Diet‐induced obesity disrupts ductal development in the mammary glands of nonpregnant mice

Mammary glands develop postnatally in response to the hypothalamic‐pituitary‐gonadal axis. Obesity‐induced changes in the local environment, however, retard mammary gland development during late pregnancy and lactation. To clarify the effects of obesity on fundamental duct development, we compared the mammary glands of nulliparous nonpregnant obese mice fed a high‐fat diet with those of lean mice fed a normal diet. Obese mice had enlarged mammary glands, reflecting fat pad size, whereas the ducts in obese mice showed a less dense distribution with less frequent branching. Additionally, the ducts were surrounded by thick collagen layers, and were incompletely lined with myoepithelium. Because leptin receptors were localized in the epithelium region and leptin that was highly expressed in the obese glands suppressed mammary epithelial cell proliferation in vitro, the present results suggest that obesity disrupts mammary ductal development, possibly by remodeling the mammary microenvironment and promoting the expression of such paracrine factors as leptin. Developmental Dynamics 238:1092–1099, 2009. © 2009 Wiley‐Liss, Inc.     

Transgenic mice expressing a dominant-negative mutant type II transforming growth factor-beta receptor exhibit impaired mammary development and enhanced mammary tumor formation

We have previously shown that expression of a dominant-negative type II transforming growth factor-beta receptor (DNIIR) in mammary epithelium under control of the MMTV promoter/enhancer causes alveolar hyperplasia and differentiation in virgin mice. Here we show that MMTV-DNIIR female mice have accelerated mammary gland differentiation during early pregnancy with impaired development during late pregnancy and lactation followed by delayed postlactational involution. Mammary tumors, mostly carcinoma in situ, developed spontaneously in the MMTV-DNIIR mice with a long median latency (27.5 months). Crossbreeding to MMTV-transforming growth factor (TGF)-alpha mice to obtain mice expressing both transgenes resulted in mammary tumor formation with a much shorter latency more similar to those expressing only the MMTV-TGF-alpha transgene (<10 months median latency). The major difference in mammary tumors arising in MMTV-TGF-alpha compared to bigenic MMTV-DNIIR/MMTV-TGF-alpha was the marked suppression of tumor invasion by DNIIR transgene expression. Invading carcinoma cells in both MMTV-DNIIR and bigenic animals showed loss of DNIIR transgene expression as determined by in situ hybridization. The data indicate that signaling from endogenous TGF-betas not only plays an important role in normal mammary gland physiology but also can also suppress the early stage of tumor formation and contribute to tumor invasion once carcinomas have developed.     

Sinus‐like dilatations of the mammary milk ducts,Ki67 expression,and CD3‐positive T lymphocyte infiltration,in the mammary gland of wild European rabbits during pregnancy and lactation

Sinus‐like dilatations of the mammary duct are recognisable in the mammary gland of pregnant and lactating wild European rabbits. These dilatations exhibit a bilaminar epithelial lining, with luminal epithelial cells expressing basal and lateral E‐cadherin. Occasional binucleated mammary epithelial cells are present in the luminal layer. Underlying the luminal epithelial cells is a basal layer of cytokeratin 14‐positive cells, supported by a thin layer of fibrous tissue. Multi‐segmental epithelial proliferation, as indicated by Ki67 expression, is apparent in the luminal epithelial cells, suggesting a capacity for division during pregnancy and lactation. CD3‐positive T lymphocytes are present both intraepithelially, suggesting exocytosis, and in foci subjacent to the ductular epithelium. We consider that sinus‐like dilatations of the mammary duct may have the potential to give rise to a subset of the mammary gland neoplasms classified as ductal in origin. Milk accumulation in these sinus‐like dilatations is likely to provide a niche for bacterial replication in cases of mastitis in rabbits. These structures are an important component of the innate immune system of the mammary gland, both as a physical barrier and as an interface between the milk and mammary immune cells.     

