Intact Mammary Gland Research Articles

Bovine Escherichia coli Mastitis and Effects on Milk Microbiota

Escherichia coli is a microorganism that is found in the normal intestinal microbiota of humans and warm-blooded animals, causing intestinal or extra-intestinal infections. Many pathogenic E. coli strains can cause diarrhea, septicemia, neonatal meningitis, mastitis, urogenital system infections and various intra-abdominal, lung, soft tissue and skin infections in pets. Determining the genotypic and phenotypic characteristics of Escherichia coli strains isolated from animals is very important for the prevention of infections caused by E. coli. In recent years, a new animal pathotype mammary pathogenic E. coli (MPEC), which causes mammary gland infections in animals has been included in the extraintestinal pathogenic E. coli group. The fact that approximately %25-%35 of the use of antimicrobials in the treatment of Gram-negative agents is unsuccessful indicates that the use of correct diagnostic tools should take place in routine before etiological diagnosis. Profiles in bovine milk with mastitis suggest that clinical mastitis is associated with dysbacteriosis and that the microbial community in an intact mammary gland helps prevent intramammary infection. In this review, the change in bacterial diversity of milk microbiota due to antimicrobial use in E. coli-induced mastitis cases is discussed together with current studies.

ОПРЕДЕЛЕНИЕ СОДЕРЖАНИЯ ХИМИЧЕСКИХ ЭЛЕМЕНТОВ В ЗДОРОВОЙ ТКАНИ МОЛОЧНОЙ ЖЕЛЕЗЫ ЧЕЛОВЕКА МЕТОДОМ НЕЙТРОННО-АКТИВАЦИОННОГО АНАЛИЗА

Purpose: To develop the method of chemical element (ChE) content measurement in samples of breast tissue (BT) using the activation by neutrons of nuclear reactor combined with the high-resolution spectrometry of gamma-radiation of short-lived radionuclides (INAA-SLR), and to investigate ChE contents in normal human mammary gland. Material and methods: In the samples of BT taken from female with intact mammary glands (mostly died from trauma, n = 38) the contents of calcium (Ca), chlorine (Cl), iodine (I), potassium (K), magnesium (Mg), manganese (Mn), sodium (Na), and strontium (Sr) were measured. To determine these element contents, the method of INAA-SLR was developed. Results: The accuracy of the developed method and the reliability of the results obtained in the study were confirmed by the measurements of international certified reference material IAEA H-4 Animal Muscles and the good agreement with data of its certificate. The main statistic parameters, including, arithmetic mean, standard deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels was calculated for ChE contents in the normal BT. It was shown, for example, that means of mass fractions ± standard errors of means (M±SEM, mg/kg dry tissue) in normal human mammary gland were: Ca 128±14, Cl 1014±146, I 0,82±0,11, K 196±20, Mg 80,0±9,4, Mn 0,27±0,04, Na 665±71 и Sr 5,20±0,75. It was found that the Ca, Cl, I, K, Mg, Mn, Na и Sr contents in human mammary gland very differ from the normal level of these elements in whole blood, muscle and adipose tissue, particularly in contents of I and Sr. Conclusion: The method of INAA-SLR allow to obtain the representative data on Ca, Cl, I, K, Mg, Mn, Na и Sr contents in human BT specimen. It was shown, that such trace elements as I and Sr may be directly involved in the mammary gland function.

Influence of normal mammary epithelium on breast cancer progression: the protective role of early pregnancy

The microenvironment has a well recognized role in breast cancer progression. Despite different theories, the mechanism of early pregnancy protection in mammary carcinogenesis is unknown. Since pregnancy is responsible for mammary gland differentiation, we tested the hypothesis that differentiated mammary epithelial cells may inhibit breast cancer progression. In other words, the protective role of early pregnancy could be due to the inhibitory influences of the more differentiated mammary tissue. In order to test our hypothesis, we used 30 female Balb/c nude mice and MCF-7 cells of breast adenocarcinoma. The female mice were divided into two test groups, group I (GI) and group II (GII), and a control group. In GII, the animals were submitted to epithelial removal in the left fourth inguinal mammary gland at 3 weeks of age. Both groups were given continuous hormonal treatment to simulate the pregnancy development of the mammary gland. Two million MCF-7 cells were then injected into the fourth inguinal mammary gland (GI) or in the respective cleared mammary fat pad (GII). Five weeks later the mice were sacrificed and their tumors removed. Tumor development rates and tumor volumes were determined and proliferation and apoptosis were evaluated by immunohistochemistry. Tumors of GII mice had a larger mean volume than those of GI mice (P = 0.001, Mann-Whitney U-test) and an apparent increase in proliferation, demonstrated by a higher staining intensity for proliferating cell nuclear antigen (PCNA). As tumors presented caspase 8 staining, there may be apoptotic activation involved in cell death, mainly through an extrinsic pathway. These results suggest that a differentiated intact mammary gland may have an inhibitory influence on mammary tumor growth in mice.

