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MicroRNAs are small noncoding RNAs that have important roles in the lactation process and milk biosynthesis. Some polymorphisms have been studied in various livestock species from the perspective of pathology or production traits. To target variants that could be the causal variants of dairy traits, genetic variants of microRNAs expressed in the mammary gland or present in milk and localized in dairy quantitative trait loci (QTLs) were investigated in bovine, caprine, and ovine species. In this study, a total of 59,124 (out of 28 millions), 13,427 (out of 87 millions), and 4761 (out of 38 millions) genetic variants in microRNAs expressed in the mammary gland or present in milk were identified in bovine, caprine, and ovine species, respectively. A total of 4679 of these detected bovine genetic variants are located in dairy QTLs. In caprine species, 127 genetic variants are localized in dairy QTLs. In ovine species, no genetic variant was identified in dairy QTLs. This study leads to the detection of microRNA genetic variants of interest in the context of dairy production, taking advantage of whole genome data to identify microRNA genetic variants expressed in the mammary gland and localized in dairy QTLs.

International audience; The mammary gland undergoes important anatomical and physiological changes from embryogenesis through puberty, pregnancy, lactation and involution. These steps are under the control of a complex network of molecular factors, in which epigenetic mechanisms play a role that is increasingly well described. Recently, studies investigating epigenetic modifications and their impacts on gene expression in the mammary gland have been performed at different physiological stages and in different mammary cell types. This has led to the establishment of a role for epigenetic marks in milk component biosynthesis. This review aims to summarize the available knowledge regarding the involvement of the four main molecular mechanisms in epigenetics: DNA methylation, histone modifications, polycomb protein activity and non-coding RNA functions.

The mouse transplantation model remains the most relevant methodology to assess the functional capacities of mammary cells and is particularly appropriate for investigations regarding mammary stem cells, whatever the species studied. Following xenotransplantation in mice mammary fat pad, the development of the xenograft is commonly evaluated by immunohistology. Here, we present a simple and rapid method to control the species specificity of a xenograft based on genomic DNA PCR amplification. DNA is extracted from the fixed samples intended for histology, thus allowing the reuse of precious samples. Standard and digital droplet PCR (requiring low DNA quantities) methods have been used to make the present method suitable for the analysis of xenotransplanted samples.

Background Nutritional changes can affect future lactation efficiency. In a rabbit model, an obesogenic diet initiated before puberty and pursued throughout pregnancy enhances mammary differentiation, but when started during the neonatal period can cause abnormal mammary development in early pregnancy. The aim of this study was to investigate the impact of an unbalanced diet administered during the pubertal period only. Results Consuming an obesogenic diet at puberty did not affect either metabolic parameters or certain maternal reproductive parameters at the onset of adulthood. In contrast, at Day 8 of pregnancy, epithelial tissue showed a lower proliferation rate in obesogenic‐diet fed rabbits than in control‐diet fed rabbits. Wap and Cx26 genes, mammary epithelial cell differentiation markers, were upregulated although Wap protein level remained unchanged. However, the expression of genes involved in lipid metabolism and in alveolar formation was not modified. Conclusion Taken together, our results demonstrate that the consumption for 5 weeks of an obesogenic diet during the pubertal period initiates mammary structure modifications and affects mammary epithelial cell proliferation and differentiation. Our findings highlight the potentially important role played by unbalanced nutrition during critical early‐life windows in terms of regulating mammary epithelial cell differentiation and subsequent function in adulthood., Key Findings Our results demonstrate that the consumption for five weeks of an obesogenic diet during the pubertal period initiates mammary structure modifications and affects mammary epithelial cell proliferation and differentiation. Our findings highlight the potentially important role played by unbalanced nutrition during critical early‐life windows in terms of regulating mammary epithelial cell differentiation and subsequent function in adulthood.

Recent reports have shown that ingested microRNAs may be transferred to blood, accumulate in tissues and exert canonical regulation on endogenous transcripts. In spite of several attempts to replicate these findings, they have not been confirmed and several questions remain. By using a transgenic mouse model presenting a high level of miR-30b in milk, the horizontal delivery of this microRNA via oral ingestion was studied in pups. Our findings demonstrated that, although very high levels of miR-30b were found in milk and in stomach contents of the pups, we did not detect an increase in miR-30b in tissues of pups fed by transgenic females compared to pups fed by wild-type females.

