Search

International audience; In dairy cows, feed restriction is known to decrease milk yield by reducing the number of mammary epithelial cells (MEC) in the udder through a shift in the MEC proliferation-apoptosis balance, by reducing the metabolic activity of MEC, or both. The exfoliation of MEC from the mammary epithelium into milk is another process that may participate in regulating the number of MEC during feed restriction. The aim of the present study was to clarify the mechanisms that underlie the milk yield loss induced by feed restriction. Nineteen Holstein dairy cows producing 40.0 +/- 0.7 kg/d at 77 +/- 5 d in milk were divided into a control group (n = 9) and a feed-restricted group (n = 10). Ad libitum dry matter intake (DMI) was recorded during a pre-experimental period of 2 wk. For 29 d (period 1), cows were fed either 100 (control) or 80% (feedrestricted) of their ad libitum DMI measured during the pre-experimental period. Then, all cows were fed ad libitum for 35 d (period 2). Milk production and DMI were recorded daily. Blood and milk samples were collected once during the pre-experimental period; on d 5, 9, and 27 of period 1; and on d 5, 9, and 30 of period 2. Mammary epithelial cells were purified from milk using an immunomagnetic method to determine the rate of MEC exfoliation. Mammary tissue samples were collected by biopsy at the end of each period to analyze the rates of cell proliferation and apoptosis and the expression of genes involved in synthesizing constituents of milk. Feed restriction decreased milk yield by 3 kg/d but had no effect on rates of proliferation and apoptosis in the mammary tissue or on the expression of genes involved in milk synthesis. The daily MEC exfoliation rate was 65% greater in feed-restricted cows than in control cows. These effects in feed-restricted cows were associated with reduced insulin-like growth factor-1 and cortisol plasma concentrations. When all cows returned to ad libitum feeding, no significant difference on milk yield or MEC exfoliation rate was observed between feed-restricted and control cows, but refeeding increased prolactin release during milking. These results show that the exfoliation process may play a role in regulating the number of MEC in the udders of dairy cows during feed restriction without any carryover effect on their milk production.

National audience; Les microARN (miARNs) sont de petits ARNs non codants régulant l'expression génique au niveau posttranscriptionnel. Ils sont synthétisés par les cellules et sont aussi présents dans les fluides physiologiques tels que le lait, dans les vésicules extracellulaires ainsi que dans les globules gras qui sont sécrétés en grande quantité dans le lait. Si l’on considère que les miARNs contenus dans les globules gras du lait sont majoritairement le reflet de ceux du tissu mammaire, le profil de certains diffère. Ceci pose la question de l’origine des miARNs présents dans les globules gras ainsi que de leur mode de sécrétion. L'objectif de la présente étude pilote était d'évaluer l'existence d'un possible mécanisme de tri des miARNs produits par la cellule épithéliale mammaire (CEM) et de leur export ou non via les globules gras du lait. Pour cela, 12 vaches en lactation ont permis la collecte de glande mammaire (n=6) et de lait (n=6). Trois préparations de CEM, capturées par microdissection laser, ont été effectuées. Quatre miARNs (miR-125b-5p, miR-126-3p, miR-141-3p et miR-204-5p) ont été choisis sur la base de leur abondance différente dans le lait et dans le tissu mammaire. Après extraction des ARN totaux (kit miRVana), les miARNs ont été quantifiés par RTqPCR (kits TaqMan® MicroRNA Reverse Transcription et TaqMan® Advanced miRNA cDNA synthesis + TaqMan® Fast Advanced Master Mix) dans le tissu mammaire (parenchyme sécrétoire profond), dans la matière grasse du lait, et les CEM microdisséquées. Deux miARNs (miR-125b-5p et miR-141-3p) ont été détectés dans le tissu mammaire, la matière grasse et les CEM, tandis que miR-204-5p n'a été détecté que dans le tissu mammaire. Cela suggère que ce miARN pourrait être exprimé dans un type cellulaire mammaire autre que les CEM, par exemple les cellules myoépithéliales, immunitaires ou les adipocytes mammaires. Le résultat le plus intéressant concerne miR-126-3p qui a été détecté dans le tissu mammaire, les CEM, mais pas dans la matière grasse du lait, ce qui suggère une localisation différente ou un possible mécanisme d'adressage des miARNs vers les voies de sécrétion ou non dans les CEM. En conclusion, cette étude pilote met en évidence des différences qualitatives de contenu en miARNs entre les CEM et la matière grasse laitière. Ces résultats laissent penser qu’il existerait un mécanisme non aléatoire de chargement des miARNs dans les globules gras, à l'image des processus de tri décrits pour les vésicules extracellulaires. Ce mécanisme reste à élucider pour ce qui concerne les globules gras.

Background High-yielding dairy cows are prone to oxidative stress due to the high metabolic needs of homeostasis and milk production. Oxidative stress and inflammation are tightly linked; therefore, anti-inflammatory and/or natural antioxidant compounds may help improve mammary cell health. Baicalin, one of the major flavonoids in Scutellaria baicalensis, has natural antioxidant and anti-inflammatory properties in various cell types, but its effects on bovine mammary epithelial cells (BMECs) have not been investigated. Methods Explants from bovine mammary glands were collected by biopsy at the peak of lactation (approximately 60 days after the start of lactation) (n = three animals) to isolate BMECs corresponding to mature secretory cells. Cell viability, apoptosis, proliferative capacity and reactive oxygen species (ROS) production by BMECs were measured after increasing doses of baicalin were added to the culture media in the absence or presence of H2O2, which was used as an in vitro model of oxidative stress. Results Low doses of baicalin (1–10 µg/mL) had no or only slightly positive effects on the proliferation and viability of BMECs, whereas higher doses (100 or 200 µg/mL) markedly decreased BMEC proliferation. Baicalin decreased apoptosis rate at low concentrations (10 µg/mL) but increased apoptosis at higher doses. ROS production was decreased in BMECs treated with increasing doses of baicalin compared with untreated cells, and this decreased production was associated with increased intracellular concentrations of catalase and NRF-2. Irrespective of the dose, baicalin pretreatment attenuated H2O2-induced ROS production. Discussion These results indicate that baicalin exerts protective antioxidant effects on bovine mammary cells. This finding suggests that baicalin could be used to prevent oxidative metabolic disorders in dairy cows.

ObjectivesIn ruminants, the early phase of drying-off is a period of intense mammary gland involution that is due, in part, to dramatic decline prolactin (PRL) release. The speed at which the bovine mammary gland involutes following the abrupt cessation of lactation is also directly related to the risk of new intramammary infections. Thus, strategies to hasten involution following dry-off could have implications in preventing mastitis and optimizing mammary tissue regenerative processes.Materials and methodsTo assess the effect of prolactin inhibition by cabergoline on mammary gland involution, 14 Holstein dairy cows were injected with a single i.m. administration of 5.6 mg cabergoline (n=7) or placebo (n=7) within 4 hours after the last milking before the drying off at the day of drying-off (D0). Mammary secretion samples were collected using a teat-cannula once during lactation (D-6) and at D1, D2, D3, D4, D8 and D14 after the drying-off. The mammary secretion samples were used for lactoferrin and zymography analyses to detect the activity of enzymes such as MMP, matrix metalloproteinases involved in the remodelling of mammary tissue during involution. Mammary epithelial cells (MEC) were also purified from mammary secretions after centrifugation andimmunocytochemical binding in order to evaluate the MEC exfoliation. Mammary biopsy samples were collected one week before drying-off (D-6), at D1 and at D8 and used for lactoferrin immunochemistry and zymography analyses.ResultsThe activity of MMP9 increased after drying-off in mammary secretions (P < 0.001). Cabergoline increased the activity of MMP9 (1.7 fold, P < 0.05) in mammary secretions and MMP-2 in mammary tissue after drying-off (1.4 fold, P ≤ 0.01). MEC concentration progressively increased in mammary secretions after drying-off (P < 0.01). Cabergoline induced an increase in MEC concentration (P =0.04). Lactoferrin content progressively increased in mammary secretions during involution. The rise of lactoferrin content in mammary secretions was significant starting at D4 in the cabergoline treated cows (P ≤0.05) whereas it only happened at D8 in controls (P < 0.05). Overall, cabergoline treatment increased lactoferrin content of mammary secretions (P = 0.10). The total lactoferrin immunostaining in the mammary tissue increased after drying-off (P < 0.05). Compared with during lactation, this increase was observed at D1 and D8, respectively for cabergoline treated cows and control cows (P

