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. Milk is synthesized and secreted by the differentiated polarized mammary epithelial cells of the gland. Reconstructing in vitro a mammary-like structure mimicking the functional tissue represents a major challenge in mammary gland biology, especially for farm animals for which specific agronomic questions arise. This would greatly facilitate the study of mammary gland development, milk secretion processes and pathological effects of viral or bacterial infections at the cellular level, all with the objective of improving milk production at the animal level. With this aim, various 3D cell culture models have been developed such as mammospheres and, more recently, efforts to develop organoids in vitro have been considerable. Researchers are now starting to draw inspiration from other fields, such as bioengineering, to generate organoids that would be more physiologically relevant. In this chapter, we will discuss 3D cell culture systems as organoids and their relevance for agronomic research.

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 CD49f, CD24, and CD10. The pubertal period was characterized by a high proportion of CD49fpos cells corresponding to various epithelial subpopulations, notably the CD24pos subpopulations. The proportion of CD49fpos cells was weaker during lactation and dry-off, and CD24pos cells were relatively few. Of note, the (sub)population profile at dry-off appeared close to that during lactation. Using a targeted gene approach, we associated specific genes with epithelial subpopulations, their expression level varying, or not, according to physiological stages. Caseins were only expressed in the CD49fmedCD24neg subpopulation. Basal marker genes (keratin(KRT)5, KRT14 and αSMA) were found in the CD49fhighCD24neg subpopulations. Luminal gene markers (KRT7, KRT8 and KRT19, CDH1 and the PRLR) were expressed in the CD49flowCD24neg subpopulation. The CD49flowCD24pos subpopulation, only abundant at puberty, expressed luminal gene markers and KI67 at high level. In contrast to others, the CD49fhighCD24pos cells accounted for a small proportion of total cells, decreasing from puberty to dry-off. They were characterized by expression of luminal and basal gene markers and low KI67 level. Interestingly, this subpopulation showed a remarkable stability of gene expression profile throughout physiological stages and bear the hallmark of quiescence that designate them as the potential bovine mammary stem cells.

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. Here we show that the caseins are heat-stable, a feature that leads to their complete extraction from a complex cellular extract by boiling. This allows for high enrichment and direct analysis of the caseins, even when they are poorly expressed in the starting material.

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. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

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. Interestingly, the cLD population did not mimic the decline in perilipin-2. We tested several chemical modulators of regulators of Arf1 activity on the association of perilipin-2 with cLDs. QS11 and Exo2 accelerated the reduction in perilipin-2, although less than BFA. In contrast, Exo1 unexpectedly slowed down its degradation. Correlatively, BFA, QS11, and Exo2 enhanced the dissociation of perilipin-2 from cLDs, whereas Exo1 inhibited it. There was a synergistic effect of BFA with Exo2 and QS11, and of Exo2 with QS11, whereas Exo1 antagonized the effect of BFA without affecting that of Exo2 or QS11. We concluded that the Arf1 complex regulates the association of perilipin-2 with cLDs. Additionally, MG132 and BFA modified the number of cLDs over a relatively short period.

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 ß-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 ß-casein synthesis. The involvement of PRL and EGF in ADRP synthesis was explored by means of a battery of inhibitors. The Jak2 inhibitor AG490 provoked a stimulation of ADRP synthesis whereas it totally suppressed that of ß-casein. The use of AG1478, a specific inhibitor of EGF receptor phosphorylation, or of PD98059, a specific MEK inhibitor, revealed that the Ras/Raf/MEK/ERK1/2 pathway has no significant influence on ADRP levels. Inhibition of JNK was also ineffective. In contrast, incubation of the cells with SB 203580, a specific inhibitor of p38, slightly stimulated ADRP synthesis and induced a proportional dose-response inhibition of PRL-induced ß-casein synthesis. Finally, cell treatment with wortmannin or LY294002 revealed that both PRL and EGF positively regulate ADRP and ß-casein synthesis through PI3-kinase signaling. Because both the Akt inhibitor MK-2206 and the mTOR inhibitor rapamycin provoked a strong diminution of PRL-induced synthesis of the two proteins, and because oleate induced phosphorylation of Akt, we concluded that, in the mammary epithelial cell line HC11, the PI3-kinase/Akt/mTOR signaling pathway strongly participates in ß-casein synthesis and is a main regulator of ADRP expression.

