Milk secretion: The role of SNARE proteins.

During lactation, polarized mammary epithelial secretory cells (MESCs) secrete huge quantities of the nutrient molecules that make up milk, i.e. proteins, fat globules and soluble components such as lactose and minerals. Some of these nutrients are only produced by the MESCs themselves, while others are to a great extent transferred from the blood. MESCs can thus be seen as a crossroads for both the uptake and the secretion with cross-talks between intracellular compartments that enable spatial and temporal coordination of the secretion of the milk constituents. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk components remain incompletely characterized. Major milk proteins, namely caseins, are secreted by exocytosis, while the milk fat globules are released by budding, being enwrapped by the apical plasma membrane. Prolactin, which stimulates the transcription of casein genes, also induces the production of arachidonic acid, leading to accelerated casein transport and/or secretion. Because of their ability to form complexes that bridge two membranes and promote their fusion, SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are involved in almost all intracellular trafficking steps and exocytosis. As SNAREs can bind arachidonic acid, they could be the effectors of the secretagogue effect of prolactin in MESCs. Indeed, some SNAREs have been observed between secretory vesicles and lipid droplets suggesting that these proteins could not only orchestrate the intracellular trafficking of milk components but also act as key regulators for both the coupling and coordination of milk product secretion in response to hormones.

Effects of dietary fish oil and corn oil on rat mammary tissue.

The effects, on the maternal mammary gland, of diets containing similar lipid percentages but differing in composition of polyunsaturated fatty acids (PUFA) have been assessed in rats during pregnancy and lactation. For this purpose, tuna fish oil (an n-3-PUFA-enriched oil) and corn oil (an n-6-PUFA-enriched oil) were included in diets at ratios such that the caloric inputs were the same as that of the control diet. As expected, the maternal diet affected the tissue composition of dams. Unexpectedly, only the tuna fish oil diet had an effect on pup growth, being associated with the pups being underweight between the ages of 11 and 21 days. The maternal mammary gland of rats fed the tuna fish oil diet displayed two main modifications: the size of cytoplasmic lipid droplets was increased when compared with those in control rats and the mammary epithelium showed an unusual formation of multilayers of cells. These results show that the tuna fish oil diet, during pregnancy and lactation, exerts specific effects on mammary cells and on the formation of lipid droplets. They suggest that this maternal diet affects the functioning of the mammary tissue.

[Prolactin and its cleaved 16 kDa fragment]. / La prolactine et son fragment 16 kDa dans les tissus de mammifères.

Prolactin, owing to its origins, actions and molecular forms, is an ubiquitous and pleiotropic hormone. Indeed prolactin, initially thought to be essentially synthesized in the hypophysis, is also produced by several tissues in mammals. It is involved in more than 300 different biological activities, such as reproduction, developmental immunity and behaviour. It is also described under several molecular forms resulting from co- or post-translational modifications and enzymatic cleavage. Among these, the 16 kDa form, derived from native prolactin, has received particular attention because of its inhibitory effect on angiogenesis. Recent results have suggested an important role of tissue enzymes in the production of this form in several tissues (retina, myocardium and mammary gland). The cleavage leading to the production of 16 kDa prolactin may occur outside the cells, in the interstitial medium and therefore in the vicinity of blood capillaries. This process implies tissue-specific mechanisms of regulation. A better knowledge of the location of the cleavage and of the regulation of these activities of the cleaving enzymes is now essential for controlling the processes. This knowledge will allow a better understanding of the relationships between some pathologies (cardiomyopathy, pre-eclampsia, retinopathy) and modification of the production of the anti-angiogenic form of prolactin.

Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells.

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.

Prolactin promotes the secretion of active cathepsin D at the basal side of rat mammary acini.

Cathepsin D (CD), a lysosomal aspartic protease present in mammary tissue and milk in various molecular forms, is also found in the incubation medium of mammary acini in molecular forms that are proteolytically active on prolactin at a physiological pH. Because prolactin controls the vesicular traffic in mammary cells, we studied, in vivo and in vitro, its effects on the polarized transport and secretion of various forms of CD in the rat mammary gland. CD accumulated in vesicles not involved in endocytosis in the basal region of cells. Prolactin increased this accumulation and the release of endosomal active single-chain CD at the basal side of acini. The CD-mediated proteolysis of prolactin, leading to the antiangiogenic 16-kDa form, at a physiological pH, was observed only in conditioned medium but not milk. These data support the novel concept that an active molecular form of CD, secreted at the basal side of the mammary epithelium, participates in processing blood-borne prolactin outside the cell, this polarized secretion being controlled by prolactin itself.

Localisation of caveolin in mammary tissue depends on cell type.

