Novel Insights into the Bovine Polled Phenotype and Horn Ontogenesis in Bovidae

Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae

Contribution à l étude fonctionnelle des effets du PACAP sur l ontogénèse du cervelet chez la souris in vivo

Le cervelet adulte, constitue, chez les mammifères, la région la plus volumineuse de l encéphale après les hémisphères cérébraux et représente avant tout un centre moteur qui contribue au contrôle des contractions des muscles squelettiques nécessaires à la coordination motrice, au maintien de la posture et de l équilibre. Toutefois, il a été démontré que cette structure intervient également dans l'apprentissage et la mémorisation. Le cervelet est constitué de deux lobes flocculo-nodulaires et de deux hémisphères encadrant la région médiane du vermis. L'ensemble est recouvert par une structure laminaire, appelée cortex cérébelleux, qui est constituée de quatre couches distinctes, à savoir la couche granulaire externe (CGE), la couche moléculaire (CMol), la couche des cellules de Purkinje (CP) et la couche granulaire interne (CGI). Chez la souris, pour laquelle l apprentissage de la marche s effectue graduellement au cours de la période postnatale, le cervelet est immature à la naissance et son ontogenèse se poursuit pendant les premières semaines de la vie. Au cours de cette phase développementale, le cortex cérébelleux est sous l influence de divers facteurs. Parmi ceux-ci, le pituitary adenylate cyclase-activating polypeptide (PACAP) s'est révélé être un neuropeptide neurotrophique important. En effet, une forte expression de PACAP et de son récepteur PAC-1 a été observée dans les différentes couches cérébelleuses au cours des périodes pré- et postnatales. De plus, de nombreuses études réalisées in vivo et in vitro ont démontré que le PACAP exerce des effets neurotrophiques sur les neurones en grain, favorisant notamment la différenciation, l'élongation neuritique et la survie des cellules en grain. Enfin, il est maintenant bien établi que le PACAP exerce un puissant effet anti-apoptotique sur les cellules granulaires du cervelet en culture en inhibant la voie mitochondriale et la caspase-3. L objectif de cette thèse a tout d abord été de vérifier le rôle physiologique du PACAP in vivo dans le cervelet des mammifères en caractérisant le phénotype du cervelet de souris invalidées pour le gène PACAP (PACAP-/-). Cette étude a été réalisée sur des animaux âgés de 4 et 7 jours postnataux (P4 et P7), deux stades correspondant à des étapes clés de la genèse des cellules en grain. L'organisation générale des structures cérébelleuses n est pas sensiblement affectée par l'absence de PACAP. Toutefois, une analyse morphométrique fine a mis en évidence une réduction significative de l épaisseur des couches granulaires externe à P4 et interne à P7 chez les souris invalidées, suggérant une perte neuronale dans le cortex cérébelleux. L étude de la densité cellulaire a révélé que les variations d épaisseur observées sont effectivement dues à une réduction du nombre de cellules. Par ailleurs, nous avons pu démontrer par western blot et PCR quantitative que, chez les souris PACAP-/-, l expression de la nestine, un marqueur de précurseurs neuraux, n est pas modifiée alors que celle de la synaptophysine, un marqueur de neurones différenciés, est réduite à P4 et P7. Enfin, nous avons montré que le nombre de cellules exprimant la caspase-3 clivée, enzyme effectrice de l apoptose, ainsi que l activité biologique de cette enzyme sont significativement plus importants chez les souris PACAP-/- à P4 et P7. L'ensemble de ces résultats suggère que le PACAP exerce un rôle neurotrophique in vivo, d'une part, sur la différenciation des neurones en grain en modifiant l expression de protéines associées à la maturité neuronale et, d'autre part, sur la survie cellulaire en inhibant l'activité de la caspase-3 pro-apoptotique. Il est maintenant bien établi que l'ajustement de la population neuronale au cours du développement cérébral dépend d'un équilibre entre les facteurs neurotrophiques et les facteurs de mort cellulaire. Nous avons donc émis l'hypothèse que l'absence de PACAP chez les souris PACAP-/- provoquerait un déséquilibre en faveur de l'apoptose entraînant une perte des cellules en grain matures de la CGI. Le second volet de cette thèse a donc consisté à étudier les effets du facteur Fas ligand (FasL) sur le développement du cervelet de souris sauvages et PACAP-/- afin de déterminer si le neuropeptide est en mesure de bloquer la mort cellulaire induite par FasL. En effet, il a été démontré que FasL intervient dans l induction de la mort cellulaire programmée lors de la mise en place de diverses populations cellulaires et que son effet pro-apoptotique implique l activation de la