Sorted stem/progenitor epithelial cells of pubertal bovine mammary gland present limited potential to reconstitute an organised mammary epithelium after transplantation.

The development and maintenance of mammary gland tissue depend on the proliferation and differentiation of mammary stem and progenitor cells. Here, we investigated populations of mammary epithelial cells that are potential candidates for bovine mammary gland development using xenotransplantation into mice cleared mammary fat pad. Transplanted mammary explants from 17-month-old Holstein heifers developed outgrowths exhibiting the archetypal morphology and molecular marker distributions of the bovine gland.

Mammary gland 3D cell culture systems in farm animals.

In vivo study of tissue or organ biology in mammals is very complex and progress is slowed by poor accessibility of samples and ethical concerns. Fortunately, however, advances in stem cell identification and culture have made it possible to derive in vitro 3D "tissues" called organoids, these three-dimensional structures partly or fully mimicking the in vivo functioning of organs. The mammary gland produces milk, the source of nutrition for newborn mammals.

Genomic DNA PCR analysis to assess xenograft development in mouse mammary gland.

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.

Review: the cellular mechanisms underlying mammary tissue plasticity during lactation in ruminants.

The mammary tissue is characterized by its capacity to adapt in response to a wide variety of changing conditions. This adaptation capacity is referred to as the plasticity of mammary tissue. In dairy ruminants, lactation is challenged by modifications that can either be induced on purpose, such as by modifying management practices, or occur involuntarily, when adverse environmental constraints arise.

Mammary Epithelial Cell Lineage Changes During Cow's Life.

Milk production is highly dependent on the optimal development of the mammary epithelium. It is therefore essential to better understand mammary epithelial cell growth and maintenance from the related epithelial lineage during the animal life. Here, we characterized the epithelial lineage at puberty, lactation and dry-off in bovine using the cell surface markers CD49, CD24, and CD10.

Molecular signature of the putative stem/progenitor cells committed to the development of the bovine mammary gland at puberty.

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.

Technical note: Quantification of caseins from a crude extract of mammary epithelial cells.

En masse secretion of milk proteins, notably the caseins in the form of casein micelles, is a unique feature of the milk-secreting mammary epithelial cell. Caseins are therefore specific markers of these cells and constitute an ideal tool to monitor their differentiation, as well as functional status, during the development of the gland. To use them as such, a reliable method for quantitative analysis of the caseins from mammary cells or tissue is needed.

Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

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).

Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus.

The perilipin-2 (adipophilin) coat of cytosolic lipid droplets is regulated by an Arf1-dependent mechanism in HC11 mouse mammary epithelial cells.

The cytosolic lipid droplets (cLDs) store excess intracellular lipids, and perilipin-2 is believed to protect cLDs from degradation. Here, we investigated the role of the small G-protein Arf1 and the proteasome in the fates of perilipin-2 and cLDs. In oleate-loaded cells, upon brefeldin A (BFA) treatment, perilipin-2 remained associated with cLDs for at least 30 min before significant release, and proteasomal degradation-mediated decrease was observed.

The membrane-associated form of α(s1)-casein interacts with cholesterol-rich detergent-resistant microdomains.

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.

Prolactin and epidermal growth factor stimulate adipophilin synthesis in HC11 mouse mammary epithelial cells via the PI3-kinase/Akt/mTOR pathway.

The aim of the present study is to estimate the role played by cortisol, prolactin (PRL) and epidermal growth factor (EGF) in the synthesis of adipocyte differentiation-related protein (ADRP) as compared to the well-studied regulation of β-casein synthesis by these hormones in the mammary epithelial cell line HC11. This comparison between a cytoplasmic lipid droplet-associated protein, which is strictly specific to both lipid accumulation and secretion by lactating mammary epithelial cells, and an archetypal milk protein is useful for evaluating the extent to which a mechanistic relationship exists between biosynthesis, transport and secretion of these two major milk components. We found that cortisol inhibits PRL-stimulated ADRP synthesis, as opposed to its known stimulating effect on β-casein synthesis.

Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells.

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered.

AlphaS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form.

Background: Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known.

Results: In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues.

Oleate and linoleate stimulate degradation of β-casein in prolactin-treated HC11 mouse mammary epithelial cells.

Although virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein.

R-spondin1 is required for normal epithelial morphogenesis during mammary gland development.

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.

Lipid-deprived diet perturbs O-glycosylation of secretory proteins in rat mammary epithelial cells.

Nutrition modulates both production and composition of milk. Milk composition was studied in rats chronically fed a diet without additional lipids, and therefore eating only traces of the recommended supply of essential polyunsaturated fatty acid. Despite a large decrease in milk-protein synthesis, only protein composition, but not protein concentration, was found to change in the milk of rats following a lipid-deprived diet.

[Nuclear organization and expression of milk protein genes].

Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied.

Sulphated proteins secreted by rat mammary epithelial cells.

The main sulphated proteins secreted by rat mammary gland tissue have Mr of approximately 32 000, 27 000 and 25 000 Da. In addition, there are high Mr components which have a diffuse electrophoretic mobility (Mr > 200 000) and most likely corresponded to proteoglycans. The sulphate groups in the proteins with discrete Mr are most likely all linked to carbohydrates.

Disulphide bonds in casein micelle from milk.

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants.

Interactions between the rabbit CSN1 gene and the nuclear matrix of stably transfected HC11 mammary epithelial cells vary with its level of expression.

The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated.

[The double-play of PP17/TIP47].

A missing link in the understanding of the mechanisms of transport of the mannose 6-phosphate receptors has recently been discovered, following the identification of the protein TIP47. In association with Rab9-GTP, this protein is responsible for the return of the receptors from the late endosomes back to the trans-Golgi network. Curiously, the same protein called PP17b, was described as a placental protein twenty years ago, and more recently, as a blood marker for human uterine cervical cancer.

Structure of the rabbit alphas1- and beta-casein gene cluster, assignment to chromosome 15 and expression of the alphas1-casein gene in HC11 cells.

Several casein (CSN) genes (CSN1, 2, 10 and alphas2-CSN) have been described and shown to be clustered in mouse, man and cattle. These genes are expressed simultaneously in the mammary gland during lactation, but they are silent in most mammary cell lines, even in the presence of lactogenic hormones. However, it has been shown that the CSN2 gene, and this gene only, can be induced in certain mammary cell lines, such as HC11.

Phospholipase D-dependent and -independent mechanisms are involved in milk protein secretion in rabbit mammary epithelial cells.

Phospholipase D has been implicated in membrane traffic in the secretory pathway of yeast and of some mammalian cell lines. Here we investigated the involvement of phospholipase D in protein transport at various steps of the secretory pathway of mammary epithelial cells. Treatment of rabbit mammary explants with butanol, which blocks the formation of phosphatidic acid, decreased the secretion of caseins and to a lesser extent that of whey acidic protein.