Cell proliferation in the mammary gland during late pregnancy and lactation

Nulliparous, CFW mice were injected with 25 m?c of tritiated thymidine on day 19 of pregnancy, and days 1, 2, 3, 5, 7, 10, 15 and 20 of lactation. The animals were killed one hour after injection. The inguinal mammary glands were removed and processed for paraffin sectioning. Radioautographs were prepared, using the dipping technique. Quantitation of mammary epithelial cell proliferation for the intra- and interlobular (ducts) epithelium was performed by determining the percent of labeled epithelial cells in a large sample of cells (labeling index). It was concluded that epithelial cell sample sizes of 1,000–2,000 cells were adequate to measure mammary epithelial proliferation. A wave of epithelial proliferation was observed during early lactation. In the intralobular epithelium, a peak labeling index of 11.1% was attained on day two of lactation whereas a peak labeling index of 7.9% was observed on day three of lactation in the interlobular epithelium. Cells of the connective tissue and vascular bed proliferated in response to the growth of the mammary epithelium. Myoepithelial cells were frequently labeled on days two and three of lactation.     

Immunohistochemical comparison of T-cell and macrophage populations in mammary tissue of control and Trichinella spiralis-infected rats

We have shown that antigen-specific T lymphocytes can mediate maternal-to-neonatal immunity during lactation. Present studies address the dynamics of lymphocyte accumulation in the mammary gland during normal and disease stimulated conditions. Monoclonal antibodies specific for total T cells, suppressor/cytotoxic and helper subsets, and macrophages were used in conjunction with immunohistochemistry to identify and count the individual cell types. In unstimulated mammary tissue, following a rise in T cells to maximal numbers in late pregnancy, the total number of T cells/high power field (HPF) was significantly diminished in early lactation and continued to decline to the late lactation period. Both the numbers of T cells/HPF located in the mammary alveolar epithelium and surrounding connective tissue were significantly reduced in early lactation as compared to late pregnancy. This indicates the possible passage of cells into the milk during lactation. Prior infection of the mother with Trichinella spiralis and a secondary challenge 48 h. before sacrifice caused a significant reduction in the number of T cells in the mammary tissue in early lactation as compared with unstimulated controls, indicating the possibility of an even greater outflow of T cells into milk. In controls, the T-suppressor/cytotoxic subtype showed a reduction in early lactation versus late pregnancy but showed no shifts in total cells/HPF during infection. The T-helper subtype in controls remained unchanged from late pregnancy to early lactation with a considerable decline in late lactation. However, the T-helper cells were significantly decreased in T. spiralis-treated animals as compared with noninfected controls in early lactation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leucocyte phenotypes in involuting and fully involuted mammary glandular tissues and secretions of sheep

Mammary glandular tissues and mammary secretions were obtained from sheep at 2–60 d after weaning to study the leucocyte phenotypes associated with mammary involution. From 2–4 d after weaning, neutrophils were the predominant leucocytes in the alveolar and ductal lumina. Lymphocytes were present in the alveolar and ductal epithelium, interalveolar and periductal areas. Most of the lymphocytes in the alveolar and ductal epithelium (IEL) were CD8⁺, some were CD45R⁺ and few were CD4⁺. In the periductal clusters and in the interalveolar areas most of the lymphocytes were CD4⁺. There was a significant increase (P < 0.05) in the percentages of CD45R⁺ granulated IEL from 2 to 7 d after weaning, and this paralleled the increase in the percentages of apoptotic cells in the glandular epithelium. By 7–60 d after weaning, most cells within the alveolar and ductal lumina were macrophages followed by predominantly CD8⁺ lymphocytes. CD8⁺ lymphocytes were still predominant in the alveolar and ductal epithelium while CD4⁺ cells were predominant in the interalveolar areas. Very few γδ⁺ T cells were observed at all the stages examined. The cells in the mammary secretions correlated with those observed in the alveolar and ductal lumina. At the early stages of involution, the neutrophils and macrophages were heavily laden with lipid droplets, casein and cellular debris. The most interesting feature was the presence of cells either with extensive cytoplasmic processes (LCA⁺ MHC class II⁺) or cytoplasmic veils (LCA⁺ MHC class II⁺CD1⁺), probably dendritic cells. It is concluded that the cellular constituents of the mammary gland at the latter part of involution may afford the mammary gland more resistance to infection than the lactating gland and the gland at early stages of involution. The CD45R⁺ IEL may trigger apoptotic cell death in the mammary glandular epithelium during mammary involution.     

