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Striatal-enriched protein tyrosine phosphatase (STEP) is a signal transduction protein involved in the pathogenesis of neuropathologies. A STEP inhibitor (TC-2153) has antipsychotic and antidepressant effects. Here, we evaluated the role of STEP in fear-induced aggression using Norway rats selectively bred for 90 generations for either high aggression toward humans (aggressive rats) or its absence (tame rats). We studied the effects of acute administration of TC-2153 on behavior and STEP expression in the brain of these animals and the influence of chronic treatment with TC-2153 on the behavior and STEP expression in aggressive rats in comparison with classic antidepressant fluoxetine, which is known to exert antiaggressive action. Acute TC-2153 administration decreased the aggressive reaction to humans in aggressive rats, while having no impact on the friendly behavior of tame rats. Moreover, in the elevated plus-maze test, the drug had an anxiolytic effect on both aggressive and tame rats. Aggressive rats demonstrated elevated levels of a STEP isoform (STEP46) as compared to tame animals, whereas acute TC-2153 administration significantly reduced STEP46 protein concentration in the brain of aggressive rats. Chronic treatment of aggressive rats with either TC-2153 or fluoxetine attenuated fear-induced aggression. Chronic administration of fluoxetine enhanced the exploratory activity in the elevated plus-maze test and decreased the STEP46 protein level in aggressive rats' hippocampus, whereas chronic TC-2153 administration did not affect these parameters. Thus, STEP46 can play an important role in the mechanisms of aggression and may mediate antiaggressive effects of TC-2153 and fluoxetine.

International audience; Many experimental data in several species clearly demonstrate the important genetic contribution to variations in HPA axis activity. The influence of corticosteroid hormones on adaptive processes and on production traits such as growth rate, feed efficiency, carcass composition and meat quality is a strong impetus to the search for the molecular bases of these differences for efficient genetic selection. Three main sources of genetic variability have been documented so far in farm animal species, the adrenal cortex sensitivity to ACTH regulating corticosteroid hormone production, the bioavailability of corticosteroid hormones and especially corticosteroid-binding globulin capacity, and glucocorticoid receptor function. The effect of single mutations may be dependent on the genetic background and genetic variation of cortisol levels may have different functional consequences depending on the molecular mechanisms responsible for this change. The next step will be to explore HPA axis variability from a system genetics perspective including the multiple sources of variation and their interactions. This multifactorial approach is a prerequisite to the use of the HPA axis phenotypes in the genetic selection for more productive and robust animals, with a high level of production of quality products.

Pig husbandry is known as an intensive breeding system, piglets being submitted to multiple stressful events such as early weaning, successive mixing, crowding and shipping. These stressors are thought to impair immune defences and might contribute, at least partly, to the prophylactic use of antibiotics. Robustness was recently defined as the ability of an individual to express a high-production potential in a wide variety of environmental conditions. Increasing robustness thus appears as a valuable option to improve resilience to stressors and could be obtained by selecting piglets upon their adrenocortical activity. In this study, we aimed at depicting the consequences of an acute social stress on the immune capacity of piglets genetically selected upon divergent hypothalamic-pituitary-adrenocortical (HPA) axis activity. For this purpose, we monitored neuroendocrine and immune parameters, in high- (HPA(hi)) and low- (HPAI(lo)) responders to ACTH, just before and immediately after a one-hour mixing with unfamiliar conspecifics. As expected, stressed piglets displayed higher levels of circulating cortisol and norepinephrine. Blood cell count analysis combined to flow cytometry revealed a stress-induced leukocyte mobilization in the bloodstream with a specific recruitment of CD8 alpha(+) lymphocytes. Besides, one-hour mixing decreased LPS-induced IL-8 and TNF alpha secretions in whole-blood assays (WBA) and reduced mononuclear cell phagocytosis. Altogether, our data demonstrate that acute social stress alters immune competence of piglets from both groups, and bring new insights in favour of good farming practices. While for most parameters high- and low-responders to ACTH behaved similarly, HPA(hi) piglets displayed higher number of CD4(+) CD8 alpha(-) T cells, as well as increased cytokine production in WBA (LPS-induced TNFa and PIL-induced IL-8), which could confer them increased resistance to pathogens. Finally, a principal component analysis including all parameters highlighted that overall stress effects were less pronounced on piglets with a strong HPA axis. Thus, selection upon adrenocortical axis activity seems to reduce the magnitude of response to stress and appears as a good tool to increase piglet robustness.

International audience; Selection for feed efficiency is questioned as reducing the ability of farm animals to face stress and overcome challenges. The hypothalamic-pituitary-adrenocortical (HPA) axis activity, measured by the cortisol level in plasma after ACTH injection, has been proposed as an indicator of robustness. The objectives of this study were to evaluate (1) if a modified cortisol level in pigs alters their feed efficiency and their production performance; and (2) if alternative dietary resources would affect these responses. Parallel trials including divergent lines for plasma cortisol 1 hour after ACTH injection and divergent lines for residual feed intake (RFI) were run during growth with a conventional diet and a diet with high fibres, low energy and low protein content. Selection for higher or lower plasma cortisol levels after stimulation of the adrenal gland did not impact growth and feed intake traits, but it had a significant impact on body composition and carcass yield, with improved composition in the high cortisol animals. The two lines had similar responses to an alternative diet, with decreased growth rate and feed intake, and increased feed conversion ratio. On the other hand, lines selected for divergent RFI had different responses to the alternative diet, the more efficient line having a more reduced growth rate with the diet with lower energy and AA contents. However, in terms of FCR it remained more efficient. The initial hypothesis of decreased efficiency associated to increased cortisol was not validated. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant no. 633531.

