oseikria, Valérie Labas, M Dupont, Sébastien Elis, E Briant, Virginie Maillard, L. Le Berre, O Ghazouani, Sandrine Freret, Ana-Paula Teixeira-Gomes, Alice Desmarchais, D. Le Bourhis, Pascal Salvetti, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Station de Phénotypage, Allice, Unité Expérimentale de Physiologie Animale de l‘Orfrasiére (UE PAO), Institut National de la Recherche Agronomique (INRA), Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours, Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), and Elis, Sébastien

Subjects
0301 basic medicine, Embryology, [SDV]Life Sciences [q-bio], Embryonic Development, Fertilization in Vitro, Biology, 03 medical and health sciences, Endocrinology, Animal science, Ovulation Induction, Fatty Acids, Omega-3, medicine, Animals, [INFO]Computer Science [cs], Blastocyst, Fertilisation, 2. Zero hunger, chemistry.chemical_classification, Transvaginal oocyte retrieval, 0402 animal and dairy science, Obstetrics and Gynecology, food and beverages, 04 agricultural and veterinary sciences, Cell Biology, Embryo, Mammalian, Fish oil, Oocyte, 040201 dairy & animal science, Follicular fluid, Diet, In vitro maturation, 030104 developmental biology, medicine.anatomical_structure, Reproductive Medicine, chemistry, Dietary Supplements, Oocytes, Cattle, Female, Polyunsaturated fatty acid
Abstract

Beneficial effects of n-3 polyunsaturated fatty acid (PUFA) supplementation on dairy cow reproduction have been previously reported. The objectives of the present study were to assess whether n-3 PUFA supplementation would affect in vitro embryo production (IVP) after ovarian stimulation. Holstein cows received a diet with 1% dry matter supplementation of either n-3 PUFA (n = 18, microencapsulated fish oil) or a control, n-6 PUFA (n = 19, microencapsulated soy oil). Both plasma and follicular fluid FA composition showed integration of total PUFA through the diet. All cows underwent an IVP protocol consisting of ovarian stimulation, ultrasound-guided transvaginal oocyte retrieval (ovum pick-up, OPU, five per cow) followed by in vitro maturation, fertilisation and 7 days of embryo development. A tendency toward an increase in the blastocyst rate (diet effect, P = 0.0865) was observed in n-3 cows, with 49.6 ± 5.5% vs 42.3 ± 5.5% in control n-6 cows. A significant increase (diet effect, P = 0.0217) in the good-quality blastocyst rate (freezable blastocysts) was reported in n-3 cows (42.2 ± 7.7%) compared to control n-6 cows (32.7 ± 7.7%). A significant difference in lipid composition was shown in the oocytes recovered by OPU from n-3 and n-6 treated cows, by intact single-oocyte MALDI-TOF mass spectrometry. The 42 differentially abundant identified lipids were mainly involved in cell membrane structure. In conclusion, n-3 PUFA supplementation enhanced oocyte quality and modified their lipid composition. Further studies are necessary to investigate the potential link of these lipid modifications with enhanced oocyte quality.

Published
2019

49. Proteomic analysis of uterine fluid of fertile and subfertile hens before and after insemination

Author

Audrey Gargaros, Grégoire Harichaux, Luiz Cordeiro, Aurélien Brionne, Valérie Labas, Cindy Riou, Nadine Gérard, Joël Gautron, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Biologie des Oiseaux et Aviculture (BOA), Institut National de la Recherche Agronomique (INRA)-Université de Tours, Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT)

Subjects
Male, Embryology, endocrine system, Proteome, [SDV]Life Sciences [q-bio], Uterus, Oviducts, Biology, Metallopeptidase activity, Insemination, Andrology, 03 medical and health sciences, 0302 clinical medicine, Endocrinology, medicine, Animals, [INFO]Computer Science [cs], Eggshell, HSPA8, Insemination, Artificial, 030304 developmental biology, fertility, 0303 health sciences, insemination, [SDV.GEN]Life Sciences [q-bio]/Genetics, urogenital system, Mucin, Obstetrics and Gynecology, Cell Biology, Spermatozoa, Sperm, Epithelium, Avian uterine, genital, [SDV.GEN.GA]Life Sciences [q-bio]/Genetics/Animal genetics, medicine.anatomical_structure, Gene Expression Regulation, Reproductive Medicine, Infertility, 030220 oncology & carcinogenesis, Female, Chickens
Abstract

International audience; Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen’s genital tract, and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content, and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F- hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity, and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.

Published
2019

50. Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos

Author

Emilie Corbin, Marie Saint-Dizier, Daniel Le Bourhis, Pascal Salvetti, Pascal Mermillod, Daniel Tomas, Florine Dubuisson, Julie Beurois, Valérie Labas, Charles Banliat, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Allice, French National Institute for Agricultural Research (INRA), APIS‐GENE, Grant/Award Number: Embryomimetisme, and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Phospholipid, Embryonic Development, embryo, Fertilization in Vitro, Oviducts, Biology, cryopreservation, Cryopreservation, Andrology, Embryo Culture Techniques, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Human fertilization, Organ Culture Techniques, Genetics, medicine, Animals, Blastocyst, Gonadal Steroid Hormones, 030219 obstetrics & reproductive medicine, Zygote, [SDV.BA]Life Sciences [q-bio]/Animal biology, Embryo, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Cell Biology, membrane phospholipids, In vitro, In Vitro Oocyte Maturation Techniques, 030104 developmental biology, medicine.anatomical_structure, chemistry, ICM-MS, embryonic structures, lipidomics, Cattle, Female, Developmental Biology, Hormone
Abstract

International audience; The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification-warming, exposure to P4 improved the proportion of embryos that re-expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI-TOF mass spectrometry, a total of 242 phospholipid masses of 400-1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

Published
2019

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