Regulation and functional relevance of milk fat globules and their components in the mammary gland

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.     

Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution

Mammary gland development is dependent on macrophages, as demonstrated by their requirement during the expansion phases of puberty and pregnancy. Equally dramatic tissue restructuring occurs following lactation, when the gland regresses to a state that histologically resembles pre-pregnancy through massive programmed epithelial cell death and stromal repopulation. Postpartum involution is characterized by wound healing-like events, including an influx of macrophages with M2 characteristics. Macrophage levels peak after the initial wave of epithelial cell death, suggesting that initiation and execution of cell death are macrophage independent. To address the role of macrophages during weaning-induced mammary gland involution, conditional systemic deletion of macrophages expressing colony stimulating factor 1 receptor (CSF1R) was initiated just prior to weaning in the Mafia mouse model. Depletion of CSF1R(+) macrophages resulted in delayed mammary involution as evidenced by loss of lysosomal-mediated and apoptotic epithelial cell death, lack of alveolar regression and absence of adipocyte repopulation 7 days post-weaning. Failure to execute involution occurred in the presence of milk stasis and STAT3 activation, indicating that neither is sufficient to initiate involution in the absence of CSF1R(+) macrophages. Injection of wild-type bone marrow-derived macrophages (BMDMs) or M2-differentiated macrophages into macrophage-depleted mammary glands was sufficient to rescue involution, including apoptosis, alveolar regression and adipocyte repopulation. BMDMs exposed to the postpartum mammary involution environment upregulated the M2 markers arginase 1 and mannose receptor. These data demonstrate the necessity of macrophages, and implicate M2-polarized macrophages, for epithelial cell death during normal postpartum mammary gland involution.     

Knocking Off SOCS Genes in the Mammary Gland

Suppressors of cytokine signaling (SOCS) proteins are critical regulators of cytokinemediatedresponses in diverse tissues. In the mammary gland, signal transductionpathways elicited by cytokines and hormones have been shown to control distinct stagesof development. In vivo evidence points to essential roles for Socs1 and Socs2 as keyphysiological attenuators of prolactin receptor (PRLR) signaling during pregnancy andlactogenesis. Recently, Socs3 has been shown to be a critical regulator of involution, thecoordinated process of programmed cell death and tissue remodelling that is initiatedafter the cessation of lactation. This review will predominantly focus on the antiapoptoticfunction of Socs3 during mammary gland involution in which it acts as a keyattenuator of Stat3-mediated signal transduction. Perturbation of this pathway leads to anincrease in the levels of c-myc and its likely target genes, p53, bax and E2F-1, providingevidence that c-myc is a central effector of apoptosis during involution.     

乳腺重构的过程及其调节因素

受到妊娠周期的影响,乳腺组织在雌性哺乳动物一生中经历着妊娠-哺乳-退化的周期性发育变化.在乳腺退化到再次泌乳的过程中,乳腺细胞经历凋亡和更新,从而实现乳腺组织的自我更新和修复,即乳腺重构.重构期间乳腺在组织结构和生理过程中发生显著变化,但该过程物种间差异较大.乳用家畜为维持泌乳,妊娠期和干奶期重叠,展示出独特的再生性乳...     

Suckling effects in sows: importance for mammary development and productivity

An understanding of the mechanisms regulating milk yield in sows is crucial for producers to make the best management decisions during lactation. Suckling of mammary glands by piglets is one factor that is essential for development of these glands during lactation and for the maintenance of lactation in sows. The process of mammary development is not static as the majority of it takes place in the last third of gestation, continues during lactation, is followed by involution at weaning and starts over again in the next gestation. During involution, the mammary glands undergo a rapid and drastic regression in parenchymal tissue, and this can also occur during lactation if a gland is not suckled regularly. Indeed, the pattern of regression is similar for glands that involute at weaning or during lactation. Suckling during 12 to 14 h postpartum is insufficient to maintain lactation and the process of involution that occurs in early lactation is reversible within 1 day of farrowing but is irreversible if a gland is not used for 3 days. However, milk yield from a gland which is ‘rescued’ within the first 24 h remains lower throughout lactation. Suckling does not only affect milk yield in the ongoing lactation, but it also seems to affect that of the next lactation. Indeed, non-suckling of a mammary gland in first-parity sows decreased development and milk yield of that gland in second parity. Nursing behaviour of piglets in early lactation was also affected, where changes were indicative of piglets in second parity being hungrier when suckling glands that were not previously used. It is not known, however, if the same effects would be seen between the second and third lactation. Furthermore, the minimum suckling period required to ensure maximal milk yield from a gland in the next lactation is not known. This review provides an update on our current knowledge of the importance of suckling for mammary development and milk yield in swine.     