Control of husbandry management is essential to produce female goat kids that fully express their milk potential during their career. It involves the determination of key periods during growth, particularly for the mammary gland development. The objective of this thesis was to determinate the effects of early weaning combined with different post-weaning feeding strategies on the performances of alpine goat kids as well as on their mammary gland development. On the one hand, we showed that early weaning at 10 kg of body weight did not affect body development, reproductive parameters or milk production (MP). On the other hand, a high concentrate intake during the rearing period increased body development and the weight of mammary glands.However, this did not impact the development of mammary parenchyma during the pre-pubertal phase or at the beginning of gestation, nor even the proportion of Mammary Epithelial Cells (MEC). The proliferation of MEC was identical regardless of the amount of concentrate ingested. MP was not affected by increasing concentrate intake. Modifying the energy and protein concentration of the concentrate in the pre-pubertal period had no effect on growth, reproductive performance, mammary gland development or mammary parenchyma development. Therefore, goats seem to adapt to changes in feeding strategies without a negative effect of the increase in growth on their ability to produce milk.; La maîtrise de la conduite d’élevage est essentielle pour produire des chevrettes qui expriment pleinement leur potentiel laitier au cours de leur carrière. Elle implique la détermination de phases clés au cours de la période de croissance, notamment pour la mise en place de la glande mammaire. L’objectif de cette thèse était de déterminer les effets d’un sevrage précoce combiné à différentes conduites alimentaires post-sevrage sur les performances zootechniques de chevrettes de race alpine ainsi que sur le développement de leur glande mammaire. Tout d’abord, nous avons montré qu’un sevrage précoce à 10 kg de poids vif n’affectait ni le développement corporel, ni les performances de reproduction, ni même la production laitière (PL) des chevrettesPar contre, un apport plus élevé de concentré pendant la phase d’élevage a augmenté le développement corporel et le poids de la glande mammaire. Toutefois, cela n’a impacté ni le développement du parenchyme mammaire au cours de la phase pré-pubère ou en début de gestation, ni même la proportion de Cellule Epithéliale Mammaire (CEM). La prolifération des CEM était identique quelle que soit la quantité de concentré ingérée. La PL n’a pas été modifiée par l’augmentation de l’ingestion de concentré. La modification de la concentration en énergie et en protéines du concentré en phase pré-pubère n’a eu d’effet ni sur les performances de croissance ou de reproduction, ni sur le développement de la glande mammaire. La chèvre semble donc s’adapter aux modifications de la conduite alimentaire sans pour autant que l’augmentation de croissanc

Objectives[br/] In ruminants, the early phase of drying-off is a period of intense mammary gland involution that is due, in part, to dramatic decline prolactin (PRL) release. The speed at which the bovine mammary gland involutes following the abrupt cessation of lactation is also directly related to the risk of new intramammary infections. Thus, strategies to hasten involution following dry-off could have implications in preventing mastitis and optimizing mammary tissue regenerative processes.[br/] Materials and methods[br/] To assess the effect of prolactin inhibition by cabergoline on mammary gland involution, 14 Holstein dairy cows were injected with a single i.m. administration of 5.6 mg cabergoline (n=7) or placebo (n=7) within 4 hours after the last milking before the drying off at the day of drying-off (D0). Mammary secretion samples were collected using a teat-cannula once during lactation (D-6) and at D1, D2, D3, D4, D8 and D14 after the drying-off. The mammary secretion samples were used for lactoferrin and zymography analyses to detect the activity of enzymes such as MMP, matrix metalloproteinases involved in the remodelling of mammary tissue during involution. Mammary epithelial cells (MEC) were also purified from mammary secretions after centrifugation and immunocytochemical binding in order to evaluate the MEC exfoliation. Mammary biopsy samples were collected one week before drying-off (D-6), at D1 and at D8 and used for lactoferrin immunochemistry and zymography analyses.[br/] Results[br/] The activity of MMP9 increased after drying-off in mammary secretions (P < 0.001). Cabergoline increased the activity of MMP9 (1.7 fold, P < 0.05) in mammary secretions and MMP-2 in mammary tissue after drying-off (1.4 fold, P ≤ 0.01). MEC concentration progressively increased in mammary secretions after drying-off (P < 0.01). Cabergoline induced an increase in MEC concentration (P =0.04). Lactoferrin content progressively increased in mammary secretions during involution. The rise of lactoferrin content in mammary secretions was significant starting at D4 in the cabergoline treated cows (P ≤0.05) whereas it only happened at D8 in controls (P < 0.05). Overall, cabergoline treatment increased lactoferrin content of mammary secretions (P = 0.10). The total lactoferrin immunostaining in the mammary tissue increased after drying-off (P < 0.05). Compared with during lactation, this increase was observed at D1 and D8, respectively for cabergoline treated cows and control cows (P

National audience; Chez les vaches laitières, des désordres métaboliques interviennent après la parturition et entraînent des phénomènes inflammatoires. Les cytokines pro-inflammatoires (TNFα, IL1β, IL6) pourraient avoir un effet direct sur la consommation alimentaire, la répartition des nutriments et être liées à une diminution de la production laitière. Différentes molécules à effet anti-inflammatoire, comme le salicylate, utilisées quelques jours après vêlage ont un effet positif à long terme sur la production laitière. Néanmoins, l’utilisation d’anti-inflammatoires dans l’alimentation animale pour stimuler la production de lait n’est pas autorisée. L’utilisation d’extraits de flavonoïdes a permis de montrer qu’ils avaient des effets anti-inflammatoires in vitro et in vivo. Il a également été montré que des extraits de Scutellaria baïcalensis réduisaient, de façon dose-dépendante, les niveaux de TNFα, d’IL1β et d’IL6 chez les animaux traités. Chez la vache laitière, l’extrait de Scutellaria baïcalensis apporté post-vêlage pendant 60 jours dans la ration augmente la production de lait de 230 litres sur l’ensemble de la lactation. Le principe actif de l’extrait de Scutellaria baïcalensis est la baïcaline. Les mécanismes d’action et les cibles cellulaires ne sont pour l’instant pas connus. Le but de cette étude était donc de caractériser in vitro les effets de la baïcaline sur des cellules épithéliales mammaires bovines (CEM) primaires. Des échantillons de tissus mammaires bovins ont été prélevés au pic de lactation (environ 60 jours de lactation) par biopsie, digérés et dissociés pour isoler les CEM correspondant à des cellules sécrétoires matures. Après mise en culture, les CEM ont été soumises à différentes doses de baïcaline en l'absence ou en présence de peroxyde d'hydrogène (H2O2). L'ajout de baïcaline à faibles doses (1 à 10 μg/mL) n'a eu que peu ou pas d'effets positifs sur la prolifération et la viabilité des CEM. A des doses plus élevées (> 50 μg/mL), la baïcaline a diminué la prolifération des CEM (-50%). La baïcaline a également eu un effet anti-apoptotique à faibles concentrations, mais provoqué une apoptose accrue à des doses plus élevées (> 100 μg/mL). Indépendamment des doses de baïcaline utilisées, la production de dérivés réactifs à l’oxygène (ROS) a été diminuée chez les CEM pré-traitées à la baïcaline par rapport aux cellules non traitées (-80%). Ces résultats ont clairement démontré les effets anti-oxydants de la baïcaline sur les CEM. Ceci suggère que la baïcaline à faibles doses (1 à 10 μg / mL) pourrait être utilisée pour prévenir les troubles métaboliques oxydatifs chez la vache laitière.