caspase-3. Avant toute étude physiologique, nous avons vérifié par immunohistochimie la présence de Fas, le récepteur du FasL, dans le cervelet, démontrant ainsi pour la première fois sa localisation sur les cellules en grain et les cellules de Purkinje de souris. Nous avons ensuite réalisé des expériences ex vivo sur des tranches organotypiques de cervelet de souriceaux âgés de 7 jours qui ont révélé que le FasL (5 ng/ml) induit une augmentation significative de l activité de la caspase-3 et que cet effet est inhibé par le PACAP (10-7 M). Ces données montrent que le PACAP est capable de s'opposer aux actions pro-apoptotiques du FasL via la caspase-3. Afin de transposer ce travail dans des conditions in vivo, nous avons effectué des injections sous-durales de FasL (0.1 g/ l) en l absence ou en présence de PACAP (0.3 g/ l) au niveau du cortex cérébelleux chez des souris sauvages et PACAP-/-. Les résultats indiquent que les animaux traités avec le FasL seul présentent une diminution de l épaisseur de la CGI à P8. Il semble donc que, malgré la présence du récepteur Fas sur les neurones en grain et les cellules de Purkinje, le FasL agisse spécifiquement sur les cellules granulaires. Cette hypothèse est étayée par une étude immunohistochimique qui a révélé que l'administration de FasL dans le cervelet augmente le nombre de cellules exprimant la caspase-3 clivée dans la CGI chez les animaux sauvages et PACAP-/-. Par ailleurs, nos résultats démontrent que la co-injection de PACAP prévient la réduction de la CGI, partiellement chez les souris sauvages et totalement chez les animaux PACAP-/-. Les tests comportementaux réalisés sur des souris sauvages traitées par le FasL et le PACAP n'ont pas révélé de déficits comportementaux significatifs si ce n est un défaut d équilibre transitoire à P8 chez les souris traitées simultanément par le FasL et le PACAP. Par ailleurs, aucune altération morphologique du cervelet et aucun trouble moteur n ont été observés entre les différents groupes chez l adulte, suggérant la mise en place de mécanismes de compensation au cours du temps. En résumé, cette étude réalisée sur un modèle de souris invalidées démontre pour la première fois que le PACAP exerce in vivo des effets neurotrophiques et anti-apoptotiques. Ainsi, nous avons observé que le PACAP stimule l expression de protéines associées à la maturité neuronale telle que la synaptophysine et inhibe l'activité de la caspase-3 dans les cellules en grain matures de la CGI de souris. De plus, le PACAP est capable de s'opposer aux effets pro-apoptotiques de FasL, indiquant que sa présence au cours de l'ontogenèse du cervelet participe à l'équilibre entre les facteurs neurotrophiques et les facteurs de mort cellulaire. Enfin, l'administration de FasL chez des souriceaux n'affecte pas le comportement moteur à l'âge adulte, suggérant l'implication d'autres facteurs dans la mise en place d'un mécanisme de compensation au cours de la croissance.The adult cerebellum represents, in mammals, the most voluminous brain region after the cerebral hemispheres and constitutes mainly a motor centre, implicated in the control of skeletal muscle contractions, which is required for motor coordination, posture and equilibrium. In addition, it has been shown that the cerebellum plays a role in learning and memory. The cerebellum is composed of two flocculo-nodular lobes and two hemispheres flanking the median region called the vermis. The entire structure is covered by a laminar coating named cerebellar cortex which is organized in four different layers, namely the external granule cell layer (EGL), molecular cell layer (Mol), Purkinje cell layer (PL) and internal granule cell layer (IGL). In mouse, in which walking ability appears gradually during the postnatal period, the cerebellum is immature at birth and its ontogenesis carries on during the first weeks of life. During this developmental phase, the cerebellar cortex is under the influence of various factors. Among those, pituitary adenylate cyclase-activating polypeptide (PACAP) has been identified as an important trophic neuropeptide. High concentrations of PACAP and its receptor PAC-1 have been actually detected in different layers of the cerebellar cortex during the pre- and postnatal periods. Moreover, several in vivo and in vitro studies have shown that PACAP exerts neurotrophic effects on granule neurons, notably in promoting differentiation, neurite outgrowth and survival. Finally, it is now well established that PACAP exerts a potent anti-apoptotic effect on cultured cerebellar granule cells through inhibition of the mitochondrial pathway and caspase 3. The aim of this Ph D thesis was first to confirm the physiological role of PACAP in vivo in the cerebellum of mammals by characterizing the