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.     

Correlation between nuclear glucocorticoid receptor levels and casein gene expression in murine mammary gland in vitro

The relationship between nuclear binding of glucocorticoid-receptor complex and casein gene expression was studied in organ culture of the whole mammary gland of the mouse. Pyridoxal 5'-phosphate was used as a modulatory agent for measuring nuclear binding of the receptor complex. Addition of 2 mM and 5mM pyridoxal-5'-P in the medium (Waymouth's MB752/1) resulted in 4- and 12-fold increase of its concentration in the glands incubated with insulin, prolactin, and hydrocortisone. Pyridoxal-5'-P also caused a 52% and 92% inhibition of nuclear binding of [3H]dexamethasone in the glands at 2 mM and 5 mM concentration in the presence of the same hormones in the medium. Corresponding to the reduced nuclear binding of the receptor complex casein mRNA levels, measured by a specific cDNA probe was reduced 86% and over 90% in the glands exposed to 2 mM and 5 mM pyridoxal-5'-P, respectively, in presence of insulin, prolactin, and hydrocortisone in the medium. Withdrawal of pyridoxal-5'-P from the medium restored nuclear binding of the receptor complex near the level of control glands incubated only with the hormones. mRNA casein levels also increased in the gland in the pyridoxal-5'-P-free medium containing the same hormones. This indicates that pyridoxal-5'-P does not alter the specific hormone responsiveness of the mammary cells and its action mediated at the level of the glucocorticoid receptor can influence hormone-inducible expression of the casein genes. Thus, glucocorticoid plays a major role in the multiple hormone regulation of the milk protein gene(s). The findings also suggest that the breast tissue concentration of the vitamin B6 derivative may influence the physiology of lactation in nursing mothers.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, beta-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-x(L)were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R(3)-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues.     

Overexpression of des(1-3) insulin-like growth factor 1 in the mammary glands of transgenic mice delays the loss of milk production with prolonged lactation

During prolonged lactation, the mammary gland gradually loses the capacity to produce milk. In agricultural species, this decline can be slowed by administration of exogenous growth hormone (GH), which is believed to act through insulin-like growth factor 1 (IGF1). Our previous work demonstrated delayed natural mammary gland involution in des(1-3)IGF1-overexpressing transgenic mice (Tg[Wap-des{1-3}IGF1]8266 Jmr), hereafter referred to as WAP-DES mice. The present study tested the hypothesis that overexpressed des(1-3)IGF1 would delay the loss of milk production during prolonged lactation. Accordingly, we examined lactational performance in WAP-DES mice by artificially prolonging lactation with continual litter cross-fostering. Over time, lactational capacity and mammary development declined in both WAP-DES and control mice. However, the rate of decline was 40% slower in WAP-DES mice. Mammary cell apoptosis increased by 3-fold in both groups during prolonged lactation but was not different between genotypes. Plasma concentrations of murine IGF1 were decreased in WAP-DES mice, while those of the transgenic human IGF1 were elevated during prolonged lactation. Phosphorylation of the mammary IGF1 receptor was increased in the WAP-DES mice, but only during prolonged lactation. Plasma prolactin decreased with prolonged lactation in nontransgenic mice but remained high in WAP-DES mice. The WAP-DES mice maintained a higher body mass and a greater lean body mass during prolonged lactation. These data support the conclusion that overexpressed des(1-3)IGF1 enhanced milk synthesis and mammary development during prolonged lactation through localized and direct activation of the mammary gland IGF1 receptor and through systemic effects on prolactin secretion and possibly nutrient balance.     