International audience; The urge to reduce antimicrobials use in dairy farming has prompted a search for alternative solutions. As infections of the mammary gland is a major reason for antibiotic administration to dairy ruminants, mammary probiotics have recently been presented as a possible alternative for the treatment of mastitis. To assess the validity of this proposal, we performed a general appraisal of the knowledge related to probiotics for mammary health by examining their potential modes of action and assessing the compatibility of these mechanisms with the immunobiology of mammary gland infections. Then we analyzed the literature published on the subject, taking into account the preliminary in vitro experiments and the in vivo trials. Preliminary experiments aimed essentially at exploring in vitro the capacity of putative probiotics, mainly lactic acid bacteria (LABs), to interfere with mastitis-associated bacteria or to interact with mammary epithelial cells. A few studies used LABs selected on the basis of bacteriocin production or the capacity to adhere to epithelial cells to perform in vivo experiments. Intramammary infusion of LABs showed that LABs are pro-inflammatory for the mammary gland, inducing an intense influx of neutrophils into milk during lactation and at drying-off. Yet, their capacity to cure mastitis remains to be established. A few preliminary studies tackle the possibility of using probiotics to interfere with the teat apex microbiota or to prevent the colonization of the teat canal by pathogenic bacteria. From the analysis of the published literature, it appears that currently there is no sound scientific foundation for the use of probiotics to prevent or treat mastitis. We conclude that the prospects for oral probiotics are not promising for ruminants, those for intramammary probiotics should be considered with caution, but that teat apex probiotics deserve further research.

Mammary epithelial cells (MEC) are exfoliated from the epithelium into milk, influencing the number of MEC present in the udder. This process is associated with epithelium integrity. The release of oxytocin (OT) induced by milking causes myoepithelial cell contraction, which, in turn, may stimulate MEC exfoliation through mechanical forces. To investigate the role of OT in MEC exfoliation, we inhibited or induced myoepithelial cell contraction by injecting the OT receptor antagonist atosiban (Ato) or a supraphysiological dose of OT, respectively. Eight cows were assigned to 2 treatments during 2 milkings according to a crossover experimental design: Control+OT (cows were first milked to collect standard milk and then received 5 IU of OT to collect residual milk through a second milking) and Ato + OT (cows were injected with Ato (50 μg/kg of body weight) and milked to collect cisternal milk, then received 5 IU of OT to collect alveolar milk through a second milking). Milk MEC were purified to determine their concentration and number in milk. Mammary epithelium integrity was assessed by measuring the kinetics of plasma lactose concentration. Inhibiting myoepithelial cell contraction by Ato injection decreased the number of exfoliated MEC in milk. In contrast, OT injection increased the concentration of MEC in the residual milk and the number of MEC in the alveolar milk. Ato injection reduced plasma lactose concentration, whereas, in both treatments, OT injections increased it. Our results suggested that myoepithelial cell contraction caused by OT could stimulate MEC exfoliation into milk and was associated with epithelium disruption.

Milk production is highly dependent on the extensive development of the mammary epithelium, which occurs during puberty. It is therefore essential to distinguish the epithelial cells committed to development from the related epithelial hierarchy. Using cell phenotyping and sorting, we highlighted four cell sub-populations within the bovine mammary gland at puberty. The CD49fhighCD24neg cells expressing CD10, KRT14, vimentin and PROCR corresponded to cells committed to the basal lineage. The CD49flow sub-population contained two cell subsets (CD49flowCD24neg and CD49flowCD24pos). Both subsets expressed hormone receptors including ER, PR and PRLR, as well as ALDH1 activity but only the CD49flowCD24pos subset expressed ELF5. These data indicated that the CD49flow sub-population is mainly composed of cells displaying a luminal phenotype and that this population comprises two luminal cell subsets, namely the CD24neg and CD24pos cells, likely committed to ductal and alveolar lineage, respectively. The putative mammary stem cell (MaSC) fraction was recovered in the CD49fhighCD24pos sub-population which were shown to form mammospheres in vitro. These cells differentially expressed CD10, KRT14 and KRT7, suggesting the existence of several putative MaSC sub-fractions. In-depth characterization of these epithelial sub-populations provides new insights into the bovine mammary epithelial cell lineage and suggests a common developmental lineage in mammals.

Le lait est produit par les cellules épithéliales mammaires (CEM). La quantité de lait produit est déterminée par le nombre de CEM et leur activité métabolique. Le nombre de CEM dépend de l’équilibre entre la prolifération cellulaire et l’apoptose. Le processus d’exfoliation, défini comme le décrochage des CEM de l’épithélium mammaire et leur évacuation dans le lait, a été proposé comme participant aussi à la régulation du nombre de CEM. Les objectifs de cette thèse étaient d’identifier les mécanismes biologiques impliqués dans la régulation de ce processus et d’étudier son rôle dans la régulation du nombre de CEM et son influence sur la production laitière. Nos résultats montrent qu’une partie des CEM est exfoliée entre deux traites consécutives. Cependant, la majorité des CEM sont exfoliées au moment de la traite suite à la contraction des cellules myoépithéliales et à la perte d’intégrité de l’épithélium induites par la décharge d’ocytocine. Le cortisol, au contraire, participerait à la restauration de l’intégrité de l’épithélium mammaire après la fin de la traite et limiterait l’exfoliation. Nous avons montré que les variations du taux d’exfoliation étaient opposées aux variations de production laitière dans le cas d’une restriction alimentaire et après la fin d’un traitement à l’hormone de croissance mais pas dans le cas d’un changement de fourrage, de l’inhibition de la prolactine et pendant un traitement à l’hormone de croissance. Le processus d’exfoliation des CEM participe donc à la régulation de la production laitière mais pas de façon systématique., Milk is synthesized by mammary epithelial cells (MEC). Milk yield is determined by the number of MEC in the mammary gland and the metabolic activity of these cells. It is well known that MEC number depends on the balance between cell proliferation and apoptosis. The MEC exfoliation process, defined as the shedding of MEC from the mammary epithelium into milk, is another process that might participate in the regulation of MEC number in the udder and thus in milk yield variations. The aims of this thesis were to identify the mechanisms that regulate the exfoliation process and to study the potential role of this process in regulating the number of MEC and milk yield. Our results showed that some MEC are exfoliated between milkings. Most of the MEC are, however, exfoliated during milking as a consequence of the myoepithelial cell contraction and the disruption of mammary epithelium integrity, both of which are caused by milking‐induced oxytocin release. Cortisol may play a role in limiting MEC exfoliation by restoring mammary epithelium integrity after milking. We showed that the exfoliation process participates in regulating milk yield during feed restriction and after a treatment with bovine growth hormone but did not participate in regulating milk yield when forage in the ration was changed, when prolactin secretion was inhibited, or during a treatment with bovine growth hormone. These results suggest that the MEC exfoliation process likely participates in regulating milk yield but not systematically.