Lymphocyte homing and Ig secretion in the murine mammary gland

In mice the majority of the immunoglobulins (Ig) in milk belongs to the IgA class. Prior to its transepithelial transportation into the milk, dimeric IgA (dIgA) is bound to the transmembrane form of the secretory component or polymeric Ig receptor (SC/pIgR). The latter is synthesized in the epithelial cells lining the ducts and alveoli of the mammary gland. A candidate for playing the role of adhesion molecule to primed lymphocytes present in the murine mammary gland might be the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). We studied the correlation between the levels of IgA in colostrum and milk, the number of IgA producing plasma cells in the mammary gland and the expression of MAdCAM-1 in mammary gland endothelial cells during pregnancy and lactation. The relation between the IgA levels in the milk and the expression levels of pIgR in mammary gland epithelial cells was also investigated. We found that the expression of MAdCAM-1 and pIgR starts in early-mid pregnancy; the number of IgA-producing plasma cells and the IgA concentration in milk increase from early lactation onwards. The MAdCAM-1 expression declines during lactation whereas the pIgR levels and IgA-producing plasma cell numbers rise until the end of lactation. Because the MAdCAM-1 level starts to rise several days before the rise of the IgA-producing plasma cell level, MAdCAM-1 cannot be the rate determining factor governing extravasation of primed B cells to the mammary gland. We also conclude that the pIgR is present in sufficient amounts to enable increasing S-IgA secretion into the milk during lactation.     

Adipose tissue: a vital in vivo role in mammary gland development but not differentiation.

Development and differentiation of the mammary gland occurs by means of critical stromal-epithelial interactions. Although many studies have attempted to understand these complex interactions, it has been difficult to demonstrate the essential role of adipose tissue in the development and function of the mammary gland. By using the A-ZIP/F-1 transgenic mice lacking in white adipose tissue (WAT), we have studied the role of adipocytes in mammary gland development and differentiation. In the absence of WAT, rudimentary mammary anlagen form but are unable to grow and branch normally, resulting in a few, short, severely distended ducts. However, during pregnancy, a tremendous amount of epithelial cell division and alveolar cell formation occurs even in the absence of adipocytes, illustrating that adipose tissue is not required for mammary gland differentiation. Mammary gland transplantation revealed that epithelial cells from these transgenic mice possess the potential for normal growth and differentiation when placed into a normal stromal environment. These experiments clearly demonstrate that the absence of adipocytes in the mammary gland results in disruption of stromal-epithelial interactions that prevent normal mammary gland development. The rudimentary epithelial anlage, however, contain mammary stem cells, which are fully capable of alveolar differentiation.     

Transduction pathways of GH in ovine mammary acini involving regulated and functional growth hormone receptors

We have investigated the localization and regulation of growth hormone (GH) receptor-related proteins in the ovine mammary gland. Using a new rabbit polyclonal antibody (7122A) directed against the recombinant extracellular domain of GH receptor (GHR-ECD) for western blot assays, we found two bands with apparent molecular weights of 70,000 and 50–60,000?Da in ovine mammary gland solubilized proteins. The 70,000-protein was consistent with a membrane GH receptor form deprived of post-translational modifications such as phosphorylation, glycosylation or ubiquitin binding. The 50–60,000?Da was consistent with soluble GH binding protein, generated by the cleavage of membrane GH receptor. The intensity of related GHR proteins increased slightly throughout mammary gland development and was correlated with the amount of GHR immunoreactivity observed in the mammary gland sections. Moreover, a temporal and spatial regulation of GHR immunoreactivity was found in alveolar epithelial cells. Clearly, marked GHR immunoreactivity was associated with the apical membranes of alveolar epithelial cells at lactation. The up-regulation of related GHR proteins during the differentiation of mammary tissue supports the hypothesis that GH may act specifically via its own receptors. In ovine mammary cells, GH was able to promote a time-dependent activation of MAP kinases such as prolactin (Prl) and placental lactogen (PL). GH was also able to promote slight and transient Stat5 DNA-binding activity. Differences in the time dependence of Stat5 DNA-binding activation by the three different ligands, GH, Prl and PL, were found. All these results emphasize the direct action of GH on ovine mammary cells and highlight the specificity of action of this ligand.     

