Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.     

Characterization of milk composition in narrow‐ridged finless porpoises (Neophocaena asiaeorientalis) at different lactation stages

Lactation plays a vital role in reproductive success, evolving with ecological adaptations of mammalian life histories. Knowledge of the lactation process in odontocetes is scarce and limited to a handful of species. We investigated the changes in milk composition across different lactation stages, including prepartum mammary secretion and early, mid‐ and late lactation of narrow‐ridged finless porpoises. Prepartum mammary secretion was greenish and characterized by high protein, low sugar, and negligible fat contents. In contrast, milk fat became predominant over protein and sugar contents throughout all lactation stages. At the early lactation stage, the contents of all milk constituents except water varied greatly. At the mid‐late lactation stage, the gross milk composition was relatively stable and was composed of, on average, 67.34% ± 4.13% water, 9.96% ± 0.75% protein, 21.40% ± 3.24% fat, and 1.72% ± 0.73% sugar. Our findings provide new insight into milk composition at different lactation stages in the narrow‐ridged finless porpoise.     

The effect of level of feeding, genetic merit, body condition score and age on biological parameters of a mammary gland model

An evolutionary algorithm was applied to a mechanistic model of the mammary gland to find the parameter values that minimised the difference between predicted and actual lactation curves of milk yields in New Zealand Jersey cattle managed at different feeding levels. The effect of feeding level, genetic merit, body condition score at parturition and age on total lactation yields of milk, fat and protein, days in milk, live weight and evolutionary algorithm derived mammary gland parameters was then determined using a multiple regression model. The mechanistic model of the mammary gland was able to fit lactation curves that corresponded to actual lactation curves with a high degree of accuracy. The senescence rate of quiescent (inactive) alveoli was highest at the very low feeding level. The active alveoli population at peak lactation was highest at very low feeding levels, but lower nutritional status at this feeding level prevented high milk yields from being achieved. Genetic merit had a significant linear effect on the active alveoli population at peak and mid to late lactation, with higher values in animals, which had higher breeding values for milk yields. A type of genetic merit × feeding level scaling effect was observed for total yields of milk and fat, and total number of alveoli produced from conception until the end of lactation with the benefits of increases in genetic merit being greater at high feeding levels. A genetic merit × age scaling effect was observed for total lactation protein yields. Initial rates of differentiation of progenitor cells declined with age. Production levels of alveoli from conception to the end of lactation were lowest in 5- to 8-year-old animals; however, in these older animals, quiescent alveoli were reactivated more frequently. The active alveoli population at peak lactation and rates of active alveoli proceeding to quiescence were highest in animals of intermediate body condition scores of 4.0 to 5.0. The results illustrate the potential uses of a mechanistic model of the mammary gland to fit a lactation curve and to quantify the effects of feeding level, genetic merit, body condition score, and age on mammary gland dynamics throughout lactation.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

The Ca/H antiporter TMEM165 expression,localization in the developing,lactating and involuting mammary gland parallels the secretory pathway Ca ATPase (SPCA1)

Plasma membrane Ca²⁺-ATPase 2 (PMCA2) knockout mice showed that ∼60% of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca²⁺-ATPase’s 1 and/or 2 (SPCA1 and 2). However, another secretory pathway calcium transporter was recently described. The question becomes whether this Golgi Ca²⁺/H⁺ antiporter (TMEM165) is expressed sufficiently in the Golgi of lactating mammary tissue to be a relevant contributor to secretory pathway mammary calcium transport. TMEM165 shows marked expression on day one of lactation when compared to timepoints prepartum. At peak lactation TMEM165 expression was 25 times greater than that of early pregnancy. Forced cessation of lactation resulted in a rapid ∼50% decline in TMEM165 expression at 24 h of involution and TMEM165 expression declined 95% at 96 h involution. It is clear that the timing, magnitude of TMEM165 expression and its Golgi location supports a role for this Golgi Ca2⁺/H⁺ antiporter as a contributor to mammary Golgi calcium transport needs, in addition to the better-characterized roles of SPCA1&2.     

Mitosis in Milk Secreting Epithelial Cells of Mammary Gland An Ultrastructural Study

In all stages of lactation mitotic configurations were observed in mammary gland epithelial cells of rats. An electron microscopic study is presented which shows that ultrastructure of such mitotic stages is normal and that mitotic cells contain typical products of milk secreting cells such as casein micelle-containing vesicles and milk fat droplets. Such secretory products can even be observed in the immediate vicinity of the chromosomes and microtubules of the spindle apparatus. The endoplasmic reticulum of mitotic cells appeared altered in that it did not show typical cisternal stacks characteristic of interphase cells. While the numbers of such mitotic cells were very low, especially from the second week of lactation on (always less than 0.1% of the milk secreting epithelial cells encountered), the observations clearly demonstrate that differentiation for milk secretory activity and cells division are not mutually exclusive. We conclude that postpartum growth of mammary gland epithelium and replacement of epithelial cells lost during desquamation into the milk liquids can occur by division of existing differentiated milk secreting cells and does not require mitotic activity of non-lactating 'stem cells' which are not observed in lactating alveoli.     