In the context of sow hyper-prolificacy, the amount of milk available per piglet may be limiting for piglet survival, health and growth performance, especially for the lightest piglets. Findings from an in vivo study suggested that inflammation occurring the days after farrowing had a negative impact on sow milk production in hyperprolific sows. Therefore, any feed ingredients with anti-inflammatory properties may be helpful to increase milk production and piglet performance. Recently, Scutellaria baïcalensis, a widely spread labiate in Russia, China and Japan, has been shown to increase piglet growth rate from birth to weaning. Baicalin, the major compound found in S. baïcalensis extract, is a flavone glycoside with strong antioxidant and anti-inflammatory effects. The aim of the present study was to characterize the effects of baicalin on porcine mammary epithelial cells (MEC). MEC were purified from mammary glands collected collected in sows at peak lactation and cultured in the presence of increasing concentrations of baicalin (from 0 up to 200 μg/ml). We demonstrated that baicalin stimulated MEC proliferation when added to culture media at low concentrations (+60% at 10 μg/ml, P

International audience; Diet, in term of quantity and quality, is one of the main factors that affect milk yield and composition in dairy cows. The mammary epithelial cell (MEC) exfoliation process, together with the cell proliferation/apoptosis balance, participates in regulating the MEC number in the mammary gland and thus milk yield. Little is known about the effect of diet in the regulation of MEC number in the mammary tissue. This trial aimed to investigate the effect of feeding level and nature of forage on the MEC exfoliation rate. Sixteen multiparous dairy cows producing 37.0 ± 0.68 kg at 99 ± 4.6 DIM were divided into 4 groups of 4 cows each according to their diet. After a pre-experimental period of 3 wk, cows were fed either 100 or 75% of their ad libitum DMI with diets based on corn silage or conserved grass for 3 wk (Period 1). The feeding level was then reversed for the 3 following weeks (Period 2). Milk samples were collected at the end of the pre-experimental period and 15 d after the beginning of each period for milk MEC purification using an immunomagnetic method to assessthe daily MEC exfoliation rate. The percentage of apoptotic MEC in milk was determined by flow cytometry after annexin V/propidium iodide labeling. Mammary epithelium integrity was assessed by milk Na:K ratio determination. Data were analyzed using the mixed procedure of SAS. After 15 d of treatment, feed restriction decreased milk yield by 10% (P < 0.05) without affecting the MEC exfoliation rate. The Na:K ratio was greater with feed restriction, suggesting a loss of mammary epithelium integrity (P < 0.05). The nature of forage affected neither milk yield nor mammary epithelium integrity, but the daily MEC exfoliation rate was greater incows fed with the corn silage–based diet (P < 0.01). When cows were fed 100%, the percentage of apoptotic cells among exfoliated MEC was lower with the corn silage–based diet than with conserved grass–based diet (P < 0.05). These results show that the MEC exfoliation rate was not affected by a 15-d feed restriction whereas it was affected by the nature of forage. Mammary epithelial cell exfoliation rates did not varyalong with variations of milk yield. The effect of the duration of feed restriction on the MEC exfoliation rate and the effect of the nature of forage on the cell proliferation/apoptosis balance need to be investigated further.

In dairy cows, feed restriction is known to decrease milk yield by reducing the number of mammary epithelial cells (MEC) in the udder, due to an increased rate of MEC apoptosis. The exfoliation of MEC from the mammary epithelium into milk is another process that could participate in regulating MEC number. We hypothesized that the decreased number of MEC in the udder of feed-restricted cows might also result froman increase in MEC exfoliation rate. Nineteen Holstein dairy cows producing 40 ± 0.7 kg at 80 ± 4 DIM were divided into 2 groups, control (n = 9) and feed restricted (n = 10). Ad libitum DMI was recorded during a pre-experimental period of 2 wk. For 29 d (period 1), cows were fed either 100 or 80% of their DMI measured during the pre-experimental period. Then, all cows were fed ad libitum for 35 d (period 2). Feed intake andmilk production were recorded daily. Blood and milk samples were collected once during the pre-experimental period and 5, 9, and 27 d after the beginning of periods 1 and 2. Mammary epithelial cells were purified from milk samples using an immunomagnetic method. Lipid mobilization was assessed by plasma NEFA concentration. Mammary epithelium integrity was assessed by plasma lactose concentration, milk Na, andmilk Na:K ratio. Mammary tissue samples were collected by biopsy at the end of each period in order to analyze the rate of cell proliferation and apoptosis. Data were analyzed using the mixed procedure of SAS. Compared with control feeding, feed restriction induced a decrease in DMI by 20% (P < 0.001), an increase in plasma concentration of NEFA (P < 0.001), and a 3-kg/d milk yield loss (P < 0.001) but had no effect on the rate of proliferation and apoptosis in the mammary tissue. Daily MEC exfoliation rate was 65% greater in feed-restricted cows compared with control cows (P < 0.05). Feed restriction had no effect on plasma lactose concentration and milk Na:K ratio but increased milk Na concentration, which does not allow conclusions about the integrity of the mammary epithelium. In period 2, when all cows returned to ad libitum feeding, no significant difference was observed on DMI, milk yield, MEC exfoliation rate, and mammary epithelium integrity between feed-restricted and control cows. These results show that the exfoliation process could play a role in regulating the number of MEC in the udder of dairy cows during a feed restriction without a carryover effect on their milk production.

NADPH oxidase (NOX) complexes (a family of seven isoforms) drive cellular ROS production in patho-logical processes such as cancer. NOX-driven ROS production is involved in cell mechanisms from signalling to oxidative stress. Leptin, an adipokine overexpressed in obese patients, has been investigated in studies on breast carcinogenesis, but its effects on oxidative stress remain largely unexplored, especially in breast cancer. The study used three human mammary epithelial cell models presenting different neoplastic status (healthy primary HMECs, neoplastic MCF-7 cells and neoplastic MDA-MB-231 cells) to determine the effects of leptin on short-term ROS production and to characterize the enzymes involved. All three cell models significantly expressed NADPH oxidase isoform 5 (NOX5) in our culture conditions. All models showed induced ROS production regardless of leptin concentration (10 ng/ml mimicking good health, 100 ng/ml mimicking obesity). Cell treatment with either siRNA against NOX5, NOX inhibitor DPI or a calcium channel blocker (verapamil) confirmed the putative involvement of the NOX5 isoenzyme in ROS production. Moreover, cell treatments suppressed ROS production under leptin at both concentrations. Neoplastic cells appeared unable to downregulate NOX5 mRNA expression under leptin. Leptin emerged as a potential activator of ROS production in human epithelial mammary cells, where the ROS production was apparently linked to NOX5 activation. This novel finding could shed light on the potential role of obesity-associated hyperleptinemia in mammary cells via the activation of NOX enzymes.