National audience; An unfavourable relationship between responses to stress and feed efficiency has been proposed. Parallel trials were run, including divergent lines for residual feed intake and divergent lines for plasma cortisol response 1 h after ACTH injection. At 6 weeks of age, 48 pigs from each of the four lines were injected with ACTH. Plasma cortisol was measured before injection, and 1 h and 4 h later. Growth rate, feed intake, feed efficiency, body composition and meat quality were measured during the growing-finishing period on the same pigs fed either a conventional diet (9.7 MJ net energy (NE) and 160 g crude protein/kg) or a diet with an increased proportion of dietary fibres to dilute NE and digestible amino acids (AA) by 10%. Comparison of the lines selected for feed efficiency showed that the response to ACTH was slightly stronger, and lasted longer, in the more efficient line (P < 0.05). During the feed challenge, the growth rate of the most efficient line decreased more with the diet with lower NE and AA contents than that of the less efficient line (P < 0.06). However, in terms of feed efficiency, the more efficient line remained more efficient. On the other hand, comparison of the 'response to ACTH' lines showed no difference for growth and feed intake during the growth period, but a higher carcass yield and a leaner body composition of animals with higher plasma cortisol after ACTH injection. Thus, the initial hypothesis of a negative association between feed efficiency and the HPA axis activity is not supported.; Une relation défavorable entre réponse au stress et efficacité alimentaire est souvent mise en avant. Deux lignées divergentes pour l’efficacité alimentaire, et deux lignées divergentes pour la réponse en cortisol plasmatique à une injection d’ACTH, indicateur de l’activité de l’axe corticotrope, ont été testées en parallèle. A 6 semaines d’âge, 48 porcs par lignée ont reçu une injection d’ACTH. Le cortisol plasmatique a été mesuré avant injection, 1 h et 4 h après injection. La vitesse de croissance, l’ingestion, l’efficacité alimentaire, la composition de carcasse et la qualité de la viande ont été enregistrées. La moitié des animaux a reçu un aliment croissance conventionnel (9,7 MJ d’énergie nette (EN) et 160 g de protéines brutes/kg), et l’autre moitié a été soumise à un challenge alimentaire avec un aliment contenant des fibres alimentaires pour diluer l’EN et les acides aminés digestibles dans une proportion de 10%. La réponse à l’ACTH a été légèrement plus forte, et prolongée dans le temps, dans la lignée la plus efficace (P < 0,05). La vitesse de croissance et l’ingéré de la lignée plus efficace soumise à un régime fibreux ont été plus diminués que ceux des porcs moins efficaces (P < 0,06), mais l’avantage d’efficacité alimentaire a été maintenu. D’autre part, la comparaison des lignées « réponse à l’ACTH » ne montre pas de différence pour les caractères de croissance et d’ingestion pendant la période de croissance, quel que soit l’aliment, mais montre une composition corporelle plus maigre des porcs ayant une concentration de cortisol plasmatique plus élevée. Ainsi, contrairement à l’hypothèse initiale, la sélection pour une meilleure efficacité alimentaire n’affecte pas négativement la réponse à l’ACTH, et la diminution de l’activité de l’axe corticotrope n’est pas favorablement corrélée aux performances.

International audience; Tachykinin 2 (Tac2) is expressed in a number of areas throughout the brain, including the hippocampus. However, knowledge about its function has been only well explored in the hypothalamus in the context of reproductive health. In this study, we identified and validated increased hippocampal Tac2 mRNA expression in response to chronic mild stress in mice. Expression quantitative trait locus (eQTL) analysis showed Tac2 is cis-regulated in the hippocampus. Using a systems genetics approach, we constructed a Tac2 co-expression network to better understand the relationship between Tac2 and the hippocampal stress response. Our network identified 69 total genes associated with Tac2, several of which encode major neuropeptides involved in hippocampal stress signaling as well as critical genes for producing neural plasticity, indicating that Tac2 is involved in these processes. Pathway analysis for the member of Tac2 gene network revealed a strong connection between Tac2 and neuroactive ligand-receptor interaction, calcium signaling pathway, as well as cardiac muscle contraction. In addition, we also identified 46 stress-related phenotypes, specifically fear conditioning response, that were significantly correlated with Tac2 expression. Our results provide evidence for Tac2 as a strong candidate gene who likely plays a role in hippocampal stress processing and neural plasticity.

Notice à Reprendre pas de clé UT au 14 janvier 2018; Lethal yellow (AY) mutation causes obesity and type-2 diabetes in mice. Here we studied the effect of the AYmutation on the brain and behavior. The experiments were carried out on adult (11–12 weeks old) males of AY/amice and their wild-type littermates (a/a). Mice of AY/a and a/a genotypes did not differ in their home cageactivity, sleep, food and water consumption, learning ability in the Morris water maze, anxiety in the open fieldand elevated plus-maze, as well as in the level of monoamines, metabolites and some genes expression in thebrain. At the same time, the fat mass, depressive-like immobility in the forced swim and tail suspension testswere significantly increased in AY/a mice compared with a/a ones. Magnetic resonance imaging revealed asignificant reduction of cortex volume in AY/a mice. The level of mRNA of Ptpn5 gene encoding striatal enrichedtyrosine phosphatase in the frontal cortex of AY/a mice was significantly elevated compared with their wild-typelittermates. This is the first report on the alterations in the brain and behavior in the AY/a mouse line. It istempting to speculate that this mouse line can serve as a new and useful preclinical model to study neurobehavioralcomplications associated with obesity and type-2 diabetes