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. The majority of both alphaS1- and beta-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature beta-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature alphaS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of alphaS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of alphaS1-casein with membranes. CONCLUSIONS: These experiments reveal for the first time the existence of a membrane-associated form of alphaS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that alphaS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway.

Oleate and linoleate stimulate degradation of ß-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. Unexpectedly, the cellular level of beta-casein, accumulated under lactogenic hormone treatment, decreases following treatment of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic lipid droplet proliferation or cellular beta-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of beta-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate beta-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls beta-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of beta-casein. Our data thus suggest that the degradation of beta-casein occurs via the microautophagic pathway.

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. 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.

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. 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.

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. 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.

Ca(2+)-independent phospholipase A2 participates in the vesicular transport of milk proteins.

Changes in the lipid composition of intracellular membranes are believed to take part in the molecular processes that sustain traffic between organelles of the endocytic and exocytic transport pathways. Here, we investigated the participation of the calcium-independent phospholipase A2 in the secretory pathway of mammary epithelial cells. Treatment with bromoenol lactone, a suicide substrate which interferes with the production of lysophospholipids by the calcium-independent phospholipase A2, resulted in the reduction of milk proteins secretion. The inhibitor slowed down transport of the caseins from the endoplasmic reticulum to the Golgi apparatus and affected the distribution of p58 and p23, indicating that the optimal process of transport of these proteins between the endoplasmic reticulum, the endoplasmic reticulum/Golgi intermediate compartment and/or the cis-side of the Golgi was dependent upon the production of lysolipids. Moreover, bromoenol lactone was found to delay the rate of protein transport from the trans-Golgi network to the plasma membrane. Concomitantly, membrane-bound structures containing casein accumulated in the juxtanuclear Golgi region. We concluded from these results that efficient formation of post-Golgi carriers also requires the phospholipase activity. These data further support the participation of calcium-independent phospholipase A2 in membrane trafficking and shed a new light on the tubulo/vesicular transport of milk protein through the secretory pathway.

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). 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.

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. In the present paper, we describe three overlapping bacterial artificial chromosome (BAC) clones which harbor both the rabbit CSN1 and CSN2 genes. These two genes are in a convergent orientation, separated by an intergenic region of 15 kb. DNA from one of the CSN/BAC clones was used as a probe for in situ hybridization to show that the CSN1 and CSN2 gene cluster is located on chromosome 15 band q23 and not on chromosome 12 as had been previously reported. Each of the three CSN/BAC DNAs was transfected into HC11 cells. In the presence of lactogenic hormones, the rabbit CSN1 gene was clearly expressed from all three CSN/BAC DNAs, whereas the rabbit CSN2 gene, which at the most possesses a 1 kb upstream region in one of the CSN/BAC DNAs, was not expressed at detectable levels on Northern blots. The transfected HC11 cells now express both rabbit CSN1 and mouse CSN2 genes. These transfected cells will be used as a model to study the role of CSN1 in milk protein secretion.

[The double-play of PP17/TIP47]. / Le double-jeu de la protéine 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. The sequence of PP17b/TIP47 displays not only a strong homology with those of adipophilin and the perilipins, two proteins known to be involved in the intracellular traffic of lipid droplets but also PP17b/TIP47 is associated with the later. How this ubiquitous protein could participate in processes as different as the mannose 6-phosphate receptors traffic and the formation and/or traffic of lipid droplets? A tentative hypothesis is put forward.

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. 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.

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. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling. The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.

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. These sulphated molecules were partially purified and identified to isoforms of rat alpha-lactalbumin for the 25-27 kDa bands and to kappa-casein for the 32 kDa band. This pattern of protein sulphation is, as far as we know, quite specific to rat mammary epithelial cells.

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. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.

[Nuclear organization and expression of milk protein genes]. / Organisation nucléaire et expression des gènes des protéines du lait.

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. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products.