Mammary gland is the organ of milk component synthesis that provides the nutrients for growth and development of the mammalian neonate. In addition to macronutrients like proteins, carbohydrates, and lipids known for their roles in providing substrate and energy, a new class of components has been identified notably microRNA that have signaling roles regulating a large set of biological processes. MicroRNAs, short noncoding RNAs, have been reported to act on the mammary tissues, influencing mammary development and milk component biosynthesis, and evidence is now assembling that they also signal to the infant. The expression profile of these miRNAs can be under nutritional regulation. Their presence in milk and their relative persistency through industrial treatment open new way of investigations to use them as biomarkers of animal health, as well as to evaluate their effects on the health of those consuming them. Due to the role of miRNAs on human health and diseases, their transfer from milk or milk products to infants and adults is being actively researched, though their bioavailability is not known. Research is defining their distribution in the different fractions of milk (such as cells, exosomes, fat globule, or skim milk). Indeed, the unique packaging of miRNAs could be crucial for their action through the intestinal tract. The value of milk miRNAs to diverse aspects of human health is now an emerging field of science

Preeclampsia (PE) is a common human-specific pregnancy disorder defined by hypertension and proteinuria during gestation and responsible for maternal and fetal morbimortality. STOX1 , encoding a transcription factor, was the first gene associated with PE as identified by positional cloning approaches. Its overexpression in choriocarcinoma cells mimics the transcriptional consequences of PE in the human placenta. Here, we created transgenic mouse strains overexpressing human STOX1 . Wild-type female mice crossed with transgenic male mice reproduce accurately the symptoms of severe PE: gestational hypertension, proteinuria, and elevated plasma levels of soluble fms-like tyrosine kinase 1 and soluble endoglin. Placental and kidney histology were altered. Symptoms were prevented or alleviated by aspirin treatment. STOX1-overexpressing mice constitute a unique model for studying PE, allow testing therapeutic approaches, and assessing the long-term effects of the preeclamptic syndrome.

Ajouter clé UT WOS; International audience; R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1's partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-β signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function.

BackgroundNutrition affects milk composition thus influencing its nutritional properties. Nutrition also modifies the expression of mammary genes, whose regulation is not fully understood. MicroRNAs (miRNA) are small non coding RNA which are important post-transcriptional regulators of gene expression by targeting messenger RNAs. Our goal was to characterize miRNA whose expression is regulated by nutrition in the lactating goat mammary gland, which may provide clues to deciphering regulations of the biosynthesis and secretion of milk components.Methodology/principal findingsUsing high-throughput sequencing technology, miRNomes of the lactating mammary gland were established from lactating goats fed ad libitum or deprived of food for 48 h affecting milk production and composition. High throughput miRNA sequencing revealed 30 miRNA with an expression potentially modulated by food deprivation; 16 were down-regulated and 14 were up-regulated. Diana-microT predictive tools suggested a potential role for several nutriregulated miRNA in lipid metabolism. Among the putative targets, 19 were previously identified as differently expressed genes (DEG). The functions of these 19 DEG revealed, notably, their involvement in tissue remodelling.Conclusion/significanceIn conclusion, this study offers the first evidence of nutriregulated miRNA in the ruminant mammary gland. Characterization of these 30 miRNA could contribute to a clearer understanding of gene regulation in the mammary gland in response to nutrition.

Summary: The aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt) are transcription factors that play a role in the detection of and adaptation to environmental signals. AhR-null mice are viable but show impaired lactation. Deletion of the Arnt gene from the mouse genome results in embryonic lethality. To determine the role of Arnt in mammary development and function, we inactivated the Arnt gene in mammary epithelium using Cre-loxP recombination. Inactivation of the Arnt gene during pregnancy did not disrupt alveolar development or the ability of dams to nurse their litters. In contrast, dams in which the Arnt gene had been inactivated during puberty and in ovaries were subfertile, exhibited retarded mammary development, and impaired mammary function. To distinguish defects autonomous to mammary epithelium from indirect effects controlled by ovarian hormones, we transplanted Arnt-null and AhR-null mammary epithelium into wild-type mice and evaluated development after one pregnancy. Normal mammary structures were observed in the absence of Arnt and AhR, demonstrating that neither transcription factor is necessary for mammary development. genesis 32:231–239, 2002. © 2002 Wiley-Liss, Inc.