Domestication of wild animals alters the aggression towards humans, brain monoamines and coat pigmentation. Our aim is the interplay between aggression, brain monoamines and depigmentation. The Hedlund white mutation in the American mink is an extreme case of depigmentation observed in domesticated animals. The aggressive (-2.06 ± 0.03) and tame (+3.5 ± 0.1) populations of wild-type dark brown color (standard) minks were bred during 17 successive generations for aggressive or tame reaction towards humans, respectively. The Hedlund mutation was transferred to the aggressive and tame backgrounds to generate aggressive (-1.2 ± 0.1) and tame (+3.0 ± 0.2) Hedlund minks. Four groups of 10 males with equal expression of aggressive (-2) or tame (+5) behavior, standard or with the Hedlund mutation, were selected to study biogenic amines in the brain. Decreased levels of noradrenaline in the hypothalamus, but increased concentrations of the serotonin metabolite, 5-hydroxyindoleacetic acid and dopamine metabolite, homovanillic acid, in the striatum were measured in the tame compared with the aggressive standard minks. The Hedlund mutation increased noradrenaline level in the hypothalamus and substantia nigra, serotonin level in the substantia nigra and striatum and decreased dopamine concentration in the hypothalamus and striatum. Significant interaction effects were found between the Hedlund mutation and aggressive behavior on serotonin metabolism in the substantia nigra (P < 0.001), dopamine level in the midbrain (P < 0.01) and its metabolism in the striatum (P < 0.05). These results provide the first experimental evidence of the interplay between aggression, brain monoamines and the Hedlund mutation in the American minks.

International audience; Background The effect of stress on alcohol consumption in humans is highly variable, and the underlying processes are not yet understood. Attempts to model a positive relationship between stress and increased ethanol (EtOH) consumption in animals have been only modestly successful. Our hypothesis is that individual differences in stress effects on EtOH consumption are mediated by genetics. Methods We measured alcohol consumption, using the drinking-in-the-dark (DID) paradigm in females from 2 inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), and 35 of their inbred progeny (the BXD family). A control group was maintained in normal housing and a stress group was exposed to chronic mild stress (CMS), consisting of unpredictable stressors over 7 weeks. These included predator, social, and environmental perturbations. Alcohol intake was measured over 16 weeks in both groups during baseline (preceding 5-week period), CMS (intervening 7-week period), and post-CMS (final 4-week period). Results We detected a strong effect of CMS on alcohol intake. A few strains demonstrated CMS-related increased alcohol consumption; however, most showed decreased intake. We identified 1 nearly significant quantitative trait locus on chromosome 5 that contains the neuronal nitric oxide synthase gene (Nos1). The expression of Nos1 is frequently changed following alcohol exposure, and variants in this gene segregating among the BXD population may modulate alcohol intake in response to stress. Conclusions The results we present here represent the first study to combine chronic stress and alcohol consumption in a genetic reference population of mice. Differences in susceptibility to the effects of stressful environments vis-a-vis alcohol use disorders would suggest that the differences have at least some basis in genetic constitution. We have also nominated a likely candidate gene underlying the large individual differences in effects of stress on alcohol consumption.

Selection for feed efficiency is questioned as reducing the ability of farm animals to face stress and overcome challenges. The hypothalamic-pituitary-adrenocortical (HPA) axis activity, measured by the cortisol level in plasma after ACTH injection, has been proposed as an indicator of robustness. The objectives of this study were to evaluate 1. if a modified cortisol level in pigs alters their feed efficiency and their production performance, 2. if alternative dietary resources would affect these responses. Parallel trials including divergent lines for plasma cortisol 1 hour after ACTH injection and divergent lines for residual feed intake (RFI) were run during growth with a conventional diet and a diet with high fibres, low energy and low protein content. Selection for higher or lower plasma cortisol levels after stimulation of the adrenal gland did not impact growth and feed intake traits, but it had a significant impact on body composition and carcass yield, with improved composition in the high cortisol animals. The two lines had similar responses to an alternative diet, with decreased growth rate and feed intake, and increased feed conversion ratio. On the other hand, lines selected for divergent RFI had different responses to the alternative diet, the more efficient line having a more reduced growth rate with the diet with lower energy and AA contents. However, in terms of FCR it remained more efficient. The initial hypothesis of decreased efficiency associated to increased cortisol was not validated.

International audience; The plasma cortisol levels measured one hour after injection of ACTH reflects the hypothalamic-pituitary-adrenocortical (HPA) axis activity. The test was performed on piglets at 6 weeks of age. It was investigated as a potential selection criterion to improve pig robustness. From a base population of Large White pigs, two divergent lines were derived for 3 generations of selection, one selected for a high cortisol level (H line), one for a low cortisol level (L line). All breeders, as well as unselected animals, were also genotyped on a 70kSNP chip. At the 3rd generation of selection, the divergence between the two lines was about 5 genetic standard deviations. The high heritability of the trait was confirmed (h² = 0.64 ± 0.03). The genetic correlations with individual body weights and growth rates measured during lactation, post weaning and finishing periods were very low except for the genetic correlation between cortisol level and post-weaning growth rate (rg=-0.22 ± 0.10). The genome-wide association study revealed a quantitative trait locus influencing post-ACTH cortisol level, located on chromosome 2. It was suggested that divergent selection modified the frequency of NR3C1 alleles, favouring the hypersensitive glucocorticoid receptor in the low line.

