Immunohistochemistry combined with periodic acid-Schiff for bovine mammary gland with protothecal mastitis

Immunohistochemistry (IHC) of bovine cytokeratin combined with periodic acid-Schiff (PAS) was applied to study the pathogenesis, localization and distribution of Prototheca zopfii in bovine mammary protothecosis. The standard immunohistochemical procedure using anti-bovine cytokeratin was employed before and after PAS staining to optimize this combined method. The best results were obtained when IHC procedures were performed first. Most of the epithelial cells reacted strongly with the pancytokeratin antibody. Protothecal cell walls stained well with PAS. Algal organisms were present within the lumen and between the epithelial lining and basement membrane of the affected alveoli, but not inside the positive mammary epithelial cells. This combined staining method resulted in clear alveolar epithelial detail and good contrast between the epithelial cells and algae, and contributed to studying the pathogenesis of P. zopfii in mammary protothecosis.     

Keratin-like proteins in normal and neoplastic cells of human and rat mammary gland as revealed by immunofluorescence microscopy

Normal and neoplastic human breast tissue as well as lactating and nonlactating rat mammary glands and 7,12-dimethylbenz(alpha)-anthracene-induced mammary adenocarcinomas of rat, were examined by indirect immunofluorescence microscopy using guinea pig antibodies to human and bovine epidermal prekeratin and to cytokeratin polypeptide D from mouse hepatocytes. In normal mammary glands of both species, lactating rats included, the antibodies raised against human and bovine epidermal prekeratins strongly stained ductal and myoepithelial cells, whereas antibodies to hepatic cytokeratin D revealed, in addition, fibrillar staining in cells of the alveolus-like terminal lobular units and in milk secreting cells of the rat. The presence of some finely dispersed intermediate-sized filaments of the cytokeratin type in lactating alveolar cells of rat mammary gland was also demonstrated by electron microscopy. In human intraductal mammary carcinomas the antibodies to epidermal prekeratins showed staining in myoepithelial cells and intralumenal papillary protrusions of the tumor, whereas the antibodies to hepatic cytokeratin D presented an almost complementary pattern in that they showed strongest staining in the more basally located layers of tumor cells. Intraductal adenocarcinomas of rats showed strong staining with all keratin antibodies examined. In contrast to previous studies using exclusively antisera raised against epidermal prekeratin, out results show that all types of neoplastic and non-neoplastic epithelial cells of mammary gland of both species contain-at least some-filaments of the cytokeratin type identifiable by immunologic reaction, if antibodies are used that recognize a broad range of epidermal and nonepidermal cytokeratins. Consequently, such broad range antibodies to keratin-like proteins provide adequate tools to identify and characterize neoplastic and non-neoplastic epithelial cells and to eliminate false negative immunocytochemical findings in tumor diagnosis. In addition, our observation that in the same human carcinoma two cell types can be distinguished by their reaction with two different antibodies to cytokeratins from epidermis and liver, respectively, indicates that the cells of a given carcinoma can differ in their cytoskeletal composition, thus presenting further criteria for diagnostic differentiation.     

Monoclonal antibody CD133-2 (AC141) against hematopoietic stem cell antigen CD133 shows crossreactivity with cytokeratin 18.

CD133 is an antigen expressed on hematopoietic progenitor cells and on some epithelial cells. We previously reported that a commercially available antibody against CD133, CD133-2/AC141, also reacted with an intracellular protein in placental trophoblasts. Here we show by 2D electrophoresis and mass spectroscopy that this reactivity is with cytokeratin 18, a cytokeratin present in most simple epithelia. Immunohistochemistry (IHC) with CD133-2/AC141 on a trophoblast cell line displayed a staining pattern typical for the cytoskeleton. Cryostat sections of stratified epithelia lacking cytokeratin 18 did not react with CD133-2/AC141. In conclusion, care must be taken not to misinterpret staining patterns using CD133-2/AC141 in IHC.     

Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues

A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room temperature. When bound to membrane, PAS IV was resistant to digestion by a number of proteinases, although after solubilization with non-ionic detergents, the protein was readily degraded. Amino acid analysis of the purified protein revealed a high percentage of amino acids with nonpolar residues. The location of PAS IV was determined in bovine tissues by using immunofluorescence techniques. In mammary tissue, PAS IV was located on both the apical surfaces of secretory epithelial cells and endothelial cells of capillaries. This glycoprotein was also detected in endothelial cells of heart, liver, spleen, pancreas, salivary gland, and small intestine. In addition to mammary epithelial cells, PAS IV was also located in certain other epithelial cells, most notably the bronchiolar epithelial cells of lung. The potential usefulness of this protein as a specific marker of capillary endothelial cells in certain tissues is discussed.     

PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV).

PAS IV is a 78-kDa (bovine) to 80-kDa (human) integral membrane glycoprotein of unknown function which is found in mammary epithelial cells. We now report the purification of human PAS IV and native bovine PAS IV from the milk fat globule membrane (MFGM), a preparation of apical plasmalemma from epithelial cells of lactating mammary tissue. N-Terminal sequence analyses of human and bovine PAS IV revealed homology to the N-terminal sequence of the 88-kDa human endothelial and platelet glycoprotein CD36. The similarity of MFGM PAS IV to platelet CD36 was further established by immunoblots of purified platelet CD36 and MFGM PAS IV with MFGM PAS IV specific antiserum. The removal of N-linked oligosaccharides from PAS IV and CD36 by treatment with endoglycosidase F reduced the apparent Mr of both proteins to approximately 57,000. These data suggest that PAS IV and CD36 are similar if not identical polypeptides that undergo cell type specific glycosylation.     

Identification of Cell Types in the Developing Goat Mammary Gland

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

Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells

Two monoclonal antibodies, KA 1 and KA 4, raised against human epidermis, were biochemically and immunologically characterized and were shown to react with specific cytokeratin polypeptides. On frozen sections of human mammary gland, these antibodies distinguish between myoepithelial and luminal epithelial cells. We present evidence that in these cells KA 1 antibody recognized cytokeratin 5 and KA 4 antibody cytokeratin 19. In normal mammary tissue, KA 4 antibody invariably reacted with the epithelial cells lining the lumina of acini, ductules, ducts, and sinus. In contrast, KA 1 antibody decorated only the myoepithelial and basal epithelial cells of acini, ducts, and sinus. In ductules, however, KA 1 also stained the luminal cells. All 73 invasive lobular and ductal carcinomas studied reacted with KA 4 antibody; five of these were also positive, apparently in the same tumor cells, with KA 1. The tumor cells of in situ carcinomas were also stained in a homogeneous pattern with KA 4 antibody; KA 1 antibody reacted only with the surrounding myoepithelium. In epithelial hyperplasias, the proliferating cells were decorated by KA 1 and KA 4 antibodies in a heterogeneous pattern. Other antibodies were used for comparison. The results are discussed with respect to epithelial differentiation and pathogenesis and to the application of such antibodies for immunohistodiagnosis of mammary lesions.     

牛乳腺上皮细胞的分离培养及其生物学特性

采用胶原酶消化法和胰蛋白酶选择性消化法分离、培养和纯化牛乳腺上皮细胞。形态学观察表明,培养的细胞具有典型的上皮细胞形态特征;染色体分析结果表明,培养的细胞具有正常的染色体数目。通过荧光免疫细胞染色方法鉴定了培养的细胞表达上皮细胞特异的角蛋白5和8。该细胞在添加胰岛素、氢化可的松以及羊催乳素的无血清培养液中诱导培养时,用RT-PCR方法检测到了β-酪蛋白基因的转录。这些结果表明,分离培养的细胞是乳腺上皮细胞,这些细胞在诱导培养的条件下能够转录表达β-酪蛋白。     

Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice

Systemic growth and branching stimuli, and appropriate interactions with the host stroma are essential for the development of foreign epithelia in the mammary gland of immunodeficient mice. These factors were manipulated to promote and investigate the generation of representative bovine epithelial morphology in the transplanted mouse mammary stroma. The bovine mammary epithelium is unique in its commitment to rapid proliferation and high rate of differentiation. Its morphological organization within a fibrotic stroma resembles that of the human breast, and differs significantly from the rudimentary ductal network that penetrates a fatty stroma in mice. Transplantation of bovine mammary epithelial cells into the cleared mammary fat pad of NOD-SCID mice led to continuous growth of epithelial structures. Multilayered hollow spheres developed within fibrotic areas, but in contrast to mice, no epithelial organization was formed between adipocytes. The multilayered spheres shared characteristics with the heifer gland’s epithelium, including lumen size, cell proliferation, cytokeratin orientation, estrogen/progesterone receptor expression and localization, and milk protein synthesis. However, they did not extend into the mouse fat pad via ductal morphology. Pre-transplantation of fibroblasts increased the number of spheres, but did not promote extension of bovine morphology. The bovine cells preserved their fate and rarely participated in chimeric mouse–bovine outgrowths. Nevertheless, a single case of terminal ductal lobuloalveolar unit (TDLU) development was recorded in mice treated with estrogen and progesterone, implying the feasibility of this representative bovine morphology’s development. In vitro extension of these studies revealed paracrine inhibition of bovine epithelial mammosphere development by adipocytes, which was also generalized to breast epithelial mammosphere formation. The rescue of mammosphere development by fibroblast growth factor administration evidences an active equilibrium between inhibitory and supportive effects exerted by the adipose and fibrotic regions of the stroma, respectively, which determines the development of foreign epithelium.     

牦牛DBI基因的分子特征及其在乳腺组织中的表达与定位

地西泮结合抑制因子(Diazepam binding inhibitor,DBI)与酰基辅酶A具有高亲和力,在动物组织中广泛存在,与脂肪酸代谢、类固醇激素合成密切相关。为研究DBI基因的分子特征及该基因在乳腺发育中的作用,对牦牛DBI基因编码区进行克隆,进行生物信息学分析;采用实时荧光定量PCR (Quantitative real-time PCR,qPCR)、蛋白免疫印迹技术(Western blotting,WB)和免疫组织化学(Immunohistochemistry,IHC)方法对牦牛泌乳前期、泌乳期和干乳期的乳腺组织中DBI的相对表达量和表达部位进行研究。DBI序列分析显示：牦牛DBI基因编码区序列长264 bp,编码87个氨基酸残基,与牛的同源性高达99.62％;qPCR数据表明：牦牛泌乳前期乳腺组织中DBI基因的相对表达量显著高于泌乳期和干乳期(P< 0.05);WB结果显示：牦牛泌乳前期乳腺组织中DBI蛋白的表达量最高,干乳期次之,泌乳期最低(P< 0.05);IHC结果表明：不同发育时期的牦牛乳腺组织中DBI的表达部位并无明显差异,主要表达于乳腺腺泡上皮细胞、导管上皮细胞及小叶间质细胞。DBI在不同发育时期牦牛乳腺组织中的相对表达量具有明显差异(P< 0.05),揭示DBI可能参与牦牛乳腺发育的过程,这为进一步探究DBI基因在生物体中的作用提供相应的理论参考。     

Cytokeratin expression during mouse embryonic and early postnatal mammary gland development

Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progenitor cells exist during embryonic development. Here, we use double/triple-label immunofluorescence and immunohistochemistry to systematically compare the expression of cytokeratin 5 (K5), cytokeratin 6 (K6), cytokeratin 8 (K8), cytokeratin 14 (K14) and cytokeratin 19 (K19) in embryonic and early postnatal mouse mammary glands. We show that K6⁺ and K8⁺/K14⁺ putative mammary progenitor cells arise during embryogenesis with distinct temporal and spatial distributions. Moreover, we describe a transient disconnection of the expression of K5 and K14, two cytokeratins that are often co-expressed, during the first postnatal weeks of mammary development. Finally, we report that cytokeratin expression in cultured primary mammary epithelial cells mimics that during the early stages of postnatal mammary development. These studies demonstrate an embryonic origin of putative mammary stem/progenitor cells. Moreover, they provide additional insights into the use of specific cytokeratins as markers of mammary epithelial differentiation, or the use of their promoters to direct gene overexpression or ablation in genetic studies of mouse mammary development.     

Dual immunofluorescence labeling with cell-specific markers localizes BRCA1 in both basal and luminal epithelial cells in primary outgrowth from noncancerous mammary ductal and alveolar preparations

The role played by either of the two differentiated mammary epithelial cell types in human breast cancer progression is currently not defined. This work addresses the question of whether the mammary tumor suppressor gene product BRCA1 is localized in basal and/or luminal epithelial cells in noncancerous outgrowth cultured from breast organoids. Primary epithelial cell outgrowths from ductal and alveolar preparations were directly employed to facilitate small-scale analysis under conditions closely approximating intact tissue. BRCA1 immunofluorescence was detected for the most part in cell nuclei of the epithelial outgrowth when using confocal microscopy. Nuclear staining was punctate in the cells with higher labeling intensity. Only minimal nonspecific staining was observed with mouse IgG as a negative primary antibody control or with primary antibody against the cell membrane receptor ErbB2, reported to be expressed in breast cancer, but was either not detectable or weakly expressed in normal breast tissue. Dual labeling was used to distinguish which epithelial cell type(s) stains for BRCA1. Primary monoclonal antibody against vimentin was used to identify basal cells, while antibody against cytokeratin 19 was used to identify luminal cells. Monoclonal antibody against BRCA1 was used for colabeling with each of these markers. Epifluorescence microscopy revealed BRCA1 immunoreactivity in both basal and luminal interphase cells. BRCA1 immunofluorescence was diffusely located about the chromosome mass during mitosis.     

Localization of mammary-derived growth inhibitor in capillary endothelial cells of the bovine mammary gland

Mammary-derived growth inhibitor (MDGI) has previously been localized in the mammary parencyma, dependent on the stage of differentiation of the mammary gland. Here, we have elucidated the distribution of MDGI in the mammary stroma by a combined immunohisto-and cytochemical analysis with antibodies raised against MDGI. Distinct staining of capillary endothelial cells has been revealed. Although its subcellular distribution resembles former observations in secretory epithelial cells, the expression of MDGI in capillary endothelial cells clearly precedes that in secretory epithelial cells. On the other hand, no endothelial MDGI staining has been detected in bovine heart, which contains a fatty acid-binding protein almost identical to MDGI. The localization of MDGI in the mammary capillary endothelium is discussed in terms of its possible involvement in the intracellular transport of hydrophobic ligands or in the regulation of endothelial cell proliferation.     

转溶菌酶基因山羊乳腺上皮细胞的原代培养及特性分析

采用组织块法、胰酶差速消化法分离了人溶菌酶(hLYZ)转基因山羊的乳腺上皮细胞,建立了转基因羊乳腺上皮细胞(TGMEC)的体外培养体系,并对其生长与体外分泌特性进行了分析.研究显示其体外生长曲线符合典型的S型,传代期间二倍体染色体数正常;通过免疫荧光染色、RT-PCR发现该细胞能表达上皮细胞所特有的角蛋白18,并能表达内源性乳蛋白(酪蛋白与乳球蛋白);进一步通过酶联免疫试验发现该乳腺上皮细胞在体外培养中可持续稳定地分泌重组人溶菌酶.由此获得的人溶菌酶转基因羊乳腺上皮细胞系为乳腺生物反应器研究提供了一个重要细胞膜型.     