Treatment of advanced cutaneous melanoma is still challenging and new data on melanoma biology are required. MicroRNAs (miRNAs) represent an emerging field in melanoma understanding. These small regulators of gene expression, involved in melanoma development and progression (Da cruz et al., 2012), showed promising results as biomarkers (Nair et al., 2012). As each miRNA could regulate multiple protein-coding genes and conversely, one gene could be targeted by different miRNAs (Bartel et al., 2009), the dysregulation of miRNAs could significantly impact various pathways and cells behavior.

The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various Prnp transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the Prnp-knockout-Sprn-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various Prnp and Sprn genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in Eutherians and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.

Chantier qualité GA; The biological function of the Prion protein remains largely unknown but recent data revealed its implication in early zebrafish and mammalian embryogenesis. To gain further insight into its biological function, comparative transcriptomic analysis between FVB/N and FVB/N Prnp knockout mice was performed at early embryonic stages. RNAseq analysis revealed the differential expression of 73 and 263 genes at E6.5 and E7.5, respectively. The related metabolic pathways identified in this analysis partially overlap with those described in PrP1 and PrP2 knockdown zebrafish embryos and prion-infected mammalian brains and emphasize a potentially important role for the PrP family genes in early developmental processes.

Background MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Until now their implication in the mammary gland biology was suggested by few studies mainly focusing on pathological situations allowing the characterization of miRNAs as markers of breast cancer tumour classes. If in the normal mammary gland, the expression of known miRNAs has been studied in human and mice but the full repertoire of miRNAs expressed in this tissue is not yet available. Results To extend the repertoire of mouse mammary gland expressed miRNAs, we have constructed several libraries of small miRNAs allowing the cloning of 455 sequences. After bioinformatics' analysis, 3 known miRNA (present in miRbase) and 33 new miRNAs were identified. Expression of 24 out of the 33 has been confirmed by RT-PCR. Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them. No correlation could be established between their expression pattern and evolutionary conservation. Six of them appear to be mouse specific. In several cases, multiple potential precursors of miRNA were present in the genome and we have developed a strategy to determine which of them was able to mature the miRNA. Conclusion The cloning approach has allowed improving the repertoire of miRNAs in the mammary gland, an evolutionary recent organ. This tissue is a good candidate to find tissue-specific miRNAs and to detect miRNA specific to mammals. We provide evidence for 24 new miRNA. If none of them is mammary gland specific, a few of them are not ubiquitously expressed. For the first time 6 mouse specific miRNA have been identified.

The R-spondin (Rspo) proteins constitute a novel class of ligands that induce Wnt signalling. Rspo1 knockout XX mice were previously shown to be sex-reversed, but some remain sub-fertile. These last were unable to feed their pups for some unknown reason. Using these mice and transplanted mammary tissues from Rspo1−/− virgin mice in nude mice, we report that the lack of Rspo1 expression results in the absence of duct side-branching development and subsequent alveolar formation, explaining the above mentioned phenotype. Our data demonstrate that local epithelial Rspo1 signalling is required for normal development of the mammary gland.

Chantier qualité GA; International audience; Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.

International audience; MicroRNA (miRNA) are small non-coding RNA that post-transcriptionally regulate gene expression. In humans, miRNA genes may account for 2 to 3% of the total number of genes. Although the biological functions of most miRNA are unknown, their importance for development, cell proliferation, cell death, and morphogenesis has been demonstrated in several species. One could thus speculate that miRNA should be involved in the regulation of one of the organs that can undergo cycles of cell division, differentiation and dedifferentiation in the adult, the mammary gland. In this paper we summarise several reports dealing with the potential implication of miRNA in the mammary gland, most of them focussed on pathological situations, such as the appearance of breast cancer. These data suggest an implication of miRNA on mammary gland biology. However, direct evidence of this is still lacking. Expression profile analysis of miRNA during the normal mammary gland development could help in addressing this question and in identifying miRNA potentially involved. To this aim, we undertook such an analysis on mouse mammary gland at different stages (virgin, pregnancy, lactation and involution) and will present our preliminary results.