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.

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.

[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.

Cathepsin D released by lactating rat mammary epithelial cells is involved in prolactin cleavage under physiological conditions.

The 16 kDa prolactin fragment arises from partial proteolysis of the native 23 kDa prolactin pituitary hormone. The mammary gland has been involved in this processing, although it has not been clarified whether it occurs in stroma or epithelial cells or extracellularly. Also, the processing enzyme has not been defined yet. Here we show that the incubation medium of stroma-deprived mammary acini from lactating rat contains an enzymatic activity able to cleave, in a temperature- and time-dependent fashion, the 23 kDa prolactin to generate a 16 kDa prolactin detectable under reducing conditions. This cleavage was not impaired in the presence of hirudin, a thrombin inhibitor, but strongly weakened in the presence of pepstatin A, a cathepsin D inhibitor. Cathepsin D immuno-depletion abolished the capability of acini-conditioned medium to cleave the 23 kDa prolactin. Brefeldin A treatment of acini, a condition that largely abolished the apical secretion of milk proteins, did not impair the secretion of the enzymatically active single chain of cathepsin D. These results show that mature cathepsin D from endosomes or lysosomes is released, likely at the baso-lateral site of mammary epithelial cells, and that a cathepsin D-dependent activity is required to effect, under physiological conditions, the cleavage of 23 kDa prolactin in the extracellular medium. This is the first report demonstrating that cathepsin D can perform a limited proteolysis of a substrate at physiological pH outside the cell.

Endocytic prolactin routes to the secretory pathway in lactating mammary epithelial cells.

Plasma-borne prolactin is carried from blood to milk by transcytosis across the mammary epithelial cell through the endocytic and secretory pathways. To determine the precise route of prolactin endocytosis, intracellular transport of biotinylated prolactin was monitored, in parallel with endocytosis of fluorescein isothiocyanate-conjugated dextran and IgG, by using pulse-chase experiments in lactating mammary fragments and in enzymatically dissociated acini. Biotinylated prolactin was sorted to vesiculo-tubular organelles whereas dextran was very rapidly carried to the lumen and IgG remained accumulated in the basal region of cells. To determine whether prolactin uses routes into and across the Golgi and trans-Golgi network, localisation of biotinylated prolactin was combined with the immunofluorescence detection of caseins and, respectively, p58 and TGN38. Biotinylated prolactin strongly colocalised with caseins during a chase but not all or only very little with p58 and TGN38. To characterise the organelles involved in transcytosis, gold-labelled prolactin, experimentally accumulated in late endosomes and which recovered a normal transport, was localised by electron microscopy. In mammary fragments incubated at low temperature, and in mammary fragments from rats fed with a lipid-deprived diet, transport of gold-labelled prolactin was restored by increasing the temperature and by adding arachidonic acid, respectively. These data demonstrate that a sorting occurs very rapidly between prolactin, dextran and IgG. They suggest that prolactin may reach the biosynthetic pathway after direct fusion between multivesicular bodies and secretory vesicles.

Oxytocin and milk removal: two important sources of variation in milk production and milk quality during and between milkings.

This review describes the effects of milking (routine and management) on milk yield and milk quality on dairy ruminants and examines the physiological effects of milking on the synthesis and secretion of milk. During milking, differences in the composition of milk as a result of milk ejection reflex are observed: the cisternal milk, immediately available, contains little fat, then milk ejection provokes active transport of high-fat content alveolar milk, into the cisternal compartment. Milking frequency has the capacity to affect milk production too. So, an increase in milking frequency augments milk yield whereas a decrease in milking frequency decreases milk production, with effects on milk composition. The milk ejection reflex is mediated by oxytocin, which induces myoepithelial cell contraction. Nevertheless, other actions of oxytocin may exist, such as a direct effect on proliferation and differentiation of myoepithelial cells and on secretory processes in the mammary epithelial cells.

Milk lipid and protein traffic in mammary epithelial cells: joint and independent pathways.

In mammary epithelial cells, milk lipids and proteins are synthesised in the same compartment, the endoplasmic reticulum. Lipids, carried through the cytoplasm, associate with the apical membrane which then pinches off and releases the lipid globule. Proteins, carried through membrane compartments are released in the lumen after fusion of secretory vesicles with the apical membrane. These processes assure a relatively constant composition of milk but it is not known whether lipid and protein secretion are linked. The protein composition of the milk fat globule membrane and the stimulatory effects of prolactin and oxytocin on lipid and protein secretion suggest that these processes are coupled and co-regulated. However, it is possible to observe a dissociation between the formation and the secretion of the two constituents, during differentiation and in various experimental conditions, and this suggests that coupling is not strictly required.