Lack of plasminogen leads to milk stasis and premature mammary gland involution during lactation

The extracellular serine protease, plasmin, is activated from its precursor, plasminogen (Plg), by the urokinase-type and tissue-type Plg activators (uPA and tPA respectively). One of the main plasmin substrates, fibrin, is formed from fibrinogen via thrombin activity. We have previously shown that mice deficient for Plg are strikingly less able to support a litter during lactation compared to wild type mice. Here we suggest a mechanism responsible for this lactation defect. Reduced epithelial content and increased apoptosis are observed in Plg-deficient mammary glands at lactation day 7. Immunofluorescence analysis reveals the presence of fibrin(ogen) in the stroma surrounding mammary alveoli and adipocytes and identifies fibrin(ogen) as a component of breast milk in both wild type and Plg-deficient mice. Furthermore, a large accumulation of fibrin(ogen) together with apoptotic epithelial cells is observed in the lactating mammary alveoli and ducts of some Plg-deficient mice. This suggests that fibrin plays a key role in the malfunction of mammary glands in the absence of Plg, possibly through blockade of mammary ducts inducing milk stasis, inhibiting milk expulsion and thereby inducing premature apoptosis and involution.     

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.     

Apoptotic cell death and tissue remodelling during mouse mammary gland involution.

During post-lactational mammary gland involution, the bulk of mammary epithelium dies and is reabsorbed. This massive cell death and tissue restructuring was found to be accompanied by a specific pattern of gene expression. Northern blot analysis showed that weaning resulted in a dramatic drop in ODC, a gene involved in synthesis of a component of milk, and the nearly simultaneous induction of SGP-2, a gene associated with apoptotic cell death. These changes were followed by decreases in expression of milk protein genes to basal levels and expression of genes associated with regulation of cell proliferation and differentiation, p53, c-myc and TGF-beta 1. Subsequently, additional genes implicated in stress response, tissue remodelling, and apoptotic cell death were transiently expressed, expression peaking at about 6 days post-weaning. A non-random degradation of DNA yielding the oligonucleosomal length fragmentation pattern typical of apoptotic cell death (Wyllie, 1980; Wyllie et al., 1980) was detected in association with morphological changes and gene expression. The correlations between: (a) changes in morphology, (b) pattern of gene expression and (c) changes in DNA integrity suggest that complementary programs for cell death and tissue remodelling direct post-lactational mammary gland involution.     

Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland

Milk production during lactation is a consequence of the suckling stimulus and the presence of glucocorticoids, prolactin, and insulin. After weaning the glucocorticoid hormone level drops, secretory mammary epithelial cells die by programmed cell death and the gland is prepared for a new pregnancy. We studied the role of steroid hormones and prolactin on the mammary gland structure, milk protein synthesis, and on programmed cell death. Slow-release plastic pellets containing individual hormones were implanted into a single mammary gland at lactation. At the same time the pups were removed and the consequences of the release of hormones were investigated histologically and biochemically. We found a local inhibition of involution in the vicinity of deoxycorticosterone- and progesterone-release pellets while prolactin-release pellets were ineffective. Dexamethasone, a very stable and potent glucocorticoid hormone analogue, inhibited involution and programmed cell death in all the mammary glands. It led to an accumulation of milk in the glands and was accompanied by an induction of protein kinase A, AP-1 DNA binding activity and elevated c-fos, junB, and junD mRNA levels. Several potential target genes of AP-1 such as stromelysin-1, c-jun, and SGP-2 that are induced during normal involution were strongly inhibited in dexamethasone-treated animals. Our results suggest that the cross-talk between steroid hormone receptors and AP-1 previously described in cells in culture leads to an impairment of AP-1 activity and to an inhibition of involution in the mammary gland implying that programmed cell death in the postlactational mammary gland depends on functional AP-1.     

Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland

Following the cessation of lactation, the mammary gland undergoes a physiologic process of tissue remodeling called involution in which glandular structures are lost, leaving an adipose tissue compartment that takes up a much larger proportion of the tissue. A quantitative morphometric analysis was undertaken to determine the mechanisms for clearance of the epithelial cells during this process. The involution process was set in motion by removal of pups from 14-day lactating C57BL/6 mice. Within hours, milk-secreting epithelial cells were shed into the glandular lumen. These cells became apoptotic, exhibiting exposure of phosphatidylserine residues on their surfaces, activation of effector caspase-3, staining for caspase-cleaved keratin 18, loss of internal organellar structure, and nuclear breakdown, but minimal blebbing or generation of apoptotic bodies. Clearance of residual milk and the shed epithelial cells was rapid, with most of the removal occurring in the first 72 h. Intact apoptotic epithelial cells were engulfed in large numbers by residual viable epithelial cells into spacious efferosomes. This process led to essentially complete involution within 4 days, at which point estrous cycling recommenced. Macrophages and other inflammatory cells did not contribute to the clearance of either residual milk or apoptotic cells, which appeared to be due entirely to the epithelium itself.     

Remodeling mechanisms of the mammary gland during involution

The process of post-lactational regression, or involution, of the mammary gland is a complex event characterised by extensive death of the secretory epithelium coupled with remodelling of the extracellular matrix and adipogenesis to regenerate the fat pad. Associated with these events is an inflammatory cascade and acute phase response. The critical signalling pathways that regulated involution have been defined and a wide variety of genes have been shown to modulate the various processes involved, including cell death, phagocytosis, tissue remodelling and innate immune response.     

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.     

Identification and profiling of microRNAs involved in the regenerative involution of mammary gland

Regenerative involution is important for the subsequent lactation, but molecular mechanism has not been revealed. The crucial miRNA in tissue development indicates that miRNAs might participate in regenerative involution. In the present study, the mammary tissues of the dairy goats (n = 3) were collected via biopsy at wk-8 (time to dry off), –6, –4, –1, and + 1 relative to lambing for the Hematoxylin and Eosin staining and miRNA sequencing. Alveolar structures collapsed during regenerative involution, but the structures remained intact and distended. Among the 50 miRNA expression trajectories categorized by short time-series expression miner, two significant patterns were clustered. The differentially expressed miRNAs in the two patterns were mainly related to the self-renewal of tissue and enriched in pathways containing vesical-mediated transport, tissue development, tube development, vasculature development and epithelial development. The identification of the miRNAs will help in elucidating their regulatory roles in mammary gland development.     

Mfge8 is critical for mammary gland remodeling during involution

Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling.     

Induction of mouse Ca(2+)-sensitive chloride channel 2 gene during involution of mammary gland.

To understand molecular mechanisms that regulate mammary gland involution, we identified involution-induced cDNA clones by suppression subtractive hybridization methods. Nucleotide sequencing of a clone revealed that it was 97% identical to Ca(2+)-sensitive chloride channel 1 (mCLCA1) gene that has been identified in lung tissue. We concluded that our clone was derived from different gene with mCLCA1 and named it mCLCA2. We confirmed that expression of mCLCA2 gene was predominant in mammary gland while mCLCA1 mRNA was mainly detected in lung tissues by RT-PCR. Northern analysis showed that the mCLCA2 gene was induced at involution phase compared to pregnant and lactating phases of mammary gland. Under serum starvation, HC11 mammary epithelial cells showed DNA fragmentation and induction of mCLCA2 expression.     

Secret excretion from the mouse mammary gland

Histological studies revealed that the mammary gland nipple have smooth muscle fibres along the nipple channel. These fibres infiltrate the connective tissue parallel to the skin. The ring muscles are not obvious. Delays in the milk excretion in mice may be due to specifics of allocation and functioning of the nipple smooth muscles. To obtain milk, a mechanical action upon the nipple and a synchronised release of oxitocin into the blood are necessary.