Large intestinal nutritional and physicochemical parameters from different dog sizes reshape canine microbiota structure and functions in vitro.

Different dog sizes are associated with variations in large intestinal physiology including gut microbiota, which plays a key role in animal health. This study aims to evaluate, using the CANIM-ARCOL (Canine Mucosal Artificial Colon), the relative importance of gut microbes versus physicochemical and nutritional parameters of the canine colonic environment in shaping microbiota structure and functions. CANIM-ARCOL was set up to reproduce nutrient availability, bile acid profiles, colonic pH, and transit time from small, medium, or large dogs according to in vivo data, while bioreactors were all inoculated with a fecal sample collected from medium size dogs (n = 2). Applying different dog size parameters resulted in a positive association between size and gas or SCFA production, as well as distinct microbiota profiles as revealed by 16S Metabarcoding. Comparisons with in vivo data from canine stools and previous in vitro results obtained when CANIM-ARCOL was inoculated with fecal samples from three dog sizes revealed that environmental colonic parameters were sufficient to drive microbiota functions. However, size-related fecal microbes were necessary to accurately reproduce in vitro the colonic ecosystem of small, medium, and large dogs. For the first time, this study provides mechanistic insights on which parameters from colonic ecosystem mainly drive canine microbiota in relation to dog size. The CANIM-ARCOL can be used as a relevant in vitro platform to unravel interactions between food or pharma compounds and canine colonic microbiota, under different dog size conditions. The potential of the model will be extended soon to diseased situations (e.g. chronic enteropathies or obesity).

Environmental colonic parameters (such as nutrient availability, transit time, or pH) were sufficient to drive microbiota at the functional level in the CANIM-ARCOL in vitro gut model.Size-related fecal microbes were necessary to accurately reproduce the colonic environment of small, medium, and large dogs.CANIM-ARCOL model can be used as a relevant in vitro tool to decipher the relative importance of microbiota versus environmental colonic parameters in food and pharma studies.

Conditioner application improves bedding quality and bacterial composition with potential beneficial impacts for dairy cow's health.

Recycled manure solids (RMS) is used as bedding material in cow housing but can be at risk for pathogens development. Cows spend several hours per day lying down, contributing to the transfer of potential mastitis pathogens from the bedding to the udder. The effect of a bacterial conditioner (Manure Pro, MP) application was studied on RMS-bedding and milk qualities and on animal health. MP product was applied on bedding once a week for 3 months. Bedding and teat skin samples were collected from Control and MP groups at D01, D51, and D90 and analyzed through 16S rRNA amplicon sequencing. MP application modified bacterial profiles and diversity. Control bedding was significantly associated with potential mastitis pathogens, while no taxa of potential health risk were significantly detected in MP beddings. Functional prediction identified enrichment of metabolic pathways of agronomic interest in MP beddings. Significant associations with potential mastitis pathogens were mainly observed in Control teat skin samples. Finally, significantly better hygiene and lower Somatic Cell Counts in milk were observed for cows from MP group, while no group impact was observed on milk quality and microbiota. No dissemination of MP strains was observed from bedding to teats or milk. IMPORTANCE: The use of Manure Pro (MP) conditioner improved recycled manure solids-bedding quality and this higher sanitary condition had further impacts on dairy cows' health with less potential mastitis pathogens significantly associated with bedding and teat skin samples of animals from MP group. The animals also presented an improved inflammation status, while milk quality was not modified. The use of MP conditioner on bedding may be of interest in controlling the risk of mastitis onset for dairy cows and further associated costs.

Canine Mucosal Artificial Colon: development of a new colonic in vitro model adapted to dog sizes.