The presence of mammary epithelial cells (MEC) in the milk of ruminants indicates that some MEC are shed from the mammary epithelium; however, the mechanisms that regulate the MEC exfoliation process are not known. Through the release of oxytocin, prolactin, and cortisol and through oxytocin-induced mechanical forces on the mammary epithelium, milking could participate in regulating the MEC exfoliation process. The aims of the present study were to determine the rate of MEC exfoliation throughout milking and to investigate its relationship to mammary epithelium integrity and milking-induced hormone release. Milk samples from 9 Holstein dairy cows producing 40.6 ± 1.36 kg of milk/d were collected at the beginning (after 1 and 2 min), in the middle, and at the end of milking. Milk MEC were purified using an immunomagnetic method. Blood samples were collected before, during, and after milking, and the oxytocin, prolactin, and cortisol concentrations in the samples were measured. Tight junction opening was assessed by plasma lactose concentration and the Na + :K + ratio in milk. The somatic cell count in milk varied during the course of milking; it decreased at the beginning of milking and then increased, reaching the highest values at the end of milking. Exfoliated MEC were present in all milk samples collected. The presence of MEC in the milk sample collected during min 1 of milking, likely corresponding to the cisternal milk fraction, suggests that MEC were exfoliated between milkings. The observed increase in the Na + :K + ratio in milk and in the plasma concentration of lactose indicated that disruption of mammary epithelium integrity occurred during milking. The MEC exfoliation rate at milking was not correlated with the variables describing milking-induced prolactin release but was negatively correlated with cortisol release, suggesting that cortisol may play a role in limiting exfoliation. In conclusion, milking induced a disruption of the mammary epithelial barrier. Mammary epithelial cells may be continuously exfoliated between milkings or exfoliated during milking as a consequence of the oxytocin-induced mechanical forces and the disruption of mammary epithelium integrity.

National audience

International audience

Dédifférenciation des cellules épithéliales mammaires primaires en culture : un modèle ex vivo de la plasticité du tissu mammaire. Journées d’Animation des Crédits Incitatifs du Département de Physiologie Animale et Systèmes d’Elevage (JACI Phase 2016)

The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.

Presented as part of the Lactation Biology: Mammary Gland Biology Revisited Symposium at the ADSA-ASAS Joint Annual Meeting, Orlando, Florida, July 2015.; Milk yield is partly influenced by the number of mammary epithelial cells (MEC) in the mammary gland. It is well known that variations in MEC number are due to cell proliferation and apoptosis. The exfoliation of MEC from the mammary epithelium into milk is another process that might influence MEC number in the mammary tissue. The rate of MEC exfoliation can be assessed by measuring the milk MEC content through light microscopy, flow cytometry analysis, or an immuno-magnetic method for MEC purification. Various experimental models have been used to affect milk yield and study the rate of MEC exfoliation. Reducing milking frequency from twice to once daily did not seem to have any effect on MEC loss in goat and cow milk after 7 d, but increased MEC loss per day in goats when applied for a longer period. An increase in MEC exfoliation was also observed during short days as compared with long days, or in response to an endotoxin-induced mastitis in cows. Other animal models were designed to investigate the endocrine control of the exfoliation process and its link with milk production. Suppression of ovarian steroids by ovariectomy resulted in a greater persistency of lactation and a decrease in MEC exfoliation. Administering prolactin inhibitors during lactation or at dry-off enhanced MEC exfoliation, whereas exogenous prolactin during lactation tended to prevent the negative effect of prolactin inhibitors. These findings suggest that prolactin could regulate MEC exfoliation. In most of these studies, variations of MEC exfoliation were associated with variations in milk yield and changes in mammary epithelium integrity. Exfoliation of MEC could thus influence milk yield by regulating MEC number in mammary tissue.

Immortalized bovine mammary epithelial cells (BME-UV1) and immortalized bovine mammary alveolar cells (MAC-T) have been extensively used as in vitro cell models to understand milk production in dairy cows. Precise knowledge about their phenotype and performance remains, however, unknown. This study aims to characterize MAC-T and BME-UV1 profiles when cultured in two-dimensional adherent, three-dimensional adherent (Matrigel), and three-dimensional no adherent [ultralow attachment (ULA)] supports. MAC-T and BME-UV1 were compared according to their proliferation capacities and to specific cell surface markers CD24, CD326 [epithelial cell adhesion molecule (EpCAM)], CD10, and integrin CD49f (α-6). Cytokeratin (CK14 and CK19), signal transducer and activator of transcription 5, and other proteins (occludin and cadherin-1) were analyzed. BME-UV1 in ULA support expressed higher CD49f marker. A different intensity of CD49 staining allowed the discrimination between the two cell lines in adherent condition. CD10, EpCAM, and CK19 expressions show that BME-UV1 cells have luminal capacity, while MAC-T has a myoepithelial profile with a high expression of CK14. BME-UV1 cells possess a closer committed progenitor profile due to their higher expression in aldehyde dehydrogenase and EpCAM. We observed that BME-UV1 cells have a better capacity to form spherical structures, mammospheres, in Matrigel than MAC-T, which was confirmed by the higher mammosphere area. In the ULA condition, BME-UV1 proliferated over the 6 days of culture. Taken together, our results clearly confirm the BME-UV1 luminal profile and MAC-T ductal/myoepithelial-like phenotype.

International audience; Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants’ udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition (ODM, feeding level, endocrine status, photoperiod modulation, and stage of lactation).

International audience; Le lait est produit dans la mamelle par les cellules épithéliales mammaires (CEM). L’étude de la fonction de lactation requiert de récolter du matériel biologique. La méthode classique de collecte de tissu mammaire correspond à une intervention chirurgicale par biopsie qui ne permet pas un échantillonnage simple et répété, sans endommager le tissu mammaire. En revanche, le lait contient des CEM progressivement exfoliées de l'épithélium au cours de la lactation. Le développement d’une méthode de purification des CEM à partir du lait par séparation immuno-magnétique (Boutinaud et al., 2008) permet une collecte non-invasive de matériel cellulaire. Les CEM purifiées pourraient constituer une alternative aux biopsies pour l’étude des transcrits mammaires chez les ruminants laitiers. La pertinence de l’utilisation des CEM du lait a été montrée du fait de la cohérence entre les variations des niveaux d’expression des transcrits mammaires dans les CEM du lait et les variations de production et de composition du lait chez la vache et la chèvre. En comparant nos données à celles obtenues par biopsies mammaires, nous avons montré que les CEM purifiées du lait représentent une source de transcrits mammaires pertinente pour étudier la synthèse du lait. Néanmoins, les transcrits extraits de ces cellules sont particulièrement sensibles à la dégradation. En conclusion, les CEM isolées à partir de lait sont une source pertinente et non invasive d'ARN mammaires qui peut être utilisée pour étudier l’effet de différents facteurs d’élevage (monotraite, alimentation, statut endocrinien, photopériode, stade de lactation) sur la production et la composition du lait.

Milk yield is partly influenced by the number of mammary epithelial cells (MEC) in the mammary gland. It is well known that MEC number varies due to cell proliferation and apoptosis. The exfoliation of MEC from the mammary epithelium into milk is another process which might influence MEC number in the mammary tissue. Yet, little is known about the control of MEC exfoliation process. The rate of MEC exfoliation can be assessed by measuring the milk MEC content through flow cytometry analysis or through an immuno-magnetic method for MEC purification. Various experimental models were used to affect milk yield and study the rate of MEC exfoliation. Reducing milking frequency from twice to once daily increased MEC loss per day in goat but not in cow milk. An increased daily rate of MEC exfoliation was also observed during short days as compared with long days or in response to an endotoxin-induced mastitis in cows. Other animal models were designed to investigate the endocrine control of the exfoliation process and its link with milk production. Suppression of ovarian steroids by ovariectomy resulted in a greater persistency of lactation and a decrease in MEC exfoliation.[br/] Administering prolactin inhibitors enhanced MEC loss while exogenous prolactin tended to prevent this negative effect of prolactin inhibitors. These findings suggest that prolactin could regulate MEC exfoliation. In most of these studies, variations of MEC exfoliation were associated with variations in milk yield and changes in mammary epithelium integrity. Exfoliation of MEC could be a process that regulates MEC number in the mammary tissue, and thereby could influence milk yield and lactation persistency.

