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.     

The role of estrogen receptors,erbB receptors,vascular endothelial growth factor and its receptors,and vascular endothelial growth inhibitor in the development of the rat mammary gland

We identified the localization and distribution of cell-specific epidermal growth factor receptors (EGFRs: erbB-1, erbB-2, erbB-3, erbB-4), vascular endothelial growth factor (VEGF), VEGF receptors [VEGFRs: VEGF-R1 (flt-1), VEGF-R2 (flk-1/KDR), VEGF-R3 (flt-4)], vascular endothelial growth inhibitor (VEGI), and estrogen receptor (ER), and determined whether or not these growth factors in rat mammary glands are functional. Thirty-five adult female Spraque-Dawley rats were randomly divided into five groups, each of which were at the 7th, 14th, and 21st day of pregnancy; 7th day post-delivery; and 7th day after weaning. It was determined that erbB, VEGF and its receptors, VEGI, and ER stained at different intensities. Intense staining was observed, in particular, in erbB receptors during pregnancy and involution, and also in VEGF and its receptors during lactation, while ER stained during the last periods of pregnancy and lactation. In conclusion, the expression of erbB, VEGF and its receptors, and ER were determined at varying intensities at different sites of the mammary gland during pregnancy, lactation, and involution periods.     

Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling

Background: The mammary epithelium undergoes proliferation and regression accompanied by remodeling of the fibrocellular and vascular stroma. Mast cells are abundant in these compartments and have been implicated in remodeling during wound healing and cancer progression. The purpose of this study was to test the hypothesis that mast cell abundance correlates with physiologic mammary tissue remodeling during estrous cycling, lactogenesis (pregnancy and lactation) and involution. Results: Mast cell and capillary frequency were quantified in the stroma surrounding ducts and lobules from mammary glands of rats. During estrous cycling, periductal mast cell numbers were unchanged, but lobule‐associated mast cells significantly increased in the regressive phase of diestrus II. During lactogenesis, lobular stroma mast cells peaked early in pregnancy, at D2, followed by a significant decrease throughout lactation. Involution was associated with a rapid return in mast cell numbers, similar to diestrus II. Lobular vascularization peaked during the state of metestrus, when limited secretory differentiation occurs. Lobular angiogenesis peaked at D7 of pregnancy, regressed, and then returned to high levels during lactation and early involution, when secretory differentiation is high. Conclusions: These results suggest mast cells are predominantly associated with regressive lobular remodeling during cycling and involution, whereas angiogenesis is predominantly associated with secretory differentiation. Developmental Dynamics 241:890–900, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells.

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.     

Inhibition of ductal morphogenesis in the mammary gland of WAP<Emphasis Type="Italic">-fgf4</Emphasis> transgenic mice

We previously demonstrated that expression of human FGF4 in the epithelial compartment of murine mammary glands caused hyperplasia during lactation and a dramatic delay in gland involution due to inhibition of cellular apoptosis. We now analyse the effects of transgene expression during development of the organ. Expression of WAP-Fgf4 initiate with the onset of sexual hormones (4 weeks of age), and defects in morphogenesis of the organ were already apparent at 5 weeks of age and persisted throughout all stages of post-natal development. These defects involved ductal development, but not lobuloalveolar morphogenesis, and were due to a decrease in the level of apoptosis within the terminal end buds. We also show that regulation of apoptosis by FGF4 in the mammary gland, both during development and involution, could occur via inhibition of Bcl2 expression. Overall our data demonstrate that FGF4 is a regulator of mammary epithelial cells apoptosis during all stages in which programmed cell death is an important mechanism of development, namely morphogenesis and involution. We also suggest that this growth factor could act by interfering with the Bcl2 pathway.     

Inhibition of ductal morphogenesis in the mammary gland of WAP-fgf4 transgenic mice

We previously demonstrated that expression of human FGF4 in the epithelial compartment of murine mammary glands caused hyperplasia during lactation and a dramatic delay in gland involution due to inhibition of cellular apoptosis. We now analyse the effects of transgene expression during development of the organ. Expression of WAP- Fgf4 initiate with the onset of sexual hormones (4 weeks of age), and defects in morphogenesis of the organ were already apparent at 5 weeks of age and persisted throughout all stages of post-natal development. These defects involved ductal development, but not lobuloalveolar morphogenesis, and were due to a decrease in the level of apoptosis within the terminal end buds. We also show that regulation of apoptosis by FGF4 in the mammary gland, both during development and involution, could occur via inhibition of Bcl2 expression. Overall our data demonstrate that FGF4 is a regulator of mammary epithelial cells apoptosis during all stages in which programmed cell death is an important mechanism of development, namely morphogenesis and involution. We also suggest that this growth factor could act by interfering with the Bcl2 pathway.     

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.     