National audience; The robustness of a piglet at weaning can be seen as its ability to express optimal growth without any health problems and regardless of weaning conditions. The aim of this study was to identify some biological markers measured around weaning that could predict growth of the piglet after weaning. Blood variables (n=62) describing immunity, stress, oxidative status and metabolism were measured at 26 and 33 days of age on piglets (n=288) from 16 commercial farms with contrasting health status (deteriorated: SAN- or good: SAN+). The robustness was approached by ADG from 26-47 days of age divided by live weight at 26 days (GMQR 26-47). Piglets were then classified according to the median of their farm in classes of low (GMQR-) or high (GMQR+) GMQR 26-47. The health status of the farm was associated with 37 of 67 variables measured (P < 0.05). Thus, piglets reared on SAN- farms showed higher activation of the immune system, mobilization of body reserves and oxidative stress after weaning than SAN+ piglets. Independent of the effect of health status, GMQR+ piglets showed lower mobilization of body reserves and a higher concentration in vitamin A, an antioxidant vitamin, after weaning compared to those of GMQR- piglets. Linear regression of GMQR explained 29% of the variance with 7 variables. Finally, this study shows that it is partly possible to predict the live weight 19 days after weaning from a reduced number of biological variables measured just before or 5 days after weaning (r2=0.72).; La robustesse du porcelet au sevrage peut être vue comme sa capacité à exprimer une croissance optimale sans manifester de problèmes de santé et ce quelles que soient les conditions de sevrage. L’objectif de cette étude était d’identifier des variables biologiques mesurées autour du sevrage permettant de prédire la croissance du porcelet après sevrage. Des variables sanguines (n=62) décrivant l’immunité, le stress, le statut oxydant et le métabolisme ont été mesurées à 26 et 33 jours d’âge sur des porcelets (n=288) issus de 16 élevages commerciaux de statuts sanitaires contrastés (dégradé : SAN- ou élevé : SAN+). La robustesse a été approchée par le GMQ entre 26 et 47 jours d’âge rapporté au poids à 26 jours (GMQR 26-47). Les porcelets ont été classés par rapport à la médiane de leur élevage dans les classes de GMQR 26-47 bas (GMQR-) ou haut (GMQR+). Le statut sanitaire de l’élevage était associé avec 37 des 67 variables sanguines et zootechniques (P < 0,05). Ainsi les porcelets des élevages SAN- montraient une plus grande activation de leur système immunitaire, une plus grande mobilisation de leurs réserves corporelles et un stress oxydant plus important après le sevrage que les porcelets SAN+. Indépendamment de l’effet du statut sanitaire, les porcelets GMQR+ montraient une moins forte mobilisation des réserves corporelles et une plus forte concentration en vitamine A, une vitamine antioxydante, après sevrage que les porcelets GMQR-. La régression linéaire du GMQR expliquait 29% de la variance avec 7 variables. En conclusion, ce travail montre qu’il est possible de prédire partiellement le poids 19 jours après sevrage à partir d’un nombre réduit de variables biologiques mesurées juste avant et 5 jours après le sevrage (r2=0,72).

International audience; We profiled individual differences in alcohol consumption upon initial exposure and during 5 weeks of voluntary alcohol intake in female mice from 39 BXD recombinant inbred strains and parents using the drinking in the dark (DID) method. In this paradigm, a single bottle of 20% (v/v) alcohol was presented as the sole liquid source for 2 or 4 h starting 3 h into the dark cycle. For 3 consecutive days mice had access to alcohol for 2 h followed by a 4th day of 4 h access and 3 intervening days where alcohol was not offered. We followed this regime for 5 weeks. For most strains, 2 or 4 h alcohol intake increased over the 5-week period, with some strains demonstrating greatly increased intake. There was considerable and heritable genetic variation in alcohol consumption upon initial early and sustained weekly exposure. Two different mapping algorithms were used to identify QTLs associated with alcohol intake and only QTLs detected by both methods were considered further. Multiple suggestive QTLs for alcohol intake on chromosomes (Chrs) 2, 6, and 12 were identified for the first 4 h exposure. Suggestive QTLs for sustained intake during later weeks were identified on Chrs 4 and 8. Thirty high priority candidate genes, including Entpd2, Per3, and Fto were nominated for early and sustained alcohol intake QTLs. In addition, a suggestive QTL on Chr 15 was detected for change in 2 h alcohol intake over the duration of the study and Adcy8 was identified as a strong candidate gene. Bioinformatic analyses revealed that early and sustained alcohol intake is likely driven by genes and pathways involved in signaling, and/or immune and metabolic function, while a combination of epigenetic factors related to alcohol experience and genetic factors likely drives progressive alcohol intake.

In the search for biological basis of robustness, this study aimed (i) at the determination of the heritability of the cortisol response to ACTH in juvenile pigs, using restricted maximum likelihood methodology applied to a multiple trait animal model, and (ii) at the study of the relationships between basal and stimulated cortisol levels with corticosteroid-binding globulin (CBG), IGF-I and haptoglobin, all important players in glucose metabolism and production traits. At 6 weeks of age, 298 intact male and female piglets from 30 litters (30 dams and 30 boars) were injected with 250 µg ACTH(1-24) (Synacthen). Blood was taken before ACTH injection to measure basal levels of cortisol, glucose, CBG, IGF-I and haptoglobin, and 60 min later to measure stimulated cortisol levels and glucose. Cortisol increased 2.8-fold after ACTH injection, with a high correlation between basal and stimulated levels (phenotypic correlation, r p=0.539; genetic correlation, r g=0.938). Post-ACTH cortisol levels were highly heritable (h 2=0.684) and could therefore be used for genetic selection of animals with a more reactive hypothalamic-pituitary-adrenocortical axis. CBG binding capacity correlated with cortisol levels measured in basal conditions in males only. No correlation was found between CBG binding capacity and post-ACTH cortisol levels. Basal IGF-I concentration was positively correlated with BW at birth and weaning, and showed a high correlation with CBG binding capacity with a strong sexual dimorphism, the correlation being much higher in males than in females. Basal haptoglobin concentrations were negatively correlated with CBG binding capacity and IGF-I concentrations. Complex relationships were also found between circulating glucose levels and these different variables that have been shown to be related to glucose resistance in humans. These data are therefore valuable for the genetic selection of animals to explore the consequences on production and robustness traits, but also point at pigs as a relevant model to explore the underlying mechanisms of the metabolic syndrome including the contribution of genetic factors.