Institutes in the Lead: Identifying Environmental Factors in Breast Cancer.

Institutes in the Lead: Identifying Environmental Factors in Breast Cancer.

Uncovering the stem cell hierarchy by genetic lineage tracing in the mammary gland

AbstractThe mammary gland is the distinct feature that gives the name to the class of mammals and distinguishes them from other animals. Functionally, the mammary gland is a secretory organ which main role is to produce milk to nourish the offspring. Organogenesis of the mammary gland starts during embryogenesis but occurs mainly after birth at puberty under the influence of hormonal cues. Throughout the adult life as well as during pregnancy, the mammary gland shows a remarkable regenerative ability, thus constituting an excellent model for studying stem cell biology. Although the mammary gland consists of a relatively simple epithelial structure with a luminal and a basal cell layers, these are indeed composed by distinct subsets of mammary epithelial cells. Flow cytometry and transplantation assay have identified several subpopulations of stem and/or progenitor cells in the mammary gland. Yet, physiological and developmental relevant information can only be obtained when investigating the stem cell hierarchy in the intact mammary gland. Genetic lineage tracing studies have offered unprecedented levels of information regarding the organization of the stem cell compartment and possible role of resident stem and/or progenitor cells at different stages of the mammary gland organogenesis. These studies, although creating a passionate debate, highlight the existence of heterogeneous stem cell compartment, where bipotent as well as unipotent mammary stem cells seems to co-exist. Genetic lineage tracing experiments provide relevant information on stem cells that are key for understanding both normal development as well as associated pathologies in human. It holds the promise of providing new insights into the cell-of-origin and heterogeneity of breast tumorigenesis.

Abstract B22: Analysis of Lgr5-positive cells in mouse mammary gland

Abstract Lgr5 has been identified as a stem cell marker in multiple adult tissues, and individual Lgr5+ mammary epithelial cells are capable of regenerating mouse mammary glands in transplantation assays. Here we investigated the proliferation and differentiation capabilities of murine mammary Lgr5+ cells in vitro, and their distribution in mouse mammary glands in vivo. Equal numbers of sorted Lgr5+ and Lgr5- epithelial cells were cultured in Matrigel to evaluate their stem-like properties in vitro. Compared with Lgr5- cells, Lgr5+ cells were more efficient at generating organoids in 3D culture, and the Lgr5+ cell-derived organoids grew to a larger size. Moreover, Lgr5+ cell-derived organoids survived repeated passaging and contained cells of both luminal and basal lineages, as indicated by keratin marker expression. Incidence of Lgr5+ cells in organoids was confirmed by in situ hybridization. These studies indicate that Lgr5+ cells retain the self-renewal and differentiation capabilities of stem cells ex vivo. The in vivo distribution of Lgr5+ cells was investigated using X-Gal-stained mammary glands from Lgr5-lacZ reporter mice ranging from 5 to 32 weeks old. In both adolescent and adult mice, we observed a graded distribution of Lgr5+ cells with greater abundance in the nipple-proximal (ventral) ducts than in the lymph-node proximal central region, and fewest Lgr5+ cells in the distal (dorsal) region of the glands. Lgr5+ cells from all mammary gland regions appeared capable of yielding organoids in culture. These results corroborate in organoid culture the known stem-like properties of Lgr5+ cells defined in intact mouse mammary gland. [Supported by NIH grant CA186981 and the China Scholarship Council] Citation Format: Lixing Zhang, Louise Howe, Richard Kolesnick, Anthony Brown. Analysis of Lgr5-positive cells in mouse mammary gland. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B22.