An ultrastructural and morphometric analysis of the Sertoli cell during the spermatogenic cycle of the rat

Summary The ultrastructure of Sertoli cells from selected stages of the spermatogenic cycle was assessed by morphometric analysis which showed significant changes in the morphological features of Sertoli cell cytoplasm at the commencement of the cycle (stage II) compared to the middle (stages VII-VIII) and the completion of the cycle (stages IX-XIV). Total volume and surface area of organelles (rough and smooth endoplasmic reticulum (ER), lysosomes, mitochondria and Golgi) exhibited stage-dependent and cyclic variations as did the total surface area of Sertoli cell plasma membrane. Polarization of cytoplasmic organelles to basal or columnar regions of the Sertoli cell, exhibited particularly by the Golgi, rough ER and lysosomes also showed marked cyclic fluctuations during the spermatogenic cycle. Rough and smooth ER exhibited the most dramatic stage-dependent changes in total volume and surface area the former being respectively largest and smallest in stages VII-VIII and XIII-XIV, the latter organelle presenting the reverse pattern in these two groups of stages. Similar stage-dependent alterations of lysosome volume and surface area were also noted, being maximal during stages XIII-XIV-II and reaching a nadir at stage VIII. Although the functional role of most Sertoli cell organelles and inclusions remain largely unknown, the present study suggests that the cyclic and stage-dependent variations in ultrastructure probably reflect major changes in Sertoli cell function necessary for the regulation of the spermatogenic cycle.     

Glucose 6-phosphatase activity in pregnant and lactating mammary glands of the mouse

Glucose 6-phosphatase activity was studied in the secretory epithelial cell and other cell types composing alveoli of the mammary gland (cytochemical study) and in the whole mammary gland (biochemical study) of pregnant and lactating mice. The reaction product for the enzyme activity was seen in the endoplasmic reticulum and nuclear envelope in secretory epithelial cells from all animals examined (days 7 and 14 of pregnancy, and days 0, 3, 10, and 20 of lactation. The amounts of the reaction product appeared scarce at day 7 of pregnancy, moderate at day 14 of pregnancy and day 0 of lactation, and abundant at days 3 and 10 of lactation. The reaction product, however, became generally scarce at day 20 of lactation. Biochemical activity was relatively low at days 7 and 14 of pregnancy and days 0 and 20 of lactation, while it was high at days 3 and 10 of lactation. The increased activity is probably related to functions of secretory epithelial cells in the lactating gland.     

Lactation Stage-Dependent Changes in Levels of Tumor Necrosis Factor/Cachectin in Milk

PROBLEM: Determination of lactation stage-dependent changes in levels of tumor neurosis factor (TNF) in milk. METHOD: Bioassay and immunoblocking assay were used to identify and assay tumor necrosis factor (TNF; mostly TNFα) in bovine milk at different stages of lactation. RESULTS: TNFα levels in milk started to increase steadily after the onset of drying-off (weaning/involution), peaked at 4 to 6 wk prior to parturition and precipitously decreased to undetectable levels at parturition (colostrum). Thereafter, TNFα reappeared and maintained midlevel concentration in the mature (normal) milk throughout the rest of the lactation cycle. Analysis of cells in mammary secretions by flow cytometry revealed that elevated TNFα levels coincided with an increase in macrophages in the secretion from the dry period. CONCLUSIONS: These lactation stage-dependent changes in TNFα levels reflect differential effects that TNFα have on involution and prepartum remodeling of the mammary gland of the dam and on gastrointestinal development and immunoregulatory function of the suckling.     

Cellular proliferation in the rat mammary gland during pregnancy and lactation

Cellular proliferation during different stages of pregnancy and lactation in the rat mammary gland was studied by use of autoradiography at the ultrastructural level or in combination with immunocytochemistry. The study consisted of two groups of animals, young 45-day-old rats undergoing their first pregnancy and old multiparous rats undergoing their third pregnancy. Both groups of animals were injected with [3H]thymidine, 2 hours before sacrifice. The timing of proliferative activity through pregnancy and lactation was similar in the two groups. A peak level of proliferation was observed on the 5th day of gestation and the rate of proliferation was at its lowest at the end of gestation. A second small peak was observed on the 3rd day of lactation. In virgin animals, both epithelial and myoepithelial cells showed a high level of proliferative activity. The epithelial cells continued to divide during all stages of their differentiation, throughout pregnancy and lactation. Myoepithelial cells showed active division in early pregnancy but their rate of proliferation declined as the cells acquired abundant cytoplasmic filaments for their contractile function. Intra-alveolar dendritic cells, which have been recently identified in the lactating rat breast, showed a high proliferative rate at the end of gestation.     

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.     

An ultrastructural study of normal human mammary epithelial cells in culture

The ultrastructure of normal human mammary cells cultured from post-weaning breast fluids is described. Cells from confluent monolayers in two week old cultures were studied. The epithelial nature of these cells was established by the demonstration of a well developed system of cell-to-cell interdigitation and numerous desmosomes. These cells also share with breast epithelial cells in vivo, polarity, with blunt short microvilli on the apical surface and an oriented arrangement of organelles in the basal and apical portions of the cells. The Golgi apparatus, which is the most highly developed organelle, is localized in the apical pole and contains substantial quantities of secretory material in the cisternae and vesicles. A variegated palisade of finely granular material mixed with tonofilaments is seen in the basal portion of the cells; many of these tonofilaments end in the terminal web of the desmosomes. The regular occurrence of these cells in breast fluids during the terminal phases of lactation suggests that their separation is a part of normal breast involution.     