Background Stress is a generic term used to describe non-specific responses of the body to all kinds of challenges. A very large variability in the response can be observed across individuals, depending on numerous conditioning factors like genetics, early influences and life history. As a result, there is a wide range of individual vulnerability and resilience to stress, also called robustness. The importance of robustness-related traits in breeding strategies is increasing progressively towards the production of animals with a high level of production under a wide range of climatic conditions and management systems, together with a lower environmental impact and a high level of animal welfare. The present study aims at describing blood transcriptomic, hormonal, and metabolic responses of pigs to a systemic challenge using lipopolysaccharide (LPS). The objective is to analyze the individual variation of the biological responses in relation to the activity of the HPA axis measured by the levels of plasma cortisol after LPS and ACTH in 120 juvenile Large White (LW) pigs. The kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. Results Cortisol level reaches its peak 4 h after LPS injection. The characteristic changes of white blood cell count to LPS were observed, with a decrease of total count, maximal at t=+4 h, and the mirror changes in the respective proportions of lymphocytes and granulocytes. The lymphocytes / granulocytes ratio was maximal at t=+1 h. An integrative statistical approach was used and provided a set of candidate genes for kinetic studies and ongoing complementary studies focused on the LPS-stimulated inflammatory response. Conclusions The present study demonstrates the specific biomarkers indicative of an inflammation in swine. Furthermore, these stress responses persist for prolonged periods of time and at significant expression levels, making them good candidate markers for evaluating the efficacy of anti-inflammatory drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4363-5) contains supplementary material, which is available to authorized users.

Ovarian folliculogenesis corresponds to the development of follicles leading to either ovulation or degeneration, this latter process being called atresia. Even if atresia involves apoptosis, its mechanism is not well-understood. The objective of this project was to analyse global gene expression in pig granulosa cells of ovarian follicles during atresia. The transcriptome analysis was performed using 9216 cDNAs microarray to identify gene networks and candidate genes involved in pig ovarian follicular atresia. One thousand six hundred and eighty four significantly regulated genes were differentially regulated between small healthy follicles and small atretic follicles. Among them, two hundred and eighty seven genes had a fold-change higher than 2 between the two follicle groups. Eleven genes (DKK3, GADD45A, CAMTA2, CCDC80, DAPK2, ECSIT, MSMB, NUPR1, RUNX2, SAMD4A, and ZNF628) having a fold-change higher than 5 between groups could likely serve as markers of follicular atresia. Moreover, automatic confrontation of deregulated genes with literature data enlightened 93 genes as regulatory candidates of pig granulosa cell atresia. Among these genes known to be inhibitors of apoptosis, stimulators of apoptosis or tumor suppressors INHBB, HNF4, CLU, different interleukins (IL5, IL24), TNF-associated receptor (TNFR1), and cytochrome-c oxidase (COX) were suggested as playing an important role in porcine atresia. Present study also enlists key upstream regulators in follicle atresia based on our results and on a literature review. The novel gene candidates and gene networks identified in the current study lead to a better understanding of the molecular regulation of ovarian follicular atresia.

The hypothalamic-pituitary-adrenocortical (HPA) axis is a major neuroendocrine system involved in the regulation of numerous physiological processes and in adaptation to stress. A wide range of variability can be observed in all the components of the system, and the contribution of genetic factors has been shown in the central regulation of the axis, the production of glucocorticoid hormones by the adrenal cortex, their bioavailability, and the efficiency of their action at the level of receptor and postreceptor mechanisms. Numerous molecular polymorphisms have been described that contribute to physiological variation as well as to HPA axis-related pathological conditions. Although most studies focus on single gene polymorphisms, future studies should aim to integrate the different sources of variation into a systems genetic model to take into account the strong interdependence of the different components of the axis.

Background HPA axis plays a major role in physiological homeostasis. It is also involved in stress and adaptive response to the environment. In farm animals in general and specifically in pigs, breeding strategies have highly favored production traits such as lean growth rate, feed efficiency and prolificacy at the cost of robustness. On the hypothesis that the HPA axis could contribute to the trade-off between robustness and production traits, we have designed this experiment to explore individual variation in the biological response to the main stress hormone, cortisol, in pigs. We used ACTH injections to trigger production of cortisol in 120 juvenile Large White (LW) pigs from 28 litters and the kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. Results Cortisol level reached its peak 1 h after ACTH injection. White blood cell composition was modified with a decrease of lymphocytes and monocytes and an increase of granulocytes (FDR

The exposure of animals to a variety of stressful events can induce behavioral and physiological responses, which can be modulated by anxiety levels. It is well recognized that genetic factors play a substantial role in both anxiety and stress reactivity. The present study examined the effect of exposure to 2,4,5-trimethylthiazoline (TMT), a component of fox feces, on nociception and corticosterone levels in Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rat strains (which display genetic differences in anxiety models such as the elevated plus-maze and open-field). The influence of two quantitative trait loci (QTL), named Ofil1 and Ofil2, which are known to affect emotionality in LEW versus SHR intercrosses on the responses to TMT was also investigated. LEW and SHR rats of both sexes displayed similar levels of behavioral and neuroendocrine responses after TMT exposure. As expected, TMT odor stress produced analgesia and enhanced corticosterone levels. Ofil1 on chromosome 4 affected stress-induced analgesia in males only. Ofil2 on chromosome 7 had no effect. The results suggest that behaviors measured in classical models of generalized anxiety and reactivity to stress produced by predator odors can be genetically dissociated. Finding a locus with an effect on the behavioral responses to stress represents the starting point in the search for genes responsible for stress-related traits.

