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.

Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts.

Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants' udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition (ODM, feeding level, endocrine status, photoperiod modulation, and stage of lactation).

Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling.

Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

Oestradiol enhances apoptosis in bovine mammary epithelial cells in vitro.

Ovarian steroids, oestradiol and progesterone, are required for normal mammary growth at puberty and during pregnancy. They contribute to mammary parenchyma development by stimulating mammary epithelial cell (MEC) proliferation. However several studies demonstrate that oestradiol negatively affects milk production during the declining phase of lactation, but the oestradiol effect on MEC in lactating mammary gland remains unclear. The objective of this study was to investigate the differential effect of oestradiol on bovine MECs mimicking two physiological statuses: active and early apoptotic MECs. We demonstrated that oestradiol has a major effect on early apoptotic MECs and might accelerate MEC apoptosis by activation of caspases rather than by inducing apoptosis in active MECs. Early apoptotic MECs could be compared with senescent cells in the late-lactation mammary gland. These results suggest that the negative effect of oestradiol on milk production during the declining phase of lactation would be due to an enhancement of apoptotic processes in MECs.

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.

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.