neuroanatomical and neurochemical alterations of the cerebellum in PACAP knockout (PACAP-/-) mice. This study was carried out on 4- and 7-day-old animals (P4 and P7), two stages corresponding to key periods of granule cell genesis. The global organization of the cerebellar cortex was not affected by the absence of PACAP. However, detailed morphometric analysis revealed a significant reduction of the thickness of the EGL and IGL, respectively, at P4 and P7 in knockout animals, suggesting neuronal loss in the cerebellar cortex. The study of cellular density indicated that the alteration of the thickness of the cortical layers could be ascribed to a decrease in cell number. In addition, western blot and qPCR experiments revealed that the expression of nestin, a neural marker, was not modified in PACAP-/- mice whereas the expression of synaptophysin, a marker of differentiated neurons, was reduced at P4 and P7 in knockout animals. Finally, we have shown that the number of cells expressing cleaved caspase-3, the effector enzyme of apoptosis, as well as the biologic activity of this enzyme are significantly elevated in PACAP-/- mice at P4 and P7. Taken together, these data suggest that PACAP exerts a neurotrophic role in vivo, both on neuronal differentiation by modifying the expression of proteins associated with neuronal maturity and on cell survival by inhibiting the activity of the pro-apoptotic enzyme caspase-3. It is now clearly established that the regulation of neuronal population during brain development depends on a balance between neurotrophic and cell death factors. We thus hypothesized that the lack of PACAP in knockout animals could alter the equilibrium to favour apoptosis leading to a loss of granular cells in the IGL. So, the second part of this work consisted in studying the effect of Fas ligand (FasL) on cerebellar development in wild-type and PACAP-/- mice in order to determine if the neuropeptide PACAP is able to block cell death induced by FasL. Indeed, it has been previously shown that FasL interferes in the induction of cell death during the adjustment of various cellular populations and that this pro-apoptotic effect involves the activation of caspase-3. Here, we have used an immunohistochemical approach to investigate the presence of FasL receptor, called Fas, in the cerebellum and we observed, for the first time, the occurrence of Fas on granule and Purkinje cells in mice. Then, we performed ex vivo experiments on organotypic slices of the cerebellum from 7-day-old mice which revealed that FasL (5 ng/ml) induces a significant increase of caspase-3 activity and that this effect is inhibited by PACAP (10-7 M). These data show that PACAP is able to reverse the pro-apoptotic actions of FasL via caspase-3. In order to transpose our work to in vivo conditions, we administered FasL (0.1 g/ l) in the absence or presence of PACAP (0.3 g/ l) in the subarachnoid space of the cerebellar cortex in wild-type and PACAP-/- mice. The data indicate that animals treated with FasL alone display a decrease of IGL thickness at P8, suggesting that, in spite of the presence of Fas on granule and Purkinje cells, FasL specifically affects granule neurons. This hypothesis is supported by an immunohistochemical study showing that FasL administration at the cerebellar surface increases the number of cells expressing caspase-3 in the IGL in wild-type and PACAP-/- mice. In addition, we found that co-injection of PACAP prevents FasL-induced IGL atrophy partially in wild-type mice and totally in PACAP-/- animals. Behavioral tests carried out on wild-type mice treated with FasL and/or PACAP did not reveal any locomotor alteration except a transient impairment of equilibrium at P8 in animals treated simultaneously with FasL and PACAP. Moreover, no locomotor defect or morphological alteration of the cerebellum was observed between the different groups in adult mice, suggesting the occurrence of a compensation mechanism in adulthood. To summarize, this Ph D thesis conducted on a model of knockout mice demonstrates for the first time that PACAP exerts neurotrophic and anti-apoptotic effects in vivo. Thus, we observed that PACAP stimulates the expression of neuronal differentiation markers such as synaptophysin and inhibits the activity of caspase-3 in mature granule cells in the mouse IGL. Moreover, PACAP is able to block the pro-apoptotic effect of FasL, indicating that its presence participates in the equilibrium between neurotrophic and cell death factors during ontogenesis of the cerebellum. Finally, FasL administration in young mice did not affect motor behaviour at adult stage, suggesting the implication of other factors in the setting of a compensatory mechanisms.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