Abstract 1402: A reporter mouse model reveals that human CYP19A1 (aromatase) gene expression is induced in breast cancer xenograft stroma and surrounding mammary gland by the cancer cells in vivo.

Abstract Aromatase enzyme coded by the CYP19A1 gene catalyzes the final step of estrogen biosynthesis and is of major significance in endocrine regulation of breast cancer (BCa). Human CYP19A1 gene expression is regulated through the utilization of various non-translated tissue selective first exons, each controlled by unique sets of e.g. hormones and cytokines. Extragonadal expression pattern and regulation of human CYP19A1 gene is significantly different from that of rodents, which has been a major obstacle in studies focusing on regulation of aromatase gene expression in vivo. To study the tissue-selective regulation of human CYP19A1 gene in vivo, we have generated a transgenic reporter mouse model, hARO-Luc mouse, with a construct composing of a 100-kbp-long 5’-regulatory region of the human CYP19A1 gene attached to a luciferase (Luc) reporter gene. The aim of this study was to investigate human CYP19A1 gene expression in human BCa xenografts, to determine how the BCa cells regulate CYP19A1 gene expression in tumor associated stroma in vivo. In vitro, BCa cells have been shown to induce CYP19A1 gene expression in human breast derived fibroblasts. Furthermore, in order to study the regulation of the human CYP19A1 gene expression in hARO-Luc derived undifferentiated fibroblasts, mesenchymal stromal cells were isolated from bone marrow of female reporter mice. In these cells, human CYP19A1 gene expression was induced by dexamethasone ± TNFα, and PGE2 that are among factors known to drive aromatase gene expression in human mammary fibroblasts through promoters I.4 and I.3/PII. In order to investigate human CYP19A1 expression in BCa xenografts and tumor surrounding mammary gland, the hARO-Luc reporter mice were cross-bred with athymic nude mice. Estrogen-responsive MCF-7 or T47D cells or non-estrogen-responsive MDA-MB-231 cells were injected into inguino-abdominal mammary fat pads of athymic female hARO-Luc mice, and mice that received MCF-7 or T47D cells were also implanted s.c. with estradiol releasing pellet to induce the estrogen-dependent tumor growth. The reporter gene activity was measured from tissue homogenates. MCF-7 and T47D tumors had 5-10 -fold higher Luc activity as compared with the intact mammary gland, indicating a strong up-regulation of the reporter gene in host derived tumor stroma. In contrast, in MDA-MB-231 tumor bearing mice the reporter activity in tumors was only slightly higher as compared with the intact mammary gland. Furthermore, reporter gene activity in mammary gland surrounding MDA-MB-231 tumors was 2-fold higher than in the tumor itself. In conclusion, the data indicate that human BCa cells up-regulate expression of human aromatase gene in the tumor associated stroma in vivo. Citation Format: Päivi Järvensivu, Leena Strauss, Sari Mäkelä, Matti Poutanen, Niina Saarinen. A reporter mouse model reveals that human CYP19A1 (aromatase) gene expression is induced in breast cancer xenograft stroma and surrounding mammary gland by the cancer cells in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1402. doi:10.1158/1538-7445.AM2013-1402

Quantitative Assessment of Mammary Gland Density in Rodents Using Digital Image Analysis

BackgroundRodent models have been used extensively to study mammary gland development and for studies of toxicology and carcinogenesis. Mammary gland gross morphology can visualized via the excision of intact mammary gland chains following fixation and staining with carmine using a tissue preparation referred to as a whole mount. Methods are described for the automated collection of digital images from an entire mammary gland whole mount and for the interrogation of digital data using a "masking" technique available with Image-Pro® plus image analysis software (Mediacybernetics. Silver Spring, MD).ResultsParallel to mammographic analysis in humans, measurements of rodent mammary gland density were derived from area-based or volume-based algorithms and included: total circumscribed mammary fat pad mass, mammary epithelial mass, and epithelium-free fat pad mass. These values permitted estimation of absolute mass of mammary epithelium as well as breast density. The biological plausibility of these measurements was evaluated in mammary whole mounts from rats and mice. During mammary gland development, absolute epithelial mass increased linearly without significant changes in mammographic density. Treatment of rodents with tamoxifen, 9-cis-retinoic acid, or ovariectomy, and occurrence of diet induced obesity decreased both absolute epithelial mass and mammographic density. The area and volumetric methods gave similar results.ConclusionsDigital image analysis can be used for screening agents for potential impact on reproductive toxicity or carcinogenesis as well as for mechanistic studies, particularly for cumulative effects on mammary epithelial mass as well as translational studies of mechanisms that explain the relationship between epithelial mass and cancer risk.

Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA

IntroductionDuring selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents, simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy.MethodsThe glands of 3-week-old immune-competent Balb/C female mice were used intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-Bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells that retain their template DNA. Nulliparous mice were then either injected with [3H]-thymidine (3H-TdR) to distinguish 5-BrdU label-retaining cells that enter the cell cycle and euthanized, or mated, injected with 3H-TdR, and euthanized at various days after coitus. Sections were stained for estrogen receptor-α (ER-α) or progesterone receptor (PR) with immunohistochemistry. Cells labeled with both 5-BrdU and 3H-TdR were indicative of label-retaining epithelial cells (LRECs).ResultsCells that retained a 5-BrdU label and cells labeled with [3H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labeled nuclei (LRECs) were found in the intact mammary glands of both pregnant and nulliparous mice, and in mammary glands implanted with premalignant cells. Double-labeled cells (3H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label (3H-TdR) to daughter cells, and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary-implant groups.ConclusionsThe results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.

Cold‐inducible RNA binding protein modulates proliferation in the mouse mammary epithelium

Cold‐inducible RNA binding protein (CIRP) is upregulated in response to cellular stress, either facilitating or repressing translation of its mRNA targets. We showed that CIRP is upregulated in all breast cancer cell lines examined, while others have shown CIRP overexpression in primary breast tumors. In the MCF‐7 breast cancer cell line, CIRP upregulates the expression of another stress‐induced RNA binding protein, HuR, as well as the cell cycle regulator cyclin E1. Both HuR and cyclin E1 are also overexpressed in breast cancer, with higher expression correlating with a poor prognosis. In order to determine the in vivo function of CIRP in the breast epithelium, we produced a transgenic mouse expressing CIRP in the mammary epithelium under control of the mouse mammary tumor virus (MMTV) regulatory sequences. Real‐time RT‐PCR showed maximal expression of the transgene in the pregnant mouse at gestation day 16, and a second peak of expression after birth, at lactation day 14. Immunohistochemistry for the proliferation marker Ki‐67 showed decreased proliferation in the mammary epithelium of CIRP transgenic mice compared to wild type littermates at gestation day 16 (45% CIRP vs. 56% wild type, p=0.05) and lactation day 1 (7% CIRP vs. 25% wild type, p=0.0068). These results suggest that CIRP negatively regulates proliferation in the intact mammary gland. We are currently examining effects of CIRP expression on apoptosis in the involuting mammary gland. This work was supported by a grant from the National Cancer Institute‐National Institutes of Health to RSH (R01CA095898).

Genes Regulating the Progression of Human Ductal Carcinoma In Situ to Invasive Breast Cancer.

Abstract Rationale: The progression of human ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC) represents a major potentially life-threatening event. Our understanding of this process is rudimentary. Improved understanding could lead to better ability to predict and new therapeutic strategies to prevent progression, which would be major contributions to health care. Objectives: To identify genes involved in regulating the progression of human DCIS to IBC, and to confirm their involvement utilizing relevant in vitro and in vivo model systems. Methods: Gene expression profiles were evaluated in five complimentary cohorts of human DCIS and IBC (total n=59 in each category) using cDNA microarray technology. Supervised statistical comparisons were used to identify differentially expressed genes in each cohort. The genes were distilled to top priority candidates by the following vetting strategy: (1) expression changed in same direction in 2 or more cohorts; and (2) they possessed known biological functions which might reasonably be involved in regulating tumor invasion; and (3) they had not been studied in the context of breast cancer invasion. Initial studies have focused on candidate invasion-suppressor genes (down-regulated in IBC vs. DCIS). A human DCIS-like cell line (DCIS.COM) was stably transduced with shRNAi against the genes (3-5 hairpins each) and evaluated in vitro and in vivo for changes in invasion-related behaviors vs. controls (growth and migration in 2D and 3D cell cultures, invasion in 3D culture, and a human-in-mouse [HIM} DCIS xenograft model). The latter is a novel method we developed where cells are injected into the nipple duct of intact mammary glands of immune-compromised mice. They initially grow as DCIS which progress to IBC at varying rates depending on the cells. Results: Two novel invasion-suppressor genes (CSTA and FAT1) were identified based on demonstrating significantly increased growth, migration, and invasion in the assays described. In particular, shCSTA (n=22) and shFAT1 (n=22) HIM DCIS xenografts progressed to large IBCs by 10 weeks or less, significantly (P<.0001) greater than observed in controls (n=26) (67%, 64%, vs. 26%, respectively). Conclusions: We have identified two genes (CSTA and FAT1) whose normal functions appear to suppress the progression of human DCIS to IBC. These results could lead to new therapeutic strategies to prevent or suppress progression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6141.