Quantitative and ultrastructural characteristics of immunocompetent cells in the mammary gland during pregnancy and lactation

The quantity and ultrastructure of immunocompetent cells (ICC) were evaluated in the of rat mammary gland in dynamics of pregnancy and lactation. ICC found between the alveolar epithelial cells were represented by monocytes, small and medium lymphocytes, including some granule-containing lymphocytes. The quantity of ICC was found to increase in dynamics of pregnancy and to reach a its maximum on day 3 of lactation. This was accompanied by the appearance of macrophages and plasma cells. Similar changes were found in stromal ICC. It is suggested that quantitative and structural dynamics of mammary ICC is associated with provision of immune reactivity of the newborn and its adaptive immunity.     

成年大鼠不同发育时期乳腺上皮小亮细胞CD24的免疫组化研究

目的观察成年大鼠乳腺不同发育时期CD24阳性表达的乳腺上皮小亮细胞部位、数量的变化。方法制备成年大鼠乳腺不同发育时期（选取静止期、孕期、哺乳期、退化期）组织标本，采用免疫组织化学和光镜技术检测CD24的表达。结果CD24在导管小亮细胞中的染色程度普遍高于腺泡；相比于静止期和退化期，CD24更多地表达于孕期和哺乳期的小亮细胞；静止期和退化期表达无显著性差异（P〉0．05）；孕期和哺乳期表达也无显著性差异（P〉0．05）。结论CD24蛋白参与了正常成年大鼠乳腺组织的发育过程，且可能是鉴定乳腺干细胞（MaSCs）及观察MaSCs发育与分化过程的重要分子。     

Cell-matrix interactions during development and apoptosis of the mouse mammary gland in vivo.

Epithelial cell survival is dependent on extracellular signals provided by both soluble factors and by adhesion. In the mammary gland, extensive apoptosis of epithelial cells occurs rapidly when lactation ceases, but the mechanism of apoptosis induction is not known. In tissue culture, mammary epithelial cells require laminin as a survival ligand and specific beta1 integrins are necessary to suppress apoptosis. To explore the possibility that dynamic changes in cell-matrix interactions contribute to the onset of apoptosis during mammary involution in vivo, a detailed immunohistochemical analysis of the expression of integrin subunits and their extracellular matrix ligands during mouse mammary gland development has been performed. The kinetics of apoptosis were determined by using tissue samples obtained from virgin, pregnant, lactating, and involuting gland. The maximal elevation of apoptosis occurred within 24 hr of weaning as determined by histologic analysis and caspase-3 staining. A wide variety of laminin subunits, together with nidogen-1 and -2, and perlecan were identified within the basement membrane region of epithelial ducts, lobules, and alveoli in both human and mouse mammary gland. However, no change in the distribution of any of the basement membrane proteins or their cognate integrin receptors was observed during the transition from lactation to apoptosis. Instead, we discovered that altered ligand-binding conformation of the beta1 integrin to a nonbinding state coincided with the immediate onset of mammary apoptosis. This finding may provide a novel dynamic mechanism for inhibiting the transduction of extracellular matrix survival signals, thereby contributing to the onset of apoptosis in a developmental context in vivo.     

Stromal regulation of neoplastic development: age-dependent normalization of neoplastic mammary cells by mammary stroma

There is mounting evidence that the stroma plays a crucial role in mammary gland carcinogenesis. Here, we report that mammary gland stroma from mature and multiparous rats prevents neoplastic development and encourages normal ductal growth of grafted epithelial cancer cells. Fifty thousand epithelial cancer cells were injected into the cleared fat pads of virgin hosts at 24, 52, 80, and 150 days of age and of hosts that had undergone two cycles of pregnancy, lactation, and involution. Six months after inoculation, tumor incidence was 75%, 100%, 50%, and 18.2% in 24-, 52-, 80-, and 150-day-old virgin rats, respectively, and 0% in the twice-parous animals. Most remarkably, these neoplastic cells appeared to form normal ducts in all hosts-Ha-ras-1 mutation served as a marker to identify the tumor origin of the outgrowths. The tumor development pattern suggests a parallel to the phenomenon of age- and reproductive state-dependent susceptibility and resistance to chemical carcinogens. As susceptibility to carcinogenesis decreases, the ability of the stroma to reprogram neoplastic epithelial cells increases. Thus, the neoplastic phenotype is context-dependent, and it therefore offers the intriguing possibility that the process of carcinogenesis is amenable to normalization or cure once the mechanisms of stroma-mediated normalization are elucidated and manipulated.