Differences in dog breed sizes are an important determinant of variations in digestive physiology, mainly related to the large intestine. In vitro gut models are increasingly used as alternatives to animal experiments for technical, cost, societal, and regulatory reasons. Up to now, only one in vitro model of the canine colon incorporates the dynamics of different canine gut regions, yet no adaptations exist to reproduce size-related digestive parameters. To address this limitation, we developed a new model of the canine colon, the CANIne Mucosal ARtificial COLon (CANIM-ARCOL), simulating main physiochemical (pH, transit time, anaerobiosis), nutritional (ileal effluent composition), and microbial (lumen and mucus-associated microbiota) parameters of this ecosystem and adapted to three dog sizes (i.e., small under 10 kg, medium 10-30 kg, and large over 30 kg). To validate the new model regarding microbiota composition and activities, in vitro fermentations were performed in bioreactors inoculated with stools from 13 dogs (4 small, 5 medium, and 4 large). After a stabilization period, microbiota profiles clearly clustered depending on dog size. Bacteroidota and Firmicutes abundances were positively correlated with dog size both in vitro and in vivo, while opposite trends were observed for Actinobacteria and Proteobacteria. As observed in vivo, microbial activity also increased with dog size in vitro, as evidenced from gas production, short-chain fatty acids, ammonia, and bile acid dehydroxylation. In line with the 3R regulation, CANIM-ARCOL could be a relevant platform to assess bilateral interactions between food and pharma compounds and gut microbiota, capturing inter-individual or breed variabilities. KEY POINTS: • CANIM-ARCOL integrates main canine physicochemical and microbial colonic parameters • Gut microbiota associated to different dog sizes is accurately maintained in vitro • The model can help to move toward personalized approach considering dog body weight.

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids.

Intestinal organoids are innovative in vitro tools to study the digestive epithelium. The objective of this study was to generate jejunum and colon organoids from suckling and weaned piglets in order to determine the extent to which organoids retain a location-specific and a developmental stage-specific phenotype. Organoids were studied at three time points by gene expression profiling for comparison with the transcriptomic patterns observed in crypts in vivo. In addition, the gut microbiota and the metabolome were analyzed to characterize the luminal environment of epithelial cells at the origin of organoids. The location-specific expression of 60 genes differentially expressed between jejunum and colon crypts from suckling piglets was partially retained (48%) in the derived organoids at all time point. The regional expression of these genes was independent of luminal signals since the major differences in microbiota and metabolome observed in vivo between the jejunum and the colon were not reproduced in vitro. In contrast, the regional expression of other genes was erased in organoids. Moreover, the developmental stage-specific expression of 30 genes differentially expressed between the jejunum crypts of suckling and weaned piglets was not stably retained in the derived organoids. Differentiation of organoids was necessary to observe the regional expression of certain genes while it was not sufficient to reproduce developmental stage-specific expression patterns. In conclusion, piglet intestinal organoids retained a location-specific phenotype while the characteristics of developmental stage were erased in vitro. Reproducing more closely the luminal environment might help to increase the physiological relevance of intestinal organoids.

Effect of live yeast supplementation in sow diet during gestation and lactation on sow and piglet fecal microbiota, health, and performance.

Feeding probiotics like live yeast Saccharomyces cerevisiae var. boulardii (SB) in pig diets has been suggested to preserve health and reduce antibiotic use during critical periods like weaning. This study was conducted to determine whether SB added to the diet of sows during the last 2 mo of gestation and the 4 wk of lactation may contribute to support the health and performance of piglets before and after weaning through changes in sow physiology, milk composition, and fecal microbiota. Crossbred sows (n = 45) from parity 1 to 9 were allocated to two dietary treatments: Control (n = 23) and SB (n = 22). Sows in the SB group were fed the same standard gestation and then lactation diet as the Control sows but with the addition of SB at 1 × 109 colony-forming units/kg of feed. Piglets were weaned under challenging conditions consisting of mixing of litters, no pen cleaning, and a 2-h period of nonoptimal temperature exposure. Blood and feces were collected from sows on days 28 and 113 of gestation and days 6 (feces only) and 28 of lactation, and from piglets on days 6 (feces) and 28 of lactation and day 5 after weaning. Colostrum was collected during parturition and milk on day 6 of lactation. Supplementation of sow diets with SB influenced the fecal microbiota of the sows and their piglets. Five days after weaning, the alpha-diversity was lower (P < 0.05) in piglets from SB sows than in piglets from Control sows. Analysis of microbiota with partial least square discriminant analysis discriminated feces from SB sows from that of Control sows at 110 d of gestation (29.4% error rate). Piglet feces could also be discriminated according to the diet of their mother, with a better discrimination early after birth (day 6 of lactation) than after weaning (day 5 postweaning, 3.4% vs. 12.7% error rate). Five days after weaning, piglets had greater white blood cell count, plasma haptoglobin concentration, and oxidative stress than before weaning (P < 0.001). Nevertheless, SB supplementation in sow diets had no effect (P > 0.05) on most of health criteria measured in blood and growth performance of piglets during lactation and the postweaning period. Moreover, dietary supplementation of SB to sows did not elicit any changes (P > 0.05) in their reproductive performance, metabolic and health status, nor in the concentration of immunoglobulins and nutrients in colostrum and milk. In the present experimental conditions, feeding SB to sows influenced sow and piglet microbiota with no consequences on their health and performance.