ETUDE DES REMANIEMENTS ÉPIGÉNÉTIQUES AU MOMENT DE L'EGA CHEZ LE BOVIN À L'AIDE D'APPROCHES CORRÉLATIVES EN MICROSCOPIE OPTIQUE

International audienceLors du développement embryonnaire précoce, les génomes parentaux provenant respectivement du spermatozoïde et de l'ovocyte, sont fortement remaniés. Ces remaniements permettent une reprogrammation épigénétique de la chromatine et sont concomitants avec la mise en route de la transcription de l'embryon. Le projet vise à étudier l'impact de l'utilisation de semences de taureau ayant des profils de méthylation différents et un profil de sncRNA contrastés, sur cette reprogrammation épigénétique autour de l'activation majeure du génome embryonnaire (EGA) i.e. au stade 8-cellules. Pour cela nous avons privilégié la détection de modification post traductionnelle d'histone (H3K4me3 et H3K9me3) par immunofluorescence sur embryon in toto des stades 2-cellules à Morula. Nous avons à la fois utilisé un microscope confocal pour déterminer la localisation en 3D dans le noyau du signal de fluorescence et sur les mêmes embryons, un microscope photonique équipé d'un module Apotome pour estimer la quantité de fluorescence dans les noyaux. Ces deux approches complémentaires nous ont permis d'acquérir des données quantitatives et qualitatives pour la comparaison de la distribution de ces marques d'épigénétiques entre des taureaux ayant des profils de méthylation et de sncRNA spermatique contrastés

Integration of multi-tissues data. An example from bovine embryos.

International audienceThe increasing availability of large multi-tissue data sets which contain gene expression measurements across different tissues and individuals provided unprecedented opportunities to investigate transcriptome variation across tissues and individuals, and may reveal interactions between genes and tissues. The corresponding data set is a three-dimensional array: genes, individuals and tissues (or recording times). We present here the so-called “Partial Triadic Analysis”,(PTA), a well suited statistical tool to get a clear representation of a spatial series of matrices, one for each tissue. PTA is an extension of PCA and allows one to find a structure common to every matrix and to study its stability across tissues. PTA consists in three steps: i) the interstructure step, where are compared and analyzed the relationships between the different datasets, ii) the compromise step, where all datasets are integrated into an optimum weighted average, the compromise (or consensus) table, and iii) the intrastructure step, where the single-transcriptome are compared to the compromise in order to analyze commonalities and discrepancies. PTA was applied to transcriptomic data from the ANR (Agence Nationale de la Recherche) funded BoSexDim project, consisting in 19 embryo transcriptomes recorded at D40 (40 days after fertilization) and structured by sex (Male / Female) and type (in vivo / in vitro). These transcriptomes were recorded for four tissues (brain, liver, gonad and placenta). PTA shows a compromise structured by sex (first axis) and type (second axis). The same set of genes contribute the most to the sex structuration whatever the tissue. However, the differentiation of in vivo vs in vitro embryos was not made by the same genes according to tissues. Some genes showed an inconsistent, even contradictory behaviour, with an overexpression in one tissue and an underexpression in another one. This example highlights the power of the Partial Triadic Analysis for depicting the variability of the transcriptome structure across various tissues. Acknowledgement: This research was funded by the ANR French organization (BoSexDim project). We are grateful to the Bosexdim consortium members for producing the biological material