Growth and function of the mammary gland is regulated by cytokines and modulated by suppressor of cytokine signalling (SOCS) proteins. In vitro experiments demonstrated that SOCS3 can inhibit PRL induction of milk protein gene expression and STAT5 activation. We explored the SOCS3 expression pattern during mouse mammary development and its regulation by PRL and GH in wild-type and STAT5a-null mammary tissue. Our results suggest that, in vivo, PRL stimulates SOCS3 expression in stromal adipocytes, independently of STAT5a stimulation. In mammary epithelial cells, SOCS3 expression appears to be related to STAT3 activation. Together, our results are consistent with a role of SOCS3 in the mammary gland by promoting apoptosis of differentiated cells (adipocytes during gestation and epithelial cells during involution).

International audience; Milk fat has a large effect on nutritional, technological and sensorial properties of milk products. The milk fat content and composition are modulated by genetics and nutritional factors and imply a large number of enzymes. The regulation of their gene expression in the mammary gland still needs to be clarified. An association between the extensive polymorphism at the $\alpha_{s1}$-casein ($\alpha_{s1}$-Cas) locus and both the lipid content and the characteristics of this fraction in caprine milk has been demonstrated. In order to decipher the mechanism responsible for this impact, a quantification of the transcripts of four lipogenic key enzymes (acetyl-CoA carboxylase, fatty acid synthase, lipoprotein lipase and stearoyl-CoA desaturase)was performed using real-time RT-PCR, suggesting an absence of association between the $\alpha_{s1}$-Cas genotype and expression variability of the studied genes. This approach has been completed by a more global analysis using a first generation of ruminant macroarray gathering 400 gene probes. The comparison of the expression profiles of lactating goat $\alpha_{s1}$-Cas A/A (strong allele) and F/F (defective allele) mammary gland allowed to confirm the expected variability in the expression of known genes (such as those encoding the $\alpha_{s1}$-casein) in ruminant mammary tissues as well as to identify up- and down-regulated genes. A second generation of ruminant cDNA macroarray extended to a few thousands of genes is currently in progress and will be applied to study different factors such as the nutritional regulation of gene expression in themammary gland.

A macroarray approach used to list genes differentially expressed in goat mammary gland (gestation vs. lactation), other than milk protein genes, allowed us to detect the Glycosylation-dependent Cell Adhesion Molecule 1 (GLYCAM1) gene. GLYCAM1, a member of the glycoprotein mucin family, is a component of the milk fat globule membrane (MFGM). Its complete cDNA and gene sequences were determined and it was mapped by fluorescent in situ hybridization (FISH) on goat and cattle chromosome 5 (CHI5q21 and BTA5q21), and on sheep chromosome 3 (OAR3q21). Northern blot analyses confirmed its differential expression during the development and differentiation of the mammary gland of ruminants with a significantly higher mRNA amount during lactation than during pregnancy. An experimental in vivo induction model for lactation, developed by Kann et al., showed that the expression of GLYCAM1 is hormonally regulated in the mammary gland of ewes. Interspecies comparison of the gene promoter revealed the evolutionary conservation of a short proximal nucleotide sequence encompassing several transcription factor binding sites that could mediate the above-mentioned hormonal regulation.

Mammary anlagen are formed in the embryo as a derivative of the epidermis, a process that is controlled by Lef-1 and therefore possibly by β-catenin. To investigate the role of β-catenin signaling in mammary alveolar epithelium, we have stabilized endogenous β-catenin in differentiating alveolar epithelium through the deletion of exon 3 (amino acids 5–80) of the β-catenin gene. This task was accomplished in mice carrying a floxed β-catenin gene and a Cre transgene under control of the mammary-specific whey acidic protein (WAP) gene promoter or the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Stabilized β-catenin was obtained during the first pregnancy, and its presence resulted in the dedifferentiation of alveolar epithelium followed by a transdifferentiation into epidermal and pilar structures. Extensive squamous metaplasia, but no adenocarcinomas, developed upon β-catenin activation during pregnancy and persisted throughout involution. These data demonstrate that the activation of β-catenin signaling induces a program that results in loss of mammary epithelial cell differentiation and induction of epidermal structures.

It has previously been suggested that the mammary cell could produce prolactin (PRL). This hypothesis was investigated by incubation with [35S]methionine-cysteine followed by SDS-PAGE, immunoblotting and autoradiography of immunoprecipitated PRL, and by electron microscopic analysis after incubation without or with cycloheximide. Immunoreactive 14-, 23-, 25-, 32- and 36-kDa PRL forms were radioactive. By two-dimensional electrophoresis analysis, immunoreactive and radioactive spots, of about 25 kDa and high molecular weight, were also detected. After incubation of mammary epithelial cells with cycloheximide, immunogold electron microscopy showed a drastic decrease of labelling in organelles involved in synthesis and secretion, compared to those incubated in control medium. These results make it possible to conclude that lactating mammary tissue is able to synthesize PRL.