Feeding live yeast Saccharomyces cerevisiae var. boulardii (SB) in pig diets is recommended to promote a better health and reduce antibiotic use during critical periods like weaning. Our study was conducted to determine if SB added to the diet of sows during the last 2 mo of gestation and the 4 wk of lactation may contribute to support the health and performance of their piglets before and after weaning. We hypothesized that live SB supplementation to the sows may help improve the health and metabolic status of the sows and consequently the quality of milk and microbiota provided to the piglets. Supplementation of sow diet with SB during gestation and lactation induced modifications in the fecal microbiota of sows and their piglets. For piglets, the effects of SB fed to their mother were still observed 5 d after weaning. These modifications were, however, associated with changes neither in piglet ability to cope with the stress of weaning nor in milk nutritional and immune composition.

A live yeast supplementation to gestating ewes improves bioactive molecule composition in colostrum with no impact on its bacterial composition and beneficially affects immune status of the offspring.

Colostrum quality is of paramount importance in the management of optimal ruminant growth and infectious disease prevention in early life. Live yeast supplementation effect during the last month of gestation was evaluated on ewes' colostrum composition. Two groups of ewes (n = 14) carrying twin lambs were constituted and twins were separated into groups (mothered or artificially fed) 12 h after birth. Nutrient, oligosaccharides (OS), IgG and lactoferrin concentrations were measured over 72 h after lambing, and bacterial community was described in colostrum collected at parturition (T0). Immune passive transfer was evaluated through IgG measurement in lamb serum. In both groups, colostral nutrient, OS concentrations and IgG concentrations in colostrum and lamb serum decreased over time (P < 0⋅01), except for lactose, which slightly increased (P < 0⋅001), and lactoferrin, which remained stable. Bacterial population was stable over time with high relative abundances of Aerococcaceae, Corynebacteriaceae, Moraxellaceae and Staphylococcaceae in T0 colostrum. No effect of supplementation was observed in nutrient and lactoferrin concentrations. In supplemented ewes, the level of colostral IgG was higher at T0 and a higher level of serum IgG was observed in lambs born from supplemented mothers and artificially fed, while no effect of supplementation was observed in the mothered lamb groups. Using a metabolomic approach, we showed that supplementation affected OS composition with significantly higher levels of colostral Neu-5Gc compounds up to 5 h after birth. No effect of supplementation was observed on bacterial composition. Our data suggest that live yeast supplementation offsets the negative impact of early separation and incomplete colostrum feeding in neonate lambs.

Saccharomyces cerevisiae boulardii CNCM I-1079 supplementation in finishing male pigs helps to cope with heat stress through feeding behaviour and gut microbiota modulation.

Pigs subjected to heat stress (HS) decrease their feed intake and growth. The objectives of the experiment were to determine the effects of live yeast (LY) supplementation (Saccharomyces cerevisiae var boulardii CNCM I-1079) on feeding behaviour, energy metabolism and faecal microbiota composition of finishing boars (n 10) housed in a respiration chamber at thermoneutrality (7 d at 22°C) or during HS (seven plus six days at 28°C). Dietary LY supplementation increased DM intake (P = 0·01) whatever the ambient temperature, whereas HS decreased feed intake whatever the dietary supplementation (P = 0·01). Dietary LY supplementation increased the number of meals (P = 0·02). Energy retention was higher with dietary LY supplementation (P < 0·01) but decreased during HS (P < 0·01). The skin temperature of the supplemented pigs was lower at thermoneutrality and increased during HS to a lesser extent than that of non-supplemented pigs (P < 0·01). Faecal microbiota composition was determined using 16S rRNA gene sequencing. Treponema, Christensenellaceae R-7, Ruminococcaceae UCG-002, Rikenellaceae RC9, Clostridium sensu stricto 1 and Romboutsia genera and some bacteria belonging to Alloprevotella, Oxalobacter and Anaeroplasma genera were more abundant under HS. LY supplementation attenuated HS effects on Romboutsia abundance, while decreasing the abundance of some bacteria from Ruminoccocus, Coprococcus, Peptococcus and Oxalobacter genera and increasing the abundance of beneficial bacteria from Lactococcus and Subdoligranulum genera. Our results suggest that higher level of the keystone species Ruminococcus bromii at thermoneutrality may be one of the causes for higher energy retention observed under subsequent HS.