Characterization of Primary Cilia Distribution and Morphology During Lactation,Stasis, and Involution in the Bovine Mammary Gland

Primary cilia are small, sensory organelles projecting from virtually all cells and are vital for cellular and tissue function. Their distribution in bovine mammary tissue has not previously been assessed, despite the potential for these organelles to provide specialized perceptive and regulatory functions to this acutely responsive and adaptive gland. The research objectives were to assess ciliary distribution and morphology during active lactation, milk stasis, and early involution using tissue samples obtained following the abrupt cessation of milk removal in nonpregnant, Friesian dairy cows at mid‐lactation. Routinely processed tissue sections were obtained at intervals from 6 to 192 hr after the last milking (N = 3 animals per group) and assigned to active lactation (6–12 hr), milk stasis (18–36 hr), and early involution (72–192 hr). Primary cilia were observed in luminal secretory epithelial cells (SECs), myoepithelial cells, and stromal cells following fluorescent immunohistochemistry and confocal microscopy. In SECs, some primary cilia appeared deflected against the apical cell membrane. The proportion of those deflected was greater during milk stasis than active lactation. Data show that primary cilia were suitably placed in three important cell types to potentially coordinate various forms of signal transduction relying on both mechanosensation and chemosensation, according to the physical and physiological state of the gland. Their cell‐type distribution and morphology provide new directions in the study of mammary regulation to enhance the understanding of how various mammary‐specific cellular responses may be initiated by biochemical or local biophysical factors. Anat Rec, 296:1943–1953, 2013. © 2013 Wiley Periodicals, Inc.     

Immunohistochemical comparison of T-cell and macrophage populations in mammary tissue of control and Trichinella spirails-infected rats

We have shown that antigen-specific T lymphocytes can mediate maternal-to-neonatal immunity during lactation. Present studies address the dynamics of lymphocyte accumulation in the mammary gland during normal and disease stimulated conditions. Monoclonal antibodies specific for total T cells, suppressor/cytotoxic and helper subsets, and macrophages were used in conjunction with immunohistochemistry to identify and count the individual cell types. In unstimulated mammary tissue, following a rise in T cells to maximal numbers in late pregnancy, the total number of T cells/high power field (HPF) was significantly diminished in early lactation and continued to decline to the late lactation period. Both the numbers of T cells/HPF located in the mammary alveolar epithelium and surrounding connective tissue were significantly reduced in early lactation as compared to late pregnancy. This indicates the possible passage of cells into the milk during lactation. Prior infection of the mother with Trichinella spiralis and a secondary challenge 48 h. before sacrifice caused a significant reduction in the number of T cells in the mammary tissue in early lactation as compared with unstimulated controls, indicating the possibility of an even greater outflow of T cells into milk. In controls, the T-suppressor/cytotoxic subtype showed a reduction in early lactation versus late pregnancy but showed no shifts in total cells/HPF during infection. The T-helper subtype in controls remained unchanged from late pregnancy to early lactation with a considerable decline in late lactation. However, the T-helper cells were significantly decreased in T. spiralis-treated animals as compared with noninfected controls in early lactation. The population of macrophages declined from late pregnancy to early lactation but remained unchanged between normal and T. spiralis-infected animals.     

An obesogenic diet started before puberty leads to abnormal mammary gland development during pregnancy in the rabbit

Alterations to the metabolic environment during puberty can impact future lactation efficiency and mammary tumorigenesis. During this study, we used a model of rabbits receiving an obesogenic diet (OD), starting before puberty and extending until mid‐pregnancy. Three months later, the body weight of OD animals was significantly higher than that of controls and their mammary glands displayed a precocious and abnormal development at mid‐pregnancy. OD mammary ducts were filled with dense products, while alveolar structures invaded most of the fat pad. The proportion of secretory epithelium was significantly higher in OD mammary tissue, which contained an abundant accumulation of milk proteins and lipids. In conclusion, an obesogenic diet started before puberty induced an accelerated development of the rabbit mammary gland, leading to an accumulation of secretory products at mid‐pregnancy. These results support the critical influence of nutrition on mammary growth and differentiation, which may be deleterious to mammary development and subsequent lactation. Developmental Dynamics 240:347–356, 2011. © 2011 Wiley‐Liss, Inc.     