Uterine defects and estradiol-dependent development of oviductal diverticula in mice lacking the SMAD4 C-terminal Mad homology 2 domain

International audienceIn female mammals, the oviduct and uterus are essential sites for female and male gamete transport, fertilization, implantation, and maintenance of a successful pregnancy. To delineate the reproductive function of Mothers against decapentaplegic homolog 4 (Smad4), we specifically inactivated Smad4 in ovarian granulosa cells and, oviduct and uterine mesenchymal cells using the Amhr2-cre mouse line. Deletion of exon 8 of Smad4 results in the production of an MH2-truncated SMAD4 protein. These mutant mice are infertile due to the development of oviductal diverticula and defects during the implantation process. The ovaries are fully functional as demonstrated in an ovary transfer experiment. The development of oviductal diverticula occurs shortly after puberty and is dependent on estradiol. The diverticula interfere with sperm migration and embryo transit to the uterus, reducing the number of implantation sites. Analysis of the uterus shows that, even if implantation occurs, decidualization and vascularization are defective resulting in embryo resorption as early as the seventh day of pregnancy. Thus, Smad4 plays an important function in female reproduction by controlling the structural and functional integrity of the oviduct and uterus

Différenciation et reprogrammation épigénétique des cellules germinales mâles dans le testicule fœtal bovin

National audienc

Integration of multi-tissues data. An example from bovine embryos

International audienceThe increasing availability of large multi-tissue data sets which contain gene expression measurements across different tissues and individuals provided unprecedented opportunities to investigate transcriptome variation across tissues and individuals, and may reveal interactions between genes and tissues. The corresponding data set is a three-dimensional array: genes, individuals and tissues (or recording times). We present here the so-called “Partial Triadic Analysis”,(PTA), a well suited statistical tool to get a clear representation of a spatial series of matrices, one for each tissue. PTA is an extension of PCA and allows one to find a structure common to every matrix and to study its stability across tissues. PTA consists in three steps: i) the interstructure step, where are compared and analyzed the relationships between the different datasets, ii) the compromise step, where all datasets are integrated into an optimum weighted average, the compromise (or consensus) table, and iii) the intrastructure step, where the single-transcriptome are compared to the compromise in order to analyze commonalities and discrepancies.PTA was applied to transcriptomic data from the ANR (Agence Nationale de la Recherche) funded BoSexDim project, consisting in 19 embryo transcriptomes recorded at D40 (40 days after fertilization) and structured by sex (Male / Female) and type (in vivo / in vitro). These transcriptomes were recorded for four tissues (brain, liver, gonad and placenta). PTA shows a compromise structured by sex (first axis) and type (second axis). The same set of genes contribute the most to the sex structuration whatever the tissue. However, the differentiation of in vivo vs in vitro embryos was not made by the same genes according to tissues. Some genes showed an inconsistent, even contradictory behaviour, with an overexpression in one tissue and an underexpression in another one. This example highlights the power of the Partial Triadic Analysis for depicting the variability of the transcriptome structure across various tissues. Acknowledgement: This research was funded by the ANR French organization (BoSexDim project). We are grateful to the Bosexdim consortium members for producing the biological material