Natural and abrupt involution of the mammary gland affects differently the metabolic and health consequences of weaning

In most mammals under natural conditions weaning is gradual. Weaning occurs after the mammary gland naturally produces much less milk than it did at peak and established lactation. Involution occurs following the cessation of milk evacuation from the mammary glands. The abrupt termination of the evacuation of milk from the mammary gland at peak and established lactation induces abrupt involution. Evidence on mice has shown that during abrupt involution, mammary gland utilizes some of the same tissue remodeling programs that are activated during wound healing. These results led to the proposition of the “involution hypothesis”. According to the involution hypothesis, involution is associated with increased risk for developing breast cancer. However, the involution hypothesis is challenged by the metabolic and immunological events that characterize the involution process that follows gradual weaning. It has been shown that gradual weaning is associated with pre-adaption to the forthcoming break between dam and offspring and is followed by an orderly reprogramming of the mammary gland tissue. As discussed herein, such response may actually protect the mammary glands against the development of breast cancer and thus, may explain the protective effect of extended breastfeeding. On the other hand, the termination of breastfeeding during the first 6 months of lactation is likely associated with an abrupt involution and thus with an increased risk for developing breast cancer. Review of the literature on the epidemiology of breast cancer principally supports those conclusions.     

Hormonal activation of mammary gland peroxidase.

Ultrastructural cytochemistry was used to detect an endogenous peroxidase in the rat mammary gland. The enzyme was identified only during the latter half of pregnancy and during lactation, indicating its possible dependence upon hormones. To test this hypothesis, specific hormones associated with the development and differentiation of the mammary gland were used both in vivo and in vitro in an effort to induce, or unmask, the activity of the enzyme. Estrogen injected into nonpregnant rats induced some peroxidase activity in the mammary glands of a few animals. Two hormone combinations tested in organ cultures of mouse mammary gland were able to activate the enzyme: (1) dexamethasone + insulin and (2) dexamethasone + insulin + prolactin.     

Mammary plasminogen activator: correlation with involution, hormonal modulation and comparison between normal and neoplastic tissue.

We have analyzed the plasminogen activator (PA) content of normal rodent mammary glands at different stages of the mammary life cycle and after exposing the animals to various hormones; we have also assessed the PA response of mammary explants to a variety of hormonal environments. Similar studies were performed on a limited number of primary mammary tumors. Plasminogen activator production was clearly correlated with mammary involution. A large but transient increase in enzyme content followed the initiation of involution in all glands, and the enzyme was produced by mammary cells, not by macrophages or granulocytes. Oxytocin, prolactin and hydrocortisone, which slowed or blocked involution, produced parallel effects on gland regression and PA synthesis. PA synthesis by explants in organ culture was induced by hormonal environments that fostered involution and repressed by those that promoted lactation. Mammary tumors produced much more PA than normal tissue both in vivo and in vitro, and distinct differences were found in the response of enzyme synthesis to hormones. The results reinforce the association of PA with tissue remodeling; show that the enzyme can be used as an indicator of cellular response to a wide range of hormones in both normal and malignant tissue; and suggest that observations of this type in organ culture may be of some value in predicting physiological responses in vivo.     

Differential expression of TGF-β isoforms during postlactational mammary gland involution

After cessation of lactation, the mammary gland undergoes involution, which is characterized by a massive epithelial cell death and proteolytic degradation of the extracellular matrix. Whereas the expression patterns and also the function of TGF-beta isoforms during mammary gland branching morphogenesis and lactation are well understood, their expression during postlactational involution and therefore a possible role in this process is poorly known. In this study we show that TGF-beta3 expression is dramatically induced (>fivefold) during mouse mammary gland involution when compared to that of virgin mouse, reaching a maximal expression level at day 4 after weaning. In contrast, other TGF-beta isoforms do not display significant increase in expression during involution (TGF-beta1, 1.3-fold and TGF-beta2, <1.5-fold) when compared to that of virgin or lactating mice. During mammary gland involution, TGF-beta3 is expressed in the epithelial layer and particularly in myoepithelial cells. A comparison of the kinetics of TGF-beta3 expression to that of programmed cell death and degradation of the basement membrane suggests that TGF-beta3 functions in the remodeling events of the extracellular matrix during the second stage of involution.     