Bacillus pumilus and Bacillus subtilis Promote Early Maturation of Cecal Microbiota in Broiler Chickens.

Mature and stable intestinal microbiota in chickens is essential for health and production. Slow development of microbiota in young chickens prolongs the precarious period before reaching mature configuration. Whether probiotics can play a role in the early maturation of intestinal microbiota is unknown. To address this, day-old chicks were assigned into six groups: NC (basal diet), PC (virginiamycin), low (BPL) and high-dose (BPH) of Bacillus pumilus, and low (BSL) and high-dose (BSH) of Bacillus subtilis. Cecal contents at days 7, 14, 28 and 42 were used to analyze the treatment and time effects on the diversity and composition of microbiota. Overall, the alpha diversity was significantly decreased in the NC group between days 7 and 14, while this decline was prevented in the Bacillus subtilis probiotic (BSL and BSH) and even reversed in the BPH group. The beta-diversity showed significant responses of microbial communities to probiotics in first two weeks of life. Analyses of the abundance of microbiota reflected that members of the family Ruminococcaceae (Ruminnococcus, Oscillospira, Faecalibacterium, Butyricicoccus, and Subdoligranulum), which were dominant in mature microbiota, were significantly higher in abundance at day 14 in the probiotic groups. Conversely, the abundance of genera within the family Lachnospiraceae (Ruminococcus, Blautia, and Coprococcus) was dominant in early dynamic microbiota but was significantly lower in the probiotic groups at day 14. The Lactobacillus and Bifidobacterium abundance was higher, while the Enterobacteriaceae abundance was lower in the probiotic groups. In summary, the probiotics efficiently helped the cecal microbiota reach mature configuration earlier in life. These results could be used for the future manipulation of microbiota from the perspective of improving poultry performance.

Analysis of viromes and microbiomes from pig fecal samples reveals that phages and prophages rarely carry antibiotic resistance genes.

Understanding the transmission of antibiotic resistance genes (ARGs) is critical for human health. For this, it is necessary to identify which type of mobile genetic elements is able to spread them from animal reservoirs into human pathogens. Previous research suggests that in pig feces, ARGs may be encoded by bacteriophages. However, convincing proof for phage-encoded ARGs in pig viromes is still lacking, because of bacterial DNA contaminating issues. We collected 14 pig fecal samples and performed deep sequencing on both highly purified viral fractions and total microbiota, in order to investigate phage and prophage-encoded ARGs. We show that ARGs are absent from the genomes of active, virion-forming phages (below 0.02% of viral contigs from viromes), but present in three prophages, representing 0.02% of the viral contigs identified in the microbial dataset. However, the corresponding phages were not detected in the viromes, and their genetic maps suggest they might be defective. We conclude that among pig fecal samples, phages and prophages rarely carry ARG. Furthermore, our dataset allows for the first time a comprehensive view of the interplay between prophages and viral particles, and uncovers two large clades, inoviruses and Oengus-like phages.

Early Inoculation of Microbial Suspension in Suckling Piglets Affects the Transmission of Maternal Microbiota and the Associated Antibiotic Resistance Genes.

Antibiotic resistance of microbes thriving in the animal gut is a growing concern for public health as it may serve as a hidden reservoir for antibiotic resistance genes (ARGs). We compared 16 control piglets to 24 piglets fed for 3 weeks with S1 or S2 fecal suspensions from two sows that were not exposed to antibiotics for at least 6 months: the first suspension decreased the erythromycin resistance gene ermB and the aminoglycoside phosphotransferase gene conferring resistance to kanamycine (aphA3), while the second decreased the tetracycline resistance gene tetL, with an unexpected increase in ARGs. Using 16S RNA sequencing, we identified microbial species that are likely to carry ARGs, such as the lincosamide nucleotidyltransferase lnuB, the cephalosporinase cepA, and the tetracycline resistance genes tetG and tetM, as well as microbes that never co-exist with the tetracycline resistance gene tetQ, the erythromycin resistance gene ermG and aphA3. Since 73% of the microbes detected in the sows were not detected in the piglets at weaning, a neutral model was applied to estimate whether a microbial species is more important than chance would predict. This model confirmed that force-feeding modifies the dynamics of gut colonization. In conclusion, early inoculation of gut microbes is an interesting possibility to stimulate gut microbiota towards a desirable state in pig production, but more work is needed to be able to predict which communities should be used.

1H-NMR metabolomics response to a realistic diet contamination with the mycotoxin deoxynivalenol: Effect of probiotics supplementation.