Distinct gene-expression profiles characterize mammary tumors developed in transgenic mice expressing constitutively active and C-terminally truncated variants of STAT5

BackgroundStat5 is a latent transcription factor that regulates essential growth and survival functions in normal cells. Constitutive activity of Stat5 and the involvement of its C-terminally truncated variant have been implicated in blood cell malignancies and mammary or breast cancer. To distinguish the individual contributions of the Stat5 variants to mammary tumorigenesis, global gene-expression profiling was performed on transgenic STAT5-induced tumors.ResultsWe identified 364 genes exhibiting differential expression in mammary tumors developed in transgenic mice expressing constitutively active STAT5 (STAT5ca) vs. its C-terminally truncated variant (STAT5Δ750). These genes mediate established Stat5 effects on cellular processes such as proliferation and cell death, as well as yet-unrelated homeostatic features, e.g. carbohydrate metabolism. A set of 14 genes linked STAT5Δ750 expression to the poorly differentiated carcinoma phenotype and STAT5ca to the highly differentiated papillary adenocarcinoma.Specifically affected genes exhibited differential expression in an individual tumor set vs. its counterpart and the intact mammary gland: 50 genes were specifically affected by STAT5ca, and 94% of these were downregulated, the latter involved in suppression of tumor suppressors and proliferation antagonistics. This substantial downregulation distinguishes the STAT5ca-induced tumorigenic consequences from the relatively equal effect of the STAT5Δ750 on gene expression, which included significant elevation in the expression of oncogenes and growth mediators.STAT5Δ750 mRNA expression was below detection levels in the tumors and the amount of STAT5ca transcript was not correlated with the expression of its specifically affected genes. Interestingly, we identified several groups of three to eight genes affected by a particular STAT5 variant with significant correlated expression at distinct locations in the clustergram.ConclusionThe different gene-expression profiles in mammary tumors caused by the STAT5Δ750 and STAT5ca variants, corroborated by the absence of a direct link to transgenic STAT5 expression, imply distinct metabolic consequences for their oncogenic role which probably initiate early in tumor development. Tumorigenesis may involve induction of growth factor and oncogenes by STAT5Δ750 or suppression of tumor suppressors and growth antagonists by STAT5ca. The list of genes specifically affected by the STAT5 variants may provide a basis for the development of a marker set for their distinct oncogenic role.

γδ-TCR<sup>+</sup> CD2<sup>-</sup> lymphocytes are recruited into bovine mammary gland after stimulation

γδ-T-Cell Receptor (TCR) lymphocytes were detected in mammary gland lavages collected from 10 clinically healthy virgin heifers before and after intramammary stimulation with synthetic muramyl dipeptide analogue. Using two-colour flow cytometry, CD2<sup>+</sup> and CD2<sup>–</sup> subsets of γδ-TCR lymphocytes were analyzed. CD2<sup>+</sup> γδ-TCR lymphocytes markedly prevailed over CD2<sup>–</sup> cells in intact mammary gland: 88.9 ± 4.9% of γδ-TCR lymphocytes were CD2<sup>+</sup>. After stimulation, neutrophils and γδ-TCR lymphocytes were recruited into the mammary gland. Among γδ-TCR lymphocytes, CD2<sup>–</sup> cells were mainly responsible for their expansion. After stimulation, 60.8 ± 13.4% of γδ-TCR lymphocytes were CD2<sup>+</sup> (P < 0.01 when compared with mammary gland lavages before stimulation). It follows from the present study that the cells seem to be involved in the first phase of a response to an infection affecting mammary gland.