Previous studies using the inbred rat strains Lewis (LEW) and spontaneously hypertensive rats (SHR) led to the mapping of two quantitative trait loci, named Ofil1 (on chromosome 4 of the rat) and Ofil2 (on chromosome 7), for open-field inner locomotion, a behavioral index of anxiety. Studies using other strains showed that the region next to Ofil1 influences measures of not only anxiety but also ethanol consumption. In view of the high prevalence of psychiatric disorders such as anxiety and alcoholism, as well as the comorbidity between them, the present study was designed to better characterize the contribution of these two loci to complex emotional and consummatory responses. Rats deriving from an F2 intercross between the LEW and the SHR strains were selected according to their genotype at markers flanking the loci Ofil1 and Ofil2 and bred to obtain lines of rats homozygous LEW/LEW or SHR/SHR for each of the two loci, thus generating four genotypic combinations. These selected animals as well as purebred LEW and SHR rats of both sexes were submitted to a battery of tests including measures of locomotor activity, anxiety, sweet and bitter taste reinforcement and ethanol intake. Lewis rats displayed more anxiety-like behavior and less ethanol intake than SHR rats. Ofil1 (on chromosome 4) affected both the activity in the center of the open field and ethanol drinking in females only. These results suggest that Ofil1 contains either linked genes with independent influences on anxiety-related responses and ethanol drinking or a pleiotropic gene with simultaneous effects on both traits.

Background: HPA axis plays a major role in physiological homeostasis. It is also involved in stress and adaptive response to the environment. In farm animals in general and specifically in pigs, breeding strategies have highly favored production traits such as lean growth rate, feed efficiency and prolificacy at the cost of robustness. On the hypothesis that the HPA axis could contribute to the trade-off between robustness and production traits, we have designed this experiment to explore individual variation in the biological response to the main stress hormone, cortisol, in pigs. We used ACTH injections to trigger production of cortisol in 120 juvenile Large White (LW) pigs from 28 litters and the kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. Results: Cortisol level reached its peak 1 h after ACTH injection. White blood cell composition was modified with a decrease of lymphocytes and monocytes and an increase of granulocytes (FDR < 0.05). Basal level of cortisol was correlated with birth and weaning weights. Microarray analysis identified 65 unique genes of which expression responded to the injection of ACTH (adjusted P < 0.05). These genes were classified into 4 clusters with distinctive kinetics in response to ACTH injection. The first cluster identified genes strongly correlated to cortisol and previously reported as being regulated by glucocorticoids. In particular, DDIT4, DUSP1, FKBP5, IL7R, NFKBIA, PER1, RGS2 and RHOB were shown to be connected to each other by the glucocorticoid receptor NR3C1. Most of the differentially expressed genes that encode transcription factors have not been described yet as being important in transcription networks involved in stress response. Their co-expression may mean co-regulation and they could thus provide new patterns of biomarkers of the individual sensitivity to cortisol. Conclusions: We identified 65 genes as biological markers of HPA axis activation at the gene expression level. These genes might be candidates for a better understanding of the molecular mechanisms of the stress response.

National audience

Chantier qualité GA; The hypothalamic-pituitary-adrenal (HPA) axis exerts a large range of effects on metabolism, the immune system, inflammatory processes, and brain functions. Together with the sympathetic nervous system, it is also the most important stress-responsive neuroendocrine system. Both systems influence production traits, carcass composition, and meat quality. The HPA axis may be a critical target for genetic selection of more robust animals. Indeed, numerous studies in various species have demonstrated the importance of genetic factors in shaping the individual HPA axis phenotype, and genetic polymorphism can be found at each level of the axis, including hormone production by the adrenal cortices under stimulation by adrenocorticotropic hormone (ACTH), hormone bioavailability, or receptor and postreceptor mechanisms. The aim of the present experiment was to extend these findings to the brain neurochemical systems involved in stress responses. To this end, a number of candidate genes were sequenced for molecular polymorphisms and their association was studied with stress neuroendocrine and production traits in a genetically diverse population consisting of 100 female pigs from an advanced intercross (F10-F12) between 2 highly divergent breeds, Large White (LW) and Meishan (MS). The LW breed has a high production potential for lean meat and a low HPA axis activity, and the MS breed has low growth rate, fat carcasses--but large litters of highly viable piglets--and a high HPA axis activity. Candidate genes were chosen in the catecholaminergic and serotonergic pathways, in the pituitary control of cortisol production, among genes previously demonstrated to be differentially expressed in ACTH-stimulated adrenal glands from LW and MS pigs, and in cortisol receptors. Sixty new polymorphisms were found. The association study with carcass and meat quality traits and with endocrine traits showed a number of significant results, such as monoamine oxidase (MAOA) polymorphisms with growth rate (P = 0.01); lean content and intramuscular fat (P < 0.01), which are the most important traits for carcass value; dopamine receptor D3 (DRD3) with carcass composition (P < 0.05); and vasopressin receptor 1B (AVPR1B) with meat quality traits (P ≤ 0.05). The effect of these polymorphisms on neuroendocrine parameters (eg DRD3 and HPA axis or AVPR1B and catecholamines) indicates information regarding their biological mechanism of action.

In recent decades, improvement of prolificacy and body composition (towards more muscle) was the main goal in pig breeding but the genetic progress has been accompanied by a substantial increase in piglet mortality before weaning. The most critical period is the perinatal period, mostly during the first 24-48 hours after birth. Piglet maturity, defined as the state of full development for survival at birth, is an important determinant of early mortality. However, evaluation of maturity at birth is difficult because of the lack of markers of this complex trait. The objective of our project (PORCINET, ANR09GENM005) is to take advantage of current knowledge about two pig breeds, Large White (LW) pigs selected for prolificacy and body composition and Meishan (MS) pigs being more robust piglets, to identify new markers of this trait. Maturity of several tissues and metabolite profiles of various fluids are analyzed on the fetuses (LW, MS and reciprocal F1) at day 90 or 110 of gestation (birth at day 114). The vitality of the piglets and the maternal behavior are analyzed during the two first weeks after birth. The 1H-NMR metabolic profiles of fetal plasma show that 1) the four genotypes are effectively different and that 2) variation in metabolite profiles amongst fetuses highly increases between day 90 and day 110 of gestation. Physiological parameters (e.g. cortisol, fructose and albumin) differed significantly between genotypes and developmental stages. These data associated with transcriptomic and proteomic data will enrich our understanding of piglet maturity determinism.