Low-level contamination of food and feed by deoxynivalenol (DON) is unavoidable. We investigated the effects of subclinical treatment with DON, and supplementation with probiotic yeast Saccharomyces cerevisiae boulardii I1079 as a preventive strategy in piglets. Thirty-six animals were randomly assigned to either a control diet, a diet contaminated with DON (3 mg/kg), a diet supplemented with yeast (4 × 109 CFU/kg), or a DON-contaminated diet supplemented with yeast, for four weeks. Plasma and tissue samples were collected for biochemical analysis,1H-NMR untargeted metabolomics, and histology. DON induced no significant modifications in biochemical parameters. However, lesion scores were higher and metabolomics highlighted alterations of amino acid and 2-oxocarboxylic acid metabolism. Administering yeast affected aminoacyl-tRNA synthesis and amino acid and glycerophospholipid metabolism. Yeast supplementation of piglets exposed to DON prevented histological alterations, and partial least square discriminant analysis emphasised similarity between the metabolic profiles of their plasma and that of the control group. The effect on liver metabolome remained marginal, indicating that the toxicity of the mycotoxin was not eliminated. These findings show that the 1H-NMR metabolomics profile is a reliable biomarker to assess subclinical exposure to DON, and that supplementation with S. cerevisiae boulardii increases the resilience of piglets to this mycotoxin.

Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike-in standard.

Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

Variability of the Ability of Complex Microbial Communities to Exclude Microbes Carrying Antibiotic Resistance Genes in Rabbits.

Reducing antibiotic use is a necessary step toward less antibiotic resistance in livestock, but many antibiotic resistance genes can persist for years, even in an antibiotic-free environment. In this study, we investigated the potential of three fecal complex microbial communities from antibiotic-naive does to drive the microbiota of kits from antibiotic-exposed dams and outcompete bacteria-carrying antibiotic-resistant genes. The fecal complex microbial communities were either orally delivered or simply added as fresh fecal pellets in four to five nests that were kept clean from maternal feces. Additionally, four nests were cleaned for the maternal feces and five nests were handled according to the common farm practice (i.e., cleaning once a week) as controls. At weaning, we measured the relative abundance of 26 antibiotic resistance genes, the proportion of Enterobacteriaceae resistant to tetracycline and sulfonamide antibiotics, and the taxonomic composition of the microbiota by sequencing the 16S rRNA genes of one kit per nest. Changing the surrounding microbes of the kits can hinder the transmission of antibiotic resistance genes from one generation to the next, but the three communities widely differed in their ability to orient gut microbes and in their impact on antibiotic resistance genes. The most efficient delivery of the microbial community reduced the proportion of resistant Enterobacteria from 93 to 9%, decreased the relative abundance of eight antibiotic resistance genes, and changed the gut microbes of the kits at weaning. The least efficient did not reduce any ARG or modify the bacterial community. In addition, adding fecal pellets was more efficient than the oral inoculation of the anaerobic suspension derived from these fecal pellets. However, we were unable to predict the outcome of the exclusion from the data of the donor does (species composition and abundance of antibiotic resistance genes). In conclusion, we revealed major differences between microbial communities regarding their ability to exclude antibiotic resistance genes, but more work is needed to understand the components leading to the successful exclusion of antibiotic resistance genes from the gut. As a consequence, studies about the impact of competitive exclusion should use several microbial communities in order to draw general conclusions.

Lipid-induced endoplasmic reticulum stress in liver cells results in two distinct outcomes: adaptation with enhanced insulin signaling or insulin resistance.

Chronically elevated fatty acids contribute to insulin resistance through poorly defined mechanisms. Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) have been implicated in lipid-induced insulin resistance. However, the UPR is also a fundamental mechanism required for cell adaptation and survival. We aimed to distinguish the adaptive and deleterious effects of lipid-induced ER stress on hepatic insulin action. Exposure of human hepatoma HepG2 cells or mouse primary hepatocytes to the saturated fatty acid palmitate enhanced ER stress in a dose-dependent manner. Strikingly, exposure of HepG2 cells to prolonged mild ER stress activation induced by low levels of thapsigargin, tunicamycin, or palmitate augmented insulin-stimulated Akt phosphorylation. This chronic mild ER stress subsequently attenuated the acute stress response to high-level palmitate challenge. In contrast, exposure of HepG2 cells or hepatocytes to severe ER stress induced by high levels of palmitate was associated with reduced insulin-stimulated Akt phosphorylation and glycogen synthesis, as well as increased expression of glucose-6-phosphatase. Attenuation of ER stress using chemical chaperones (trimethylamine N-oxide or tauroursodeoxycholic acid) partially protected against the lipid-induced changes in insulin signaling. These findings in liver cells suggest that mild ER stress associated with chronic low-level palmitate exposure induces an adaptive UPR that enhances insulin signaling and protects against the effects of high-level palmitate. However, in the absence of chronic adaptation, severe ER stress induced by high-level palmitate exposure induces deleterious UPR signaling that contributes to insulin resistance and metabolic dysregulation.