绵羊高效转基因通用型piggyBac转座子载体构建及功能验证

piggyBac (PB)是一种能在多种动物细胞中进行转座的DNA转座子,作为一种转基因工具已被广泛应用于各种哺乳动物转基因研究中。针对不同物种对PB转座子进行改造,是提升其通用性的必要手段。为构建基于绵羊细胞进行转基因操作的通用型PB转座子载体,本研究对PB转座酶(PBase)基因进行绵羊密码子偏好性优化并将其克隆到pBNW-TP1载体中,成功构建了PB转座子载体pBNW-TP2。将pBNW-TP2转染到绵羊成纤维细胞和乳腺上皮细胞中,利用G418筛选获取稳定转染细胞株;利用Tail-PCR检测稳定转染细胞株的PB转座位点,对细胞阳性克隆进行亚甲蓝染色;利用非配对t检验确认其转座效率。结果表明,pBNW-TP2成功介导了绵羊成纤维细胞和乳腺上皮细胞转基因阳性细胞株的生产;PB转座位点检测表明pBNW-TP2能特异性整合到绵羊基因组TTAA位点,其整合位点倾向于功能基因间;亚甲蓝染色统计分析结果提示pBNW-TP2介导的转基因效率显著提升。本研究成功构建了绵羊通用型PB转座子载体pBNW-TP2,并在绵羊体细胞中对其特性进行验证和分析,为PB转座子在绵羊体细胞中开展转基因相关研究提供了科学依据。     

Irregular distribution of mammary-derived growth inhibitor in the bovine mammary epithelium

Localization of a mammary-derived growth inhibitor (MDGI) in the bovine mammary gland was verified by light-and electron-microscopic methods. Expression of MDGI, which is known to inhibit the growth of mammary epithelial cell lines in vitro, was found to be highest in the late pregnant and in the lactating state. A combination of immunohistochemical and immunocytochemical methods with semi- and ultrathin resin sections revealed marked variations in MDGI staining. High MDGI levels were predominantly detectable in epithelial cells with large milk fat droplets. Distinct cell types that were almost free of label could be identified among bovine mammary epithelial cells that always exhibited high MDGI levels. Similar results were obtained when using a serum-free organ culture system in which MDGI was hormonally induced in cell types of comparable differentiation state. The specific occurrence of the growth inhibitor in developing alveoli and certain cell types points to the association between MDGI expression and functional differentiation in the normal mammary gland.     

Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium

The expression of the apoptosis-related proteins Bcl-2 and Bax was investigated by immunohistochemistry in the normal non-lactating human mammary gland in relation to cell proliferation and apoptosis. In order to characterize individual Bax/Bcl-2-immunoreactive cells, the epithelial markers cytokeratin 14 and 19 and the macrophage marker CD 68 were used. Secretory-like differentiation of epithelial cells was characterized by histochemistry and lectin staining of surface glycoconjugates. Cell proliferation was exclusively found in glandular epithelial cells with broad contact to the ductular lumen, whereas nuclei with apoptosis-related DNA fragmentation were seen predominantly in basally located glandular epithelial cells and in myoepithelial cells. Weak immunoreactivity for Bcl-2 and Bax was present throughout all epithelia, suggesting a balance between pro- and antiapoptotic effects in the majority of epithelial cells. However, specific cells showed a strong staining for Bax or Bcl-2. The strongly Bcl-2-immunoreactive epithelial cells were not identical with proliferating cells, but they resembled them in configuration and in the luminal intraepithelial position. In contrast, the strongly Bax-positive epithelial cells had no or only a narrow contact to the ductular lumen. The different patterns of Bax/Bcl-2 immunoreactivity in specific glandular epithelial cells suggest that there are also different grades of susceptibility towards apoptotic stimuli in individual glandular epithelial cells. We conclude that specific Bax/Bcl-2 expression patterns could reflect particular cell differentiation states, and that the strongly Bcl-2-positive cells in part could represent epithelial stem cells.