Alterations in the relative abundance of Haptoglobin (Hp) transcripts in total milk somatic cells during different stages of lactation cycle in high yielding cross-bred cows

The expression profile of Haptoglobin (Hp) gene in total milk somatic cells (SCC) of high-yielding cross-bred Karan Fries (KF) was studied during early, mid, and late lactation cycle. Milk samples (200 ml/animals) were collected from 10 high-yielding and 10 low-yielding cows throughout the lactation cycle (from day 7 to day 300) with an interval of one month. Relative mRNA expression profiles of Hp by RT polymerase chain reaction was studied in high-yielding cows, whereas low-yielding cows were taken as control. The folds of induction of Hp was significantly (p < 0.001) downregulated by a mean factor of 0.207 in milk SCC during early lactating cows. Whereas, it was significantly (p < 0.01) upregulated by a mean factor of 20.888 during mid lactation. The expression was unaltered during the late lactation. The study demonstrates that Hp is synthesized within the mammary gland and significantly upregulated during mid-lactation period compared to other stages of lactation cycle.     

Serotonin regulates mammary gland development via an autocrine-paracrine loop

Mammary gland development is controlled by a dynamic interplay between endocrine hormones and locally produced factors. Biogenic monoamines (serotonin, dopamine, norepinephrine, and others) are an important class of bioregulatory molecules that have not been shown to participate in mammary development. Here we show that mammary glands stimulated by prolactin (PRL) express genes essential for serotonin biosynthesis (tryptophan hydroxylase [TPH] and aromatic amine decarboxylase). TPH mRNA was elevated during pregnancy and lactation, and serotonin was detected in the mammary epithelium and in milk. TPH was induced by PRL in mammosphere cultures and by milk stasis in nursing dams, suggesting that the gene is controlled by milk filling in the alveoli. Serotonin suppressed beta-casein gene expression and caused shrinkage of mammary alveoli. Conversely, TPH1 gene disruption or antiserotonergic drugs resulted in enhanced secretory features and alveolar dilation. Thus, autocrine-paracrine serotonin signaling is an important regulator of mammary homeostasis and early involution.     

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts¹. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane². At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones^3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy². At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli^6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups ⁸. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium^9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation^12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice. Download video file.^{(54M, mov)}     

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f⁺ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further.     

Nuclear bodies in bovine mammary gland in different physiological states

The frequency of nuclear bodies was determined in epithelial cells of mammary ductules or alveoli, milk cistern and teat sinus of virgin heifers, non-lactating cows and cows in early lactation. The determined frequency was within the range 12–16% with the single exception of the epithelial cells of secretory alveoli of lactating cows, in which the frequency was 3 %. These data support the concept that nuclear bodies are normal nuclear organelles which can respond to altered physiological conditions.     

Mammary basal cells: Stars of the show

Nearly all mammals rely on lactation to support their young and to ensure the continued survival of their species. Despite its importance, relatively little is known about how milk is produced and how it is ejected from the lumen of mammary alveoli and ducts. This review focuses on the latter. We discuss how a relatively small number of basal cells, wrapping around each alveolar unit, contract to forcibly expel milk from the alveolar lumen. We consider how individual basal cells coordinate their activity, the fate of these cells at the end of lactation and avenues for future deliberation and exploration.     

Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue.

We used 35S-labeled cRNA probes to localize the sites of alpha-lactalbumin, alpha-S1-casein, and lactoferrin mRNA synthesis in sheep and forcibly weaned cattle mammary tissue. Expression of alpha-lactalbumin was absent in three of four "virgin" glands studied, present in some alveoli of "pregnant" glands but not in others, despite a similar histological appearance. In the early lactating gland, expression was high in those alveoli with few fat globules in their cells and lumen and was absent in alveoli with abundant fat globules. These observations suggest either that alpha-lactalbumin gene expression is linked to the long-term secretory activity of cells and falls once cells are resting or regressing, or that there are cyclical variations in expression, or that in the lactating gland some groups of epithelial cells are synthesizing alpha-lactalbumin and some are synthesizing fat. Expression patterns of alpha-S1-casein were similar to those of alpha-lactalbumin. Lactoferrin, in contrast, was expressed almost exclusively in the "fatty alveoli" of both species. Our results show that dramatic variations in milk gene expression can occur throughout the mammary gland of sheep and cattle and that at no stage of pregnancy, lactation, or involution can the gland be considered metabolically homogeneous.     