Normal spermatogenesis in a man with mutant luteinizing hormone.

Men with mutations in LHB, the gene encoding the beta subunit of luteinizing hormone (LHB), have azoospermia with absent or few fetal Leydig cells. We report a mutation in LHB in a man and his sister. The man presented with absence of virilization, undetectable luteinizing hormone, and a low serum testosterone level. He had complete spermatogenesis with a normal sperm count. The mutant luteinizing hormone had a low level of partial activity in vitro. We concluded that the residual luteinizing hormone activity, resulting in the expression of steroidogenic enzymes in few mature Leydig cells producing small amounts of intratesticular testosterone (20.2 ng per gram), was sufficient for complete and quantitatively normal spermatogenesis.

Oxytocin stimulates secretory processes in lactating rabbit mammary epithelial cells.

Oxytocin plays a major role in lactation mainly by its action on milk ejection via the contraction of myoepithelial cells. The effect of oxytocin on milk production and the presence of oxytocin receptors on different epithelial cells suggest that this hormone may play a role in mammary epithelial cells. To determine precisely the various roles of oxytocin, we studied localization of oxytocin receptors in lactating rabbit and rat mammary tissue and the influence of oxytocin on secretory processes in lactating rabbit mammary epithelial cells. Immunolocalization of oxytocin receptors on mammary epithelial cells by immunofluorescence and in mammary tissue by immunogold in addition to in situ hybridization showed that lactating rat and rabbit mammary epithelial cells expressed oxytocin receptors. Moreover, oxytocin bound specifically to epithelial cells. To determine whether oxytocin had an effect on lactating rabbit mammary epithelial cells, isolated mammary fragments were incubated in the presence or absence of 10(-6) i.u. ml(-1) of oxytocin. After 1 min of incubation with oxytocin, the morphology of epithelial cells and the localization of caseins and proteins associated with the secretory traffic suggested a striking acceleration of the transport leading to exocytosis, whereas the contraction of myoepithelial cells was only detectable after 7 min. Addition of 10(-8) g ml(-1) of atosiban before the addition of oxytocin prevented the oxytocin effect on secretory processes and on myoepithelial cell contraction. Addition of 10(-6) i.u. ml(-1) of vasopressin to the incubation medium did not mimic the stimulating effect of oxytocin on secretory traffic. These results show that lactating rabbit and rat mammary epithelial cells express oxytocin receptors and that oxytocin binds to these receptors. They strongly suggest that oxytocin has a dual effect on lactating mammary tissue: an acceleration of the intracellular transfer of caseins in mammary epithelial cells followed by the contraction of myoepithelial cells.

Thyrotropin receptor trafficking relies on the hScrib-betaPIX-GIT1-ARF6 pathway.

G protein-coupled receptors are regulated by ligand stimulation, endocytosis, degradation of recycling to the cell surface. Little information is available on the molecular mechanisms underlying G protein-coupled receptors recycling. We have investigated recycling of the G protein-coupled thyroid stimulating hormone receptor (TSHR) and found that it relies on hScrib, a membrane-associated PDZ protein. hScrib directly binds to TSHR, inhibits basal receptor endocytosis and promotes recycling, and thus TSHR signalling, at the cell membrane. We previously demonstrated that hScrib is associated with a betaPIX-GIT1 complex comprised of a guanine nucleotide exchange factor and a GTPase-activating protein for ADP ribosylation factors that is involved in vesicle trafficking. We used dominant-negative constructs and small interfering RNA to show that TSHR recycling is regulated by the interaction between hScrib and betaPIX, and by the activity of GIT1. In addition, ARF6, a major target for GIT1, is activated during TSH stimulation of HEK293 and FRTL-5 thyroid cells, and plays a key role in TSHR recycling. Thus, we have uncovered an hScrib-betaPIX-GIT1-ARF6 pathway devoted to TSHR trafficking and function.