Chantier qualité GA; International audience; Robustness in farm animals was defined by Knap as ‘the ability to combine a high production potential with resilience to stressors, allowing for unproblematic expression of a high production potential in a wide variety of environmental conditions’. The importance of robustness-related traits in breeding objectives is progressively increasing towards the production of animals with a high production level in a wide range of climatic conditions and production systems, together with a high level of animal welfare. Current strategies to increase robustness include selection for ‘functional traits’, such as skeletal and cardiovascular integrity, disease resistance and mortality in various stages. It is also possible to use global evaluation of sensitivity to the environment (e.g. reaction norm analysis or canalization), but these techniques are difficult to implement in practice. The hypothalamic–pituitary– adrenocortical (HPA) axis is the most important stress-responsive neuroendocrine system. Cortisol (or corticosterone) released by the adrenal cortices exerts a large range of effects on metabolism, the immune system, inflammatory processes and brain function, for example. Large individual variations have been described in the HPA axis activity with important physiopathological consequences. In terms of animal production, higher cortisol levels have negative effects on growth rate and feed efficiency and increase the fat/lean ratio of carcasses. On the contrary, cortisol has positive effects on traits related to robustness and adaptation. For instance, newborn survival was shown to be directly related to plasma cortisol levels at birth, resistance to bacteria and parasites are increased in animals selected for a higher HPA axis response to stress, and tolerance to heat stress is better in those animals that are able to mount a strong stress response. Intense selection for lean tissue growth during the last decades has concomitantly reduced cortisol production, which may be responsible for the negative effects of selection on piglet survival. One strategy to improve robustness is to select animals with higher HPA axis activity. Several sources of genetic polymorphism have been described in the HPA axis. Hormone production by the adrenal cortices under stimulation by adrenocorticotropin hormone is a major source of individual differences. Several candidate genes have been identified by genomic studies and are currently under investigation. Bioavailability of hormones as well as receptor and post-receptor mechanisms are also subject to individual variation. Integration of these different sources of genetic variability will allow the development of a model for marker-assisted selection to improve animal robustness without negative side effects on production traits.

Chantier qualité GA; International audience; The neurotransmitter serotonin (5-HT) is involved in the regulation of mouse intermale aggression. Previously, it was shown that intensity of mouse intermale aggression was positively associated with activity of the key enzyme of 5-HT synthesis - tryptophan hydroxylase 2 (TPH2) in mouse brain. The aim of the present study was to investigate the effect of pharmacological activation or inhibition of 5-HT synthesis in the brain on intermale aggression in two mouse strains differing in the TPH2 activity: C57BL/6J (B6, high TPH2 activity, high aggressiveness) and CC57BR/Mv (BR, low TPH2 activity, low aggressiveness). Administration of 5-HT precursor L-tryptophan (300 mg/kg, i.p.) to BR mice significantly increased the 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels in the midbrain as well as the number of attacks and their duration in the resident-intruder test. And vice versa, administration of TPH2 inhibitor p-chlorophenylalanine (pCPA) (300 mg/kg, i.p., for 3 consecutive days) to B6 mice dramatically reduced the 5-HT and 5-HIAA contents in brain structures and attenuated the frequency and the duration of aggressive attacks. At the same time, L-tryptophan or pCPA did not influence the percentage of aggressive mice and the attack latency reflecting the threshold of aggressive reaction. This result indicated that the intensity of intermale aggression, but not the threshold of aggressive reaction is positively dependent on 5-HT metabolism in mouse brain.

Chantier qualité GA; International audience; The concept of robustness refers to the combination of a high production potential and a low sensitivity to environmental perturbations. The importance of robustness-related traits in breeding objectives is progressively increasing toward the production of animals with a high production level in a wide range of climatic conditions and production systems, together with a high level of animal welfare. Current strategies to increase robustness include selection for "functional traits," such as skeletal and cardiovascular integrity, disease resistance, and mortality at various stages. It is also possible to use global evaluation of sensitivity to the environment (eg reaction norm analysis or canalization), but these techniques are difficult to implement in practice. The glucocorticoid hormones released by the adrenal cortex exert a wide range of effects on metabolism, the cardiovascular system, inflammatory processes, and brain function, for example. Protein catabolism toward energy production and storage (lipids and glycogen) supports their pivotal role in stress responses aiming at the adaptation and survival of individuals under strong environmental pressure. Large individual variations have been described in adrenocortical axis activity, with important physiopathological consequences. In terms of animal production, higher cortisol levels have negative effects on growth rate and feed efficiency and increase the fat:lean ratio of carcasses. On the contrary, cortisol has positive effects on functional traits and adaptation. Intense selection for lean tissue growth and more generally high protein output during the past decades has concomitantly reduced cortisol production, which may be responsible for the negative effects of selection on functional traits. In this paper, we review experimental evidence suggesting that the balance between production and functional traits was modified in favor of improved robustness by selecting animals with higher adrenocortical axis activity, as well as the molecular genetic tools that can be used to fine-tune this objective.

Background Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult. The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA. Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis. Results We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH). A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte. Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA. Conclusions The ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.