Overexpression of human Cripto-1 in transgenic mice delays mammary gland development and differentiation and induces mammary tumorigenesis

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.     

Expression and immunolocalization of the oxytocin receptor in human lactating and non-lactating mammary glands

The milk ejection reflex is mediated by the release of pituitary oxytocin and its interaction with specific receptors within the mammary gland. Although up-regulation of the oxytocin receptor during lactation has been shown for the rat mammary gland by ligand binding assay, investigation of the receptor expression in human breast at the molecular level has not yet been carried out in detail. Here we report the expression and immunolocalization of the oxytocin receptor in the human breast. It appears that the expression level of the receptor- specific mRNA is not significantly elevated during lactation and the protein remains at a relatively low level. However, this lack of increase may be only a dilution effect because of the high level of milk protein expression. Immunohistochemistry and immunoelectron microscopy using three anti-oxytocin receptor antibodies raised against different epitopes of the receptor indicated the presence of receptor immunoreactivity only to a very limited extent in the myoepithelial cells; more specific expression appeared to occur in the ductal/glandular epithelium in both the non-lactating as well as lactating breast. This finding was also confirmed in a New World monkey, the common marmoset (Callithrix jacchus). These results suggest that, at least for human and marmoset, in addition to--or even instead of--myoid cells, the ductal/glandular epithelium is also a target for oxytocin action, not only during lactation but also in the non- lactating breast. Thus, there may be other physiological effects of oxytocin besides direct myoid cell contraction in the breast.      

Changes in the cytologic distribution of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) during in vivo and in vitro mouse mammary epithelial cell expression and differentiation.

HIP/RPL29 is a small, highly basic, heparin/heparan sulfate interacting protein identical to ribosomal protein L29 and present in most adult epithelia. In the present study, we show that mouse HIP/RPL29 is ubiquitously present in adult mammary epithelia and is significantly increased during pregnancy and lactation. We observed for the first time that HIP/RPL29 intracellular expression and distribution varies, depending on the growth/differentiation state of the luminal epithelium. HIP/RPL29 was detected at low levels in mammary glands of virgin animals, increased markedly during lactation, and was lost again during involution. HIP/RPL29, preferentially found in the expanded cytoplasm of mature epithelial cells secreting milk, is present also in the nucleus of proliferating and differentiating ductal and alveolar elements. We used COMMA-D cells as an in vitro model for mammary-specific differentiation and examined similar intracellular redistribution of HIP/RPL29 associated with functional differentiation. However, no changes in HIP/RPL29 expression levels were detected in response to lactogenic hormones. Finally, the cellular distribution of HIP/RPL29 in both nuclear and cytoplasmic compartments was confirmed by transfecting a normal mammary epithelial cell line, NMuMG, with a fusion protein of HIP/RPL29 and EGFP. Collectively, these data support the idea that HIP/RPL29 plays more than one role during adult mammary gland development.     

Response of mononuclear cells to recombinant bovine interleukin‐1β during the non‐lactating period

Interleukin‐1 has been shown to play a critical role in the initial stages of lymphocyte proliferation. Recombinant bovine interleukin‐1 may be useful as an immunoenhancer of bovine mammary gland mononuclear cells to enhance resistance of the mammary gland to infection during the non‐lactating period. The objective of this study was to determine if recombinant bovine interleukin‐1β influenced proliferation of blood and mammary gland mononuclear cells obtained during the non‐lactating period. Bovine mononuclear cells were isolated from five primiparous Holstein cows at 14–18 and 28–32 days of involution, and 7–13 days prior to parturition. Proliferation of mononuclear cells in response to recombinant bovine interleukin‐1β was evaluated in the presence and absence of suboptimal concentrations of concanavalin A and pokeweed mitogen. Varying concentrations (0.78–25 ng ml^?1) of recombinant bovine interleukin‐1β did not influence blood or mammary gland mononuclear cell proliferation in the presence or absence of mitogens at any of the time periods studied. Data suggest that exogenous recombinant bovine interleukin‐1β may not be effective as an immunoenhancer of existent mammary gland mononuclear cell populations.