Growth patterns and histochemical characterization of bovine mammary corpora amylacea

Corpora amylacea in bovine mammary tissue were quantified across a range of size differentials for histochemical properties, lactation age, and lactation stage, in an attempt to characterize amyloid nucleation and growth. At all size classifications, corpora stained positively for amyloid, calcium deposits, and glycoprotein, while staining negatively for mucopolysaccharides. Prevalence of corpora amylacea among the size differentials was unrelated to age of lactating animals, although no corpora were observed in quarters of primiparous heifers at parturition. Corpora amylacea were most abundant during the later stages of lactation for all size differentials, and least abundant during late involution and early lactation. The majority of corpora were observed in alveolar lumens at all stages of lactation. Our results suggest that corpora amylacea development is not restricted to a particular stage of lactation, although their nucleation appears to occur within the alveolar lumens. Gradual increases in both size and numbers of corpora from parturition to late lactation suggest that development of these structures accelerates as lactation progresses. Morphological relationships between corpora amylacea and mammary parenchymal tissue during the later stages of lactation suggest that these structures may have a role in the involutionary process. These findings provide the foundation for additional immunocytochemical techniques to determine the origin of amyloid fibril components.     

The effect of lactogenic hormones on protein synthesis and amino acid uptake in rat mammary acinar cell culture at various physiological stages

• 1.1. The activities of protein synthesis and amino acid uptake at various physiological stages were determined by the incorporation of radioactive materials ([³H]-lysine, [¹⁴C]-cycloleucine) in rat mammary epithelial cell cultures. The activity of protein synthesis and amino acid uptake was higher in early lactation than in virgin, pregnant and late lactation stages.
• 2.2. Lactogenic hormones (prolactin, hydrocortisone and insulin) treatment related with mammary growth and differentiation increased the activities of protein synthesis and amino acid uptake. But increase of these activities was different at each physiological stage.
• 3.3. The effect of prolactin and hydrocortisone on the activities were greater in virgin, pregnant and late lactation than in early lactation. And effect of insulin was greater in pregnant and early lactation than in virgin and weanling.

     

Origin,concentration and structural features of human mammary gland cells cultured from breast secretions

Summary This study traced the origin of cells observed in human breast secretion samples obtained during lactation and describes the appearance of these cells following prolonged maintenance in vitro. Human milk contains a large number of single vacuolated foam cells and a small proportion of non-vacuolated epithelial cells in clusters. Foam cells are identified by their large size, the polarity of their cytoplasmic organelles, the variation in number and size of lipid vacuoles and the condensed chromatin of their eccentrically located nucleus. Both cell types originate by exfoliation from the mammary gland. This was established by comparing the structural characteristics of cells isolated from milk with those of the cuboidal cell linings of ducts and alveoli in lactating mammary tissue. Relatively pure populations of foam cells could be established from early lactation samples (3–7 days post/partum) while non-vacuolated epithelial cell clusters were more frequently cultured from late lactation specimens (1–10 days postweaning). Foam cells did not divide and lost cytoplasmic organization during prolonged culture. In contrast, non-vacuolated epithelium in clusters proliferated to form colonies of polygonal cells. These results, which imply that foam cells are an active form of the non-vacuolated mammary cells in clusters, call attention to one system for the study of the complex hormonal interactions necessary to induce and maintain lactation.Supported in part by NCI contract NO 1-CB-33898     

Metabolic adaptations in goat mammary tissue during pregnancy and lactation

Metabolic adaptations of goat mammary tissue during pregnancy and lactation were monitored in serial biopsies of the tissue. Changes in the synthetic capacity of secretory cells were studied by combining measurements of enzyme activities with short-term culture of mammary explants to measure lactose, casein and total protein synthesis. By these criteria, the main phase of mammary differentiation began in late pregnancy and was essentially complete by Week 5 of lactation, coinciding with the achievement of peak milk yield. While milk yield declined after Week 5, the activities of key enzymes expressed per mg DNA and the rates of lactose and casein synthesis in mammary explants were maintained over a considerable period. The results suggest that changes in the synthetic capacity of epithelial cells may account for much of the rise in milk yield in early lactation, but are not responsible for the declining phase of milk production characteristic of lactation in ruminants.