The hypothalamic-pituitary-adrenocortical (HPA) axis is a major neuroendocrine system involved in the regulation of numerous physiological processes and in adaptation to stress. A wide range of variability can be observed in all the components of the system, and the contribution of genetic factors has been shown in the central regulation of the axis, the production of glucocorticoid hormones by the adrenal cortex, their bioavailability, and the efficiency of their action at the level of receptor and postreceptor mechanisms. Numerous molecular polymorphisms have been described that contribute to physiological variation as well as to HPA axis-related pathological conditions. Although most studies focus on single gene polymorphisms, future studies should aim to integrate the different sources of variation into a systems genetic model to take into account the strong interdependence of the different components of the axis.

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Chantier qualité GA; We studied the effect of activation of serotonin 5-HT1A receptors with selective agonist 8-OH-DPAT (0.1, 0.5, and 1.0 mg/kg) on intraspecies aggression and freezing reaction (catalepsy) in male mice of catalepsy-resistant AKR/J and two catalepsy-prone strains CBA/Lac and congenic AKR. CBA-D13Mit76. The latter strain differs from AKR strain only by terminal chromosome 13 fragment transferred from CBA strain and containing a locus determining predisposition to catalepsy and a gene encoding 5-HT1A receptor. 8-OH-DPAT in a low dose (0.1 mg/kg) affecting primarily presynaptic receptors suppressed aggressive behavior in CBA mice, but had no effect on the time of cataleptic freezing. At the same time, this dose of the drug produced no significant effect on aggression in AKR and AKR. CBA-D13Mit76 mice, but significantly attenuated freezing in AKR. CBA-D13Mit76 mice. High doses of 8-OH-DPAT (0.5 and 1 mg/kg) which affected mainly postsynaptic receptors inhibited catalepsy in CBA and AKR. CBA-D13Mit76 mice and in a dose of 1 mg/kg it suppressed aggression in all tested mouse strains. We concluded that the genome of the recipient strain (AKR) modulated the involvement of 5-HT1A receptors into the regulation of aggression and catalepsy in mice.

Background Attention deficit hyperactivity disorder (ADHD) is a complex neuropsychiatric disorder with a substantial genetic component. The Spontaneously Hypertensive Rats (SHR), considered as a good animal model of ADHD, also show less anxiety-like behaviors than Lewis (LEW) rats. The use of these inbred rat strains led us to the mapping of two quantitative trait loci (QTL), named Ofil1 (on chromosome 4) and Ofil2 (on chromosome 7), related to locomotion in the central and aversive area of an open field. Herein, we examined whether LEW and SHR rats differ in the acoustic startle reflex, a test used to study the neurobiology of anxiety, and in the prepulse inhibition of the startle response, which is known to be impaired in ADHD patients. The effect of the two aforementioned loci on these behavioral responses was also studied. Methods For this latter purpose, rats deriving from an F2 intercross between the LEW and SHR strains were selected according to their genotype at markers flanking the QTLs and bred to obtain lines of rats homozygous LEW/LEW or SHR/SHR for each of the two loci, thus generating 4 genotypic combinations. Results The SHR rats displayed decreased startle and prepulse inhibition levels when compared to LEW rats. Ofil2 affected prepulse inhibition in female rats only. Conclusion The results suggest that the LEW and SHR strains are appropriate for studying mechanisms of sensorimotor gating and indicate that the locus Ofil2 on rat chromosome 7 contain genes controlling prepulse inhibition, a neuro-behavioral trait of ADHD.

The High-Ethanol Preferring line of rats (HEP), recently selected by R.D. Myers, is characterised by a high voluntary consumption of alcohol (3-4 g/kg/day for males and 6-8 g/kg/day for females, when a 10% ethanol solution is available as a choice vs. water) and a high sensitivity to taste reinforcement (saccharin, quinine). Our previous data obtained with HEP rats showed no evidence of development of dependence after long-term sustained alcohol intake. In this study, we subjected these rats to several long-term administration protocols suggested to favour the development of alcohol dependence, including multiple alcohol concentrations or sweetened alcohol solutions (ethanol 10% or 20%+saccharin), and deprivation periods. The results showed no increase in alcohol consumption, no shift of preference for alcohol solutions when offered as a free choice vs. a preferred saccharin solution, and a very limited alcohol-deprivation effect when alcohol is made available after a period of deprivation, the three criteria used to demonstrate the development of dependence. Regardless of the method used, HEP rats failed to show dependence after long-term, heavy ethanol consumption. Resistance to ethanol dependence may in fact be genetically influenced and the HEP rat appears as a valuable model to search for factors involved in the transition from alcohol abuse to dependence.

The aim of this study was to analyse the genetic basis of excessive ethanol consumption and its relationship with emotional reactivity. The high-ethanol preferring line of rats used is characterized by a: (i) high voluntary consumption of alcohol; (ii) high sensitivity to taste reinforcement (saccharine, quinine); (iii) high locomotor activity in a novel environment; and (iv) low emotional reactivity, these features being opposite in the Wistar-Kyoto (WKY) rat strain. The F2 population demonstrated a very large variability in these behavioural traits, and factor analysis revealed that these characteristics appear to be largely unrelated to each other. The molecular bases for these differences were investigated by quantitative trait loci (QTL) analysis. For this purpose, the 196 F2 rats were genotyped with regularly distributed markers on the whole genome, and genetic linkage maps were generated for all subsequent QTL analyses. A locus with a maximum LOD score of 7.6 and accounting for approximately 61% of the genetic variance of the trait in the F2 population was detected on chromosome 4 for alcohol drinking. In the same region, we found a QTL related to the reinforcement properties of saccharin, with a significant LOD score of 4.9 and explaining 46% of the variance of the trait. Other significant QTL were found for plus maze behaviour and open field activity on chromosome 1. Current research aims to identify the gene(s) involved.

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