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1)-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1)-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1)-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1)-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1)-casein. These experiments reveal that the insolubility of α(s1)-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1)-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.

absent

Session : Lactation Biology I; absent

Mechanisms of milk lipid secretion are highly controversial. Analyzing the fine protein composition of the “milk fat globule membrane” (MFGM), the triple-layered membrane surrounding milk lipid droplets (LDs) can provide mechanistic clues to better understand LD biosynthesis and secretion pathways in mammary epithelial cells (MECs). We therefore combined a high-sensitive Q-Exactive LC-MS/MS analysis of MFGM-derived peptides to the use of an in-house database intended to improve protein identification in the goat species. Using this approach, we performed the identification of 442 functional groups of proteins in the MFGM from goat milk. To get a more dynamic view of intracellular mechanisms driving LD dynamics in the MECs, we decided to investigate for the first time whether MFGM proteins were phosphorylated. MFGM proteins were sequentially digested by lysine-C and trypsin proteases and the resulting peptides were fractionated by a strong cation exchange chromatography. Titanium beads were used to enrich phosphopeptides from strong cation exchange chromatography eluted fractions. This approach lets us pinpoint 271 sites of phosphorylation on 124 unique goat MFGM proteins. Enriched GO terms associated with phosphorylated MFGM proteins were protein transport and actin cytoskeleton organization. Gained data are discussed with regard to lipid secretory mechanisms in the MECs. All MS data have been deposited in the ProteomeXchange with identifier PXD001039 (http://proteomecentral.proteomexchange.org/dataset/PXD001039).

Article en accès libre; The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy.

To differentiate between the contribution of mammary epithelial cells (MEC) and infiltrating immune cells to gene expression profiles of mammary tissue during early stage mastitis, we investigated in goats the in vivo transcriptional response of MEC to an experimental intra mammary infection (IMI) with Staphylococcus aureus, using a non-invasive RNA sampling method from milk fat globules (MFG). Microarrays were used to record gene expression patterns during the first 24 hours post-infection (hpi). This approach was combined with laser capture microdissection of MEC from frozen slides of mammary tissue to analyze some relevant genes at 30 hpi. During the early stages post-inoculation, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signalling pathway and initiate a sharp induction of innate immune genes predominantly associated with the pro-inflammatory response. At 30 hpi, MEC express genes encoding different acute phase proteins, including SAA3, SERPINA1 and PTX3 and factors, such as S100A12, that contribute directly to fighting the infection. No significant change in the expression of genes encoding caseins was observed until 24 hpi, thus validating our experimental model to study early stages of infection before the occurrence of tissue damage, since the milk synthesis function is still operative. This is to our knowledge the first report showing in vivo, in goats, how MEC orchestrate the innate immune response to an IMI challenge with S. aureus. Moreover, the non-invasive sampling method of mammary representative RNA from MFG provides a valuable tool to easily follow the dynamics of gene expression in MEC to search for sensitive biomarkers in milk for early detection of mastitis and therefore, to successfully improve the treatment and thus animal welfare.

L’intensification des pratiques d’élevage s’est accompagnée de l’émergence de pathologies de production, notamment des mammites. Il s’agit d’une inflammation de la glande mammaire, d’origine bactérienne dans la majeure partie des cas. Les mammites constituent à elles seules la première source de pertes financières des cheptels bovins laitiers en France et dans le monde. Néanmoins aucun traitement prophylactique ne permet à ce jour une action préventive à long terme. Parmi les agents étiologiques majeurs des mammites, Escherichia coli (E. coli) tient son importance du fait de sa prévalence et de son impact sur les rendements et la qualité du lait. La part des facteurs de l’hôte dans la capacité à éliminer le pathogène causal est relativement avérée, tandis que le lien entre caractéristiques bactériennes et sévérité de l’infection est plus délicat à établir. Cette étude s’attache donc à déterminer si les interactions entre E. coli et la glande mammaire, en particulier les cellules épithéliales mammaires (CEM) et les neutrophiles, peuvent expliquer des degrés de sévérité variables. L’influence du lait sur ces interactions a également été investiguée.

National audience; Cette étude in vitro a pour but de déterminer les effets de la leptine sur le statut oxydatif de trois modèles de cellules épithéliales mammaires humaines (HMEC : cellules primaires saines ; MCF-7 : lignée issue de tumeur canalaire invasive ; MDA-MB-231 : lignée issue de métastases d’adénocarcinome mammaire invasif) à deux concentrations de leptine : 10 ng/ml (situation normopondérale) et 100 ng/ml (situation d’obésité). Le statut pro-oxydant des cellules est déterminé par la mesure des espèces réactives de l’oxygène (ERO) par fluorescence (0 à 2h) et de la peroxydation lipidique à 24h. La défense anti-oxydante est déterminée entre autre par la mesure de l’expression génique et de l’activité catalytique des enzymes anti-oxydantes (Glutathion peroxydases (GPX), hème oxygénase (HMOX)) (à 1h, 6h et 24h). Quelque soit la concentration, la leptine augmente de façon significative (p

Staphylococcus aureus est un des pathogènes majeurs impliqués dans les mammites chez les ruminants. Il engendre des tableaux cliniques très variables, allant des mammites subcliniques aux mammites gangréneuses, mais les mécanismes de cette variabilité restent encore mal connus. La volonté de réduire l’utilisation d’antibiotiques en médecine vétérinaire ainsi que leurs faibles efficacités contre les mammites staphylococciques justifient les efforts consentis au développement de stratégies alternatives. L’une des particularités des infections intramammaires à S. aureus est leur chronicité et leur persistance dont l’une des principales causes est la capacité de S. aureus à adhérer et internaliser dans les tissus de l’hôte. Utiliser les propriétés inhibitrices des bactéries dites « probiotiques » d’un écosystème pour réduire, prévenir ou traiter des infections est une stratégie qui a déjà montré de bons résultats chez l’homme. Ce travail de thèse vise à mieux caractériser le microbiote mammaire et notamment la composante lactique et à évaluer son potentiel à moduler l’adhésion et l’internalisation de S. aureus dans les cellules épithéliales mammaires bovines. L’ensemble de ce travail a été réalisé avec deux souches de S. aureus très différentes au niveau des tableaux cliniques engendrés. La souche RF122, très virulente, et la souche Newbould 305, induisant des infections modérées et chroniques. Les résultats obtenus montrent que la capacité d’invasion cellulaire semble être un élément clé dans la chronicité de la souche Newbould 305. Ceci a été montré par l’analyse comparative (génomes, protéomes et phénotypes) des souches Newbould 305 et RF122. D’autre part, l’écosystème de la glande mammaire présente une composante lactique ayant des capacités probiotiques intéressantes. Ceci a été montré in vitro sur une modèle d’interaction tripartite, entre S. aureus, Lactobacillus casei et les cellules épithéliales mammaires MAC-T. Dans ce système, L. casei affecte l’adhésion et surtout l’internalisation de S. aureus sans modifier la viabilité et la morphologie des cellules en culture. L’inhibition semble dépendante d’un contact direct avec des L. casei viables. La caractérisation des bases moléculaires de ces phénomènes se poursuit dans l’équipe. Ces résultats ouvrent de nouvelles perspectives dans l’application de bactéries lactiques en tant que probiotique mammaire., Staphylococcus aureus is one of the main pathogens involved in ruminant mastitis. Severity of bovine S. aureus mastitis is highly variable, from subclinical to gangrenous mastitis. Nevertheless S. aureus factors that may explain this variability are still poorly documented. There is a great demand for the reduction of antibiotics use in veterinary medicine. Besides, these antibiotics have a low efficiency against staphylococcal mastitis. There is thus a need to develop alternative strategies. One feature of S. aureus is its ability to persist within the mammary tissue and induce chronicity of the infection. One of the main causes is the S. aureus ability to adhere to and internalize into host cells. The concept of biological control, using natural inhibitory properties of lactic acid bacteria (LAB) as probiotics, is now well established in humans and animals and is used to reduce, prevent or treat bacterial infections. This PhD work aims to better characterize the mammary microbiota in particular its LAB population, which might exert a positive effect on host health, and to determine its potential to modulate adhesion and internalization of S. aureus. This work was realized using two S. aureus strains as representatives of strains inducing severe (S. aureus RF122) or mild and chronic (Newbould 305) mastitis. Our results showed that the cellular invasion capacities seems to be key element in the chronicity observed in Newbould 305 mastitis. This was determined by genomic, proteomic and phenotypic comparison with RF122. Besides, we showed that the bovine mammary ecosystem present a LAB biodiversity and that these LAB presented interesting probiotic capacities. This was shown in an in vitro model of tripartite interaction, between S. aureus, Lactobacillus casei and the mammary epithelial cells MAC-T. In this system, L. casei impairs S. aureus adhesion and internalization without modifying the viability and the morphology of cells. The inhibition depends on a direct contact with live L. casei. Identification of the molecular basis of this inhibition are currently under investigation. These results open new perspectives in the application of lactic acid bacteria as mammary probiotics.