Chantier qualité GA; Background: MicroRNA (miRNA) are negative regulators of gene expression, capable of exerting pronounced influences upon the translation and stability of mRNA. They are potential regulators of normal mammary gland development and of the maintenance of mammary epithelial progenitor cells. This study was undertaken to determine the role of miR-30b on the establishment of a functional mouse mammary gland. miR-30b is a member of the miR-30 family, composed of 6 miRNA that are highly conserved in vertebrates. It has been suggested to play a role in the differentiation of several cell types.[br/] Methodology/Principal Findings: The expression of miR-30b was found to be regulated during mammary gland development. Transgenic mice overexpressing miR-30b in mammary epithelial cells were used to investigate its role. During lactation, mammary histological analysis of the transgenic mice showed a reduction in the size of alveolar lumen, a defect of the lipid droplets and a growth defect of pups fed by transgenic females. Moreover some mammary epithelial differentiated structures persisted during involution, suggesting a delay in the process. The genes whose expression was affected by the overexpression of miR-30b were characterized by microarray analysis.[br/] Conclusion/Significance: Our data suggests that miR-30b is important for the biology of the mammary gland and demonstrates that the deregulation of only one miRNA could affect lactation and involution.

International audience; Today, there is a shift towards a positional candidate approach in the molecular identification of genes. This study reports on an Expressed Sequence Tags (ESTs) mapping initiative in goats, based on sequence information gathered from a previous mammary gland cDNA systematic sequencing project. A total of 25 novel genes was localised cytogenetically on 16 goat chromosomes. Six of these ESTs were found to map to cattle milk QTL regions. These results made it possible to assess the use of ESTs as a shortcut to the molecular identification of some QTLs and as a valuable tool for comparative mapping.; Localisation cytogénétique de 25 ESTs caprins issus de glande mammaire. L'identification moléculaire des gènes s'oriente actuellement vers une stratégie de candidats positionnels. Cette étude, réalisée chez la chèvre, décrit la localisation cytogénétique d'étiquettes (ESTs) obtenues précédemment par séquençage d'ADNc de glande mammaire. Au total, 25 nouveaux gènes ont ainsi été localisés sur 16 chromosomes caprins. Six de ces gènes ont été cartographiés dans des régions de QTL laitiers bovins. Ces résultats ont permis d'évaluer les possibilités d'utilisation des ESTs, d'une part, pour l'identification moléculaire des QTL et d'autre part, comme outil de cartographie comparée.

Background The mammary gland is a dynamic organ that undergoes important physiological changes during reproductive cycles. Until now, data regarding the characterisation of miRNA in the mammary gland have been scarce and mainly focused on their abnormal expression in breast cancer. Our goal was to characterise the microRNA (miRNA) involved in mechanisms regulating the mammary function, with particular focus on the lactation stage. Methodology/principal findings Using high-throughput sequencing technology, the exhaustive repertoires of miRNA expressed (miRNome) in mouse and bovine mammary glands during established lactation were identified, characterized and compared. Furthermore, in order to obtain more information on miRNA loading in the RNA-induced silencing complex (RISC), the miRNome was compared with that obtained from RNA associated with the AGO2 protein (AGO2-miRNome) in mouse lactating mammary gland. This study enabled the identification of 164 and 167 miRNA in mouse and bovine, respectively. Among the 30 miRNA most highly expressed in each species, 24 were common to both species and six of them were preferentially highly expressed in lactating than non-lactating mammary gland. The potential functional roles of these 24 miRNA were deduced using DIANA-miRPath software, based on miRNA/mRNA interactions. Moreover, seven putative novel miRNA were identified. Using DAVID analysis, it was concluded that the predicted targets of two of these putative novel miRNA are involved in mammary gland morphogenesis. Conclusion/significance Our study provides an overview of the characteristics of lactating mouse and bovine mammary gland miRNA expression profiles. Moreover, species-conserved miRNA involved in this fundamental biological function were identified. These miRNomes will now be used as references for further studies during which the impact of animal breeding on the miRNA expression will be analysed.