Cell turnover and gene activities in sheep mammary glands prior to lambing to involution

Mammary glands are special tissue characterized by proliferation of the epithelium, during puberty and pregnancy and by programmed cell death, during involution. In this study, apoptosis was identified by TUNEL staining and then related to cell proliferation, as determined by Ki-67 staining. The apoptotic index was at its highest at 8 days of involution, whereas the proliferation index was at its highest during lactation. Caspase-3 was immunolocalised only in mast cells and along the basal membrane in the mammary tissue at −10 days from lambing, 150 days of lactation and at 8 days of involution. This finding could indicate that caspase-3 is not involved in sheep mammary gland apoptosis, but that other proteins – such as apoptosis inducing factor (AIF) – can trigger apoptosis, through the mitochondrial pathway, in a caspase-independent manner. The expression of genes involved in the regulation of lactation and apoptosis was also investigated and determined relatively to −10 days from lambing. The relative expression level of LALBA, reached its maximum during lactation, whereas the expressions of BCL2, BCL2L1, BAX, STAT5A, STAT3, IGFBP5 and FOXO3A, increased significantly during involution in correlation with apoptotic index.This work shows for the first time the turnover of mammary cells and the interaction of their signals during the complete lactation cycle in sheep. The data on gene expression can contribute to elucidate the mechanisms controlling milk production and cell turnover in this species.     

The mineralocorticoid receptor may compensate for the loss of the glucocorticoid receptor at specific stages of mammary gland development

To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.     

Apoptosis and mammary gland involution: reviewing the process

Apoptosis is a process of programmed cell death. Mammary gland involution is a tissue remodelling process. Mammary epithelial cell apoptosis is an integral component of tissue remodelling but it is only one element. Equally important are the factors which degrade basement membrane and extracellular matrix. Both operations are required for completion of mammary gland involution. The primary apoptotic process occurs first and is temporally distinct from the second stage of involution typified by lobular-alveolar collapse. Local factors related to milk accumulation trigger the first stage, but loss of systemic hormonal stimulation governs the second stage. Changes in the expression patterns of cell cycle control genes and bcl-2 family member genes are found in the first stage. Proteinase gene activation dominates the second stage. These findings support a two stage model of mammary gland involution. Both mammary epithelial cell apoptosis and mammary gland remodelling advance through a process which includes both loss of survival factors and gain of death factors. This review focuses on signalling pathways and genetic controls which are activated and repressed during mammary gland involution.     

Glucocorticoid alternative effects on proliferating and differentiated mammary epithelium are associated to opposite regulation of cell-cycle inhibitor expression

Glucocorticoids influence post-natal mammary gland development by sequentially controlling cell proliferation, differentiation, and apoptosis. In the mammary gland, it has been demonstrated that glucocorticoid treatment inhibits epithelial apoptosis in post-lactating glands. In this study, our first goal was to identify new glucocorticoid target genes that could be involved in generating this effect. Expression profiling, by microarray analysis, revealed that expression of several cell-cycle control genes was altered by dexamethasone (DEX) treatment after lactation. Importantly, it was determined that not only the exogenous synthetic hormone, but also the endogenous glucocorticoids regulated the expression of these genes. Particularly, we found that the expression of cell cycle inhibitors p21CIP1, p18INK4c, and Atm was differentially regulated by glucocorticoids through the successive stages of mammary gland development. In undifferentiated cells, DEX treatment induced their expression and reduced cell proliferation, while in differentiated cells this hormone repressed expression of those cell cycle inhibitors and promoted survival. Therefore, differentiation status determined the effect of glucocorticoids on mammary cell fate. Particularly, we have determined that p21CIP1 inhibition would mediate the activity of these hormones in differentiated mammary cells because over-expression of this protein blocked DEX-induced apoptosis protection. Together, our data suggest that the multiple roles played by glucocorticoids in mammary gland development and function might be at least partially due to the alternative roles that these hormones play on the expression of cell cycle regulators.