absent

The extensive polymorphism recorded at the CSN1S1 locus has been shown to influence goat milk composition and its technological properties. A deficit in alphaS1-casein is responsible for the accumulation of immature caseins in distended rough endoplasmic reticulum (ER) cisternae of mammary epithelial cells (MEC). This triggers a chronic ER stress which induces, in turn, an adaptive unfolded protein response (UPR). Our data strongly suggest: i) the existence of a membraneassociated form of alphaS1-casein, which is required for efficient export of the other caseins from the ER to the Golgi apparatus; ii) that alphaS1-casein plays a key role in the early steps of casein micelle biogenesis. A differential proteomic analysis (2D-DIGE) of milks from goats of extreme genotypes (A/A vs. O/O) at the CSN1S1 locus revealed the over representation of ER-resident proteins in O/O milks, strongly suggesting a singular secretion mechanism for this genotype. In addition, goats of O/O genotype produce fat globules smaller in size and displaying significant differences in MFGM composition, including both MFGM proteins and polar lipids. A relationship between genotypes at the CSN1S1 locus and the profile (structure and composition) of milk free oligosaccharides (OS), which are also built within the ER, has been shown, thus providing additional evidence for the impact of alphaS1-casein deficiency on the functioning of MEC.

Presenting author: Adrien Rossary The Second International Congress of Translational Research in Human Nutrition is organised by the Research Centre in Human Nutrition (CRNH) of Auvergne, of which INRA is a member, in collaboration with NuGO, European Association of universities and research institutes in the field of nutrigenomics; Nutritional status and hormonal factors, such as leptin, an adipokine highly regulated inobesity, induce cellular signaling pathways, some of which involving reactive oxygen species (ROS) asintracellular messenger. High levels of ROS contribute to oxidative stress, cellular damages andpathogenesis. That’s why ROS production associated to obesity could be a major risk factor ofmammary carcinogenesis.This study aimed to determine leptin effects on ROS production in 3 human epithelial mammary cellmodels which present different neoplasic status (healthy primary (HP) cells, MCF-7 and MDAMB-231). ROS production is measured by fluorescence in presence of two leptin concentrations (10ng/ml close to physiological values, 100 ng/ml as obesity level) with several probes at 2μM(Dichlorofluorescein (DCF) for total cellular ROS, Diaminofluorescein (DAF) for NO,Dihydrorhodamine (DHR) for mitochondrial ROS and Dihydroethidine (DHE) for cytosolic superoxydeanion (O2°-). Gene expression and catalytic activities of ROS production systems (NADPH oxidases,NO synthases, Dual oxidases) and of anti-oxidant enzymes (glutathione reductase, heme oxygenase,glutathione S-transferase) are performed.Whatever the cell model and the leptin concentration, a slight increase of total cellular ROSproduction is observed. This increase is independent of mitochondrial activity as DHR signalremained stable for HP cells (5.51 ± 0.40 RFU) and decreased for MCF7 and MDAMB-231 cells.Inversely, this ROS increase is dependent of cytosolic O2°- production as shown by DHE signalenhanced for HP cells (0.66 ± 0.01 to 0.81 ± 0.01 RFU), for MCF7 (0.79 ± 0.02 to 0.89 ± 0.03 RFU) andfor MDAMB-231 (0.82 ± 0.01 to 0.89 ± 0.02 RFU). Interestingly, this ROS production is dependent ofthe NADPH oxidase 5 (Nox5) expression and contributes to a different antioxidative response inregard to the neoplasic cell status. Leptin activates, only in HP cells, the antioxidative enzymesexpression and activities such as heme oxygenase or glutathione reductase.These data suggest that leptin could modulate the oxidative status of epithelial mammary cells indifferent ways according to the neoplasic cell status. Therefore leptin induces a similar ROSproduction for the 3 cell models whereas the anti oxidant cell response is not modified in MCF-7 andMA-MB-231 cells. This study highlights lower capacities of neoplasic cells to fight against oxidativestress.

absent

63rd European Federation of Animal Science EAAP Annual Meeting, Bratislava, Slovak Republic, 27–31 August 2012; The mammary gland is a dynamic organ that undergoes cyclic developmental and regressive changes during the lifetime of a female mammal. Mammogenesis begins during embryonic life with the development of the first mammary gland rudiments and ductal system. After birth, during the pre-pubertal period, the ductal growth of the mammary parenchyma occurs through the fat pad. In most of the ruminant species allometric mammary parenchyma development begins with the onset of cyclic ovarian secretions activity. The two main hormones secreted during an ovarian cycle are estradiol and progesterone. These steroid hormones are derived from cholesterol and are synthesized by theca and granulosa cells in ovaries. During puberty, the mammary parenchyma develops in a compact, highly arborescent parenchymal mass surrounded by a dense connective matrix. Ductal elongation and lobulo-alveolar development are accomplished during growth and pregnancy to prepare for future milk production. At the end of lactation, the mammary gland undergoes involution, which corresponds to a regression of the secretory tissue, a reduction in the alveolar size and a loss of mammary epithelial cells (MECs). Ovarian steroids (estradiol and progesterone) appear to be key regulators of the different stages of mammogenesis and mammary function. Through this review, the role and the importance of ovarian steroids on mammary gland and on MECs is described.