DNA methylation dynamics during spermatogenesis in ruminants

International audienceDNA methylation of cytosines is a critical epigenetic modification in mammals that plays crucial roles in transcriptional regulation, chromatin remodelling and genomic imprinting. Dynamic erasure and reestablishment of DNA methylation marks are required for spermatogenesis and the normal function of mature sperm. DNA methylation is catalysed by DNA methyltransferase enzymes (DNMT) providing either maintenance (DNMT1) or de novo (DNMT3A/B/L) DNA methylation processes. The DNA methylation dynamics during spermatogenesis has been previously described in mice and humans but nothing is known in productive livestock. However, these investigations in ruminants could be helpful to determine epigenetic events likely to be crucial for male fertility. Interestingly, a recent study from our laboratory pointed DNA undermethylation of bull spermatozoa compared to other mammals such as humans, mice, sheep or goats [1]. This result raises the question of the dynamic of DNA methylation in bovine male germline. We thus propose to define and compare the methylation dynamics of bovine and caprine germ cells during spermatogenesis. Analyses of 5mC immunohistochemistry were performed on adult testis sections to illustrate the presence of DNA methylation marks in various spermatogenic cells. As purification of the different spermatogenic populations is required to characterize DNA methylation more precisely, we firstly developed a flow cytometry-based method (Hoechtst-FACS) for cell sorting from goat testis. Fraction enrichments were evaluated using stage-specific markers by real time qPCR analyses. In addition, de novo DNA methyltransferase expression was studied on the different fractions of purified spermatogenic cells, giving some clues on DNA methylation dynamics. In the future, spermatogenic cells will be purified from both caprine and bovine testes, and together with gene expression analyses (RNA-sequencing), DNA methylation will be determined for each cell types by Reduced-Representation Bisulfite Sequencing (RRBS

Dynamics of cattle sperm sncRNAs during maturation, from testis to ejaculated sperm

International audienceBackground: During epididymal transit, spermatozoa go through several functional maturation steps, resulting from interactions with epididymal secretomes specific to each region. In particular, the sperm membrane is under constant remodeling, with sequential attachment and shedding of various molecules provided by the epididymal lumen fluid and epididymosomes, which also deliver sncRNA cargo to sperm. As a result, the payload of sperm sncRNAs changes during the transit from the epididymis caput to the cauda. This work was designed to study the dynamics of cattle sperm sncRNAs from spermatogenesis to final maturation. Results: Comprehensive catalogues of sperm sncRNAs were obtained from testicular parenchyma, epididymal caput, corpus and cauda, as well as ejaculated semen from three Holstein bulls. The primary cattle sncRNA sperm content is markedly remodeled as sperm mature along the epididymis. Expression of piRNAs, which are abundant in testis parenchyma, decreases dramatically at epididymis. Conversely, sperm progressively acquires miRNAs, rsRNAs, and tsR-NAs along epididymis, with regional specificities. For instance, miRNAs and tsRNAs are enriched in epididymis cauda and ejaculated sperm, while rsRNA expression peaks at epididymis corpus. In addition, epididymis corpus contains mainly 20 nt long piRNAs, instead of 30 nt in all other locations. Beyond the bulk differences in abundance of sncRNAs classes, K-means clustering was performed to study their spatiotemporal expression profile, highlighting differences in specific sncRNAs and providing insights into their putative biological role at each maturation stage. For instance, Gene Ontology analyses using miRNA targets highlighted enriched processes such as cell cycle regulation, response to stress and ubiquitination processes in testicular parenchyma, protein metabolism in epididymal sperm, and embryonic morphogenesis in ejaculated sperm. Conclusions: Our findings confirm that the sperm sncRNAome does not simply reflect a legacy of spermatogenesis. Instead, sperm sncRNA expression shows a remarkable level of plasticity resulting probably from the combination of multiple factors such as loss of the cytoplasmic droplet, interaction with epididymosomes, and more surprisingly, the putative in situ production and/or modification of sncRNAs by sperm. Given the suggested role of sncRNA in epigenetic trans-generational inheritance, our detailed spatiotemporal analysis may pave the way for a study of sperm sncRNAs role in embryo development