The mammary gland is a complex tissue which function is to ensure the offspring’s feeding by producing and secreting milk during lactation. Casein micelles and fat globules (MFGs) are essential constituents of milk and are both secreted at the apical side of mammary epithelial cells (MECs) during lactation. MFGs are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. The lactogenic hormone prolactin (PRL) not only induces the synthesis of caseins but also an increase of arachidonic acid (AA) in MECs, which may accelerate the apical transport and possibly the exocytosis of caseins (secretagogue effect of PRL). Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. By forming stable ternary complexes, SNARE (Soluble NSF Attachment Receptor) proteins constitute the core fusion machinery for cellular membrane trafficking and exocytosis events in many cell types. In addition, SNAREs have recently been described as target for AA, thus promoting exocytosis in neuroendocrine cells. We therefore attempted to identify the SNARE proteins involved in caseins secretion in the mouse mammary gland during lactation. An exciting possibility is that the SNAREs involved in caseins’ exocytosis can be the target of AA released in response to PRL.

Lipids are released in milk as fat globules, which are droplets of apolar lipids sur-rounded by a complex membrane deriving from the mammary epithelial cell (MEC) and called the milk fat globule membrane (MFGM). The structure of the MFGM is highly com-plex and closely related to the mechanisms of milk fat globule secretion in the mammary epithelial cell. Indeed, MFGM is composed of two biological membranes, a phospholipid monolayer, deriving from the endoplasmic reticulum, and a phospholipid bilayer, which originates from the apical plasma membrane of the MEC, with variable amounts of cyto-plasm trapped between. Biochemical techniques (i.e. sodium dodecyl sulphate-polyacryl-amide gel electrophoresis followed by different staining procedures) have been employed historically to characterize major MFGM proteins, namely MUC-1, fatty acid synthase, xan-thine oxidase, butyrophilin, lactadherin, and adipophilin. However, recent advances in the field of proteomics (mostly development of one-dimensional gel electrophoresis approach coupled with tandem mass spectrometry) have led to the identification of hundreds of proteins associated with the MFGM. Surprisingly, newly identified MFGM proteins were not only involved in lipid metabolic or exocytosis-related biological processes, but also in cell signalling, translation, or host defense-related mechanisms. Therefore, the milk fat globule should no longer be viewed as an inert structure only devoted to the delivery of lipids to the newborn, but rather as a dynamic and informative compartment which can contribute to the improvement of our comprehension of the mammary gland biology.

absent

Diplôme : Dr. Ing.

Diplôme : Ph. D.; La structure de la micelle de caséines détermine les caractéristiques fonctionnelles de la fraction protéique du lait et conditionne sa transformation industrielle. Cependant, la structure micellaire n'est pas élucidée. De nombreuses interrogations subsistent concernant l'agencement des caséines dans la micelle, l'existence ou non d'associations privilégiées, les interactions entre les caséines et la fraction minérale, et le rôle de chacun de ces composants dans le processus d'élaboration de cet édifice supramoléculaire. L'objectif de notre travail est de comprendre l'arrangement des caséines dans la micelle et le rôle de chacune dans l'élaboration intracellulaire de cette structure. La stratégie adoptée est donc d'étudier la formation de la micelle de caséines dans la voie de sécrétion de la cellule épithéliale mammaire (CEM). Dans un premier temps, nous avons choisi de caractériser l'état d'agrégation des caséines dans le premier compartiment de la voie de sécrétion, le réticulum endoplasmique rugueux. L'essentiel des expériences a été réalisé sur les caséines du tissu mammaire de rat et nous avons confirmé notre modèle chez un ruminant, la chèvre. Des fractions purifiées de microsomes rugueux, issues du RER du tissu mammaire, ont été perméabilisées par de la saponine dans des conditions conservant ou non la structure micellaire. L'analyse du comportement des caséines immatures des microsomes en conditions conservatives montre que la caséine beta ne semble pas agrégée dans ce compartiment alors qu'une forte proportion de la caséine alphaS1 reste associée aux microsomes. Les extractions membranaires dans différentes conditions physico-chimiques ont révélé l'existence d'une forme membranaire de la caséine alphaS1 dans les microsomes rugueux mais également dans des compartiments plus tardifs de la voie de sécrétion. De plus, nous montrons que les ponts disulfure intermoléculaires jouent un rôle important dans la liaison de la caséine alphaS1 avec les membranes. Pour caractériser les bases moléculaires de l'interaction de la caséine alphaS1 avec les phospholipides des membranes biologiques, nous avons mis en oeuvre des techniques spectroscopiques. Les formes phosphorylée et déphosphorylée de la caséine alphaS1 purifiée à partir de lait de rat ont été placées en présence de liposomes de différentes tailles (SUVs et LUVs). La fluorescence intrinsèque du tryptophane révèle des modifications conformationnelles pour la caséine alphaS1 déphosphorylée et des changements structuraux ont été mis en évidence par dichroïsme circulaire, pour les deux formes de la protéine lors de la liaison avec les phospholipides des vésicules lipidiques. L'originalité de ce travail a été de montrer l'existence, pour la première fois, d'une forme membranaire d'une caséine dans la CEM. Nos résultats suggèrent que cette forme membranaire de la caséine alphaS1 pourrait jouer un rôle clé dans l'édification de la structure micellaire et/ou dans le transport intracellulaire et la sécrétion des caséines.

absent

La production de lait dépend fortement du développement de l'épithélium mammaire. Dès la puberté, les cellules souches adultes mammaires (MaSC) et, les cellules progénitrices qui en dérivent, participent au développement de l’épithélium mammaire en donnant naissance aux cellules épithéliales matures (luminales et basales/myoépithéliales). Dans le tissu mammaire adulte, ces mêmes MaSC sont à l’origine du potentiel régénératif du tissu sécrétoire lors de chaque cycle de reproduction. Pour mieux comprendre le rôle de ces cellules dans la construction et l’homéostasie du tissu épithélial, responsable du potentiel laitier chez la vache, nous avons entrepris d’identifier et de caractériser le lignage épithélial par des approches de cytométrie en flux et de tri cellulaire. Sur la base de l’expression de deux marqueurs de surface membranaire, CD49f et CD24, nous avons mis en évidence quatre sous-populations distinctes engagées dans le développement mammaire à la puberté. A partir du phénotype et de la signature moléculaire (expression de gènes cibles) de chacune de ces sous-populations cellulaires triées, nous avons montré qu’au stade de la puberté, l’épithélium mammaire contient de rares cellules souches mammaires, des cellules progénitrices à typage mixte luminal/basal et des cellules luminales et basales. Fort de ces premiers résultats, nous avons ensuite comparé ces sous-populations épithéliales à trois stades physiologiques clés chez la vache laitière : la puberté (génisses de 17 mois), la lactation (vaches multipares produisant 35 kg de lait) et le tarissement (vaches taries). Il apparait que ces (sous)populations diffèrent soit en proportion soit en caractéristiques, voire les deux, à ces différents stades. L’ensemble de nos données montre que le lignage épithélial évolue substantiellement au cours de la vie de la vache. Nous avons concomitamment acquis de nouvelles informations sur un domaine peu étudié : la hiérarchie des cellules épithéliales de la glande mammaire chez la vache laitière. L’approche cytométrique, développée pour identifier et phénotyper les cellules épithéliales du tissu mammaire, est une approche que l’on peut appliquer à d’autres matrices que le tissu (cellules, lait,…). A partir d’analyse en cytométrie en flux de cellules marquées avec CD49f et CD24, nous avons aussi pu caractériser le phénotype de lignées cellulaires mammaires bovines couramment utilisées dans nos laboratoires. Appliquée aux cellules isolées du lait, cette technologie informe sur le type de cellules épithéliales exfoliées dans le lait ainsi que leur nombre. Concrètement, cette approche cytométrique représente un outil de phénotypage pertinent pour distinguer les types cellulaires épithéliaux, et ceci sur de nombreuses matrices.