Expression of steroid receptors in intact rat uterus, mammary gland, and liver treated with selective estrogen receptor modulators and conjugated equine estrogens

The aim of the present study was to determine the effects of conjugated equine estrogens (CEE) and selective estrogen receptor modulators (SERM) (tamoxifen [TAM] and raloxifene [RAL]) on the expression of steroid receptors-estrogen receptor (ER) and progesterone receptor (PR)-in intact rat uterus, mammary gland, and liver. A total of 24 female rats weighing 250 to 300 g were randomized into 4 groups. Groups 1, 2, 3, and 4 were respectively given conjugated equine estrogen, tamoxifen, raloxifene, and vehicle for a 28-day period. ER and PR expression was detected in tissues of the uterus, mammary gland, and liver. Uterine wet weight and serum estradiol levels were established for all groups. No statistical difference was observed between groups in the ER expression of mammary gland and liver and in the PR expression of uterus, mammary gland, and liver, but differences were noted in serum estradiol levels and uterine ER expression. Serum estradiol levels were lower in the TAM-treated group; differences between the TAM-treated group and the other groups were statistically important (P<.05). Uterine ER expression was greater in the CEE-treated group; differences between the CEE-treated group and the TAM- and RAL-treated groups were statistically important (P<.05). CEE or SERM versus vehicle treatment in controls did not seem to result in statistically important differences in ER and PR expression in intact rat uterus, mammary gland, and liver. Only ER expression in the uterus was found to be greater in the CEE-treated group than in SERM-treated groups.

The stroma as a crucial target in rat mammary gland carcinogenesis.

A complex network of interactions between the stroma, the extracellular matrix and the epithelium drives mammary gland development and function. Two main assumptions in chemical carcinogenesis of the mammary gland have been that carcinogens induce neoplasia by causing mutations in the DNA of the epithelial cells and that the alterations of tissue architecture observed in neoplasms are a consequence of this primary mutational event. Here, we use a rat mammary tissue recombination model and the chemical carcinogen N-nitrosomethylurea (NMU) to determine whether the primary target of the carcinogen is the epithelium, the stroma or both tissue compartments. Mammary epithelial cells were exposed in vitro either to the carcinogen or vehicle before being transplanted into the cleared fat pads of rats exposed to carcinogen or vehicle. We observed that neoplastic transformation of these mammary epithelial cells occurred only when the stroma was exposed in vivo to NMU, regardless of whether or not the epithelial cells were exposed to the carcinogen. Mammary epithelial cells exposed in vitro to the carcinogen formed phenotypically normal ducts when injected into a non-treated stroma. Mutation in the Ha-ras-1 gene did not correlate with initiation of neoplasia. Not only was it often found in both cleared mammary fat pads of vehicle-treated animals and intact mammary glands of untreated animals, but it was also absent in some tumors. Our results suggest that the stroma is a crucial target of the carcinogen and that mutation in the Ha-ras-1 gene is neither necessary nor sufficient for tumor initiation.

Requirement for IGF-I in epidermal growth factor-mediated cell cycle progression of mammary epithelial cells.

Induction of cyclin proteins is required for progression of cells through the G(1)-S and G(2)-M cell cycle checkpoints and is a primary mechanism by which mitogens regulate cell cycle progression. IGF-I and the epidermal growth factor (EGF)-related ligands are mitogens for mammary epithelial cells in vitro and are essential for growth of the mammary epithelium during development. We report here that IGF-I in combination with EGF or TGFalpha is synergistic in promoting DNA synthesis in mammary epithelial cells in the intact mammary gland cultured in vitro. We further investigated the role of IGF-I and EGF in cyclin expression and cell cycle progression in the mammary gland and demonstrate that IGF-I and EGF induce expression of early G(1) cyclins. However, we show that IGF-I, but not EGF, induces late G(1) and G(2) cyclins and is required for mammary epithelial cells to overcome the G(1)-S checkpoint. These data demonstrate that IGF-I is essential for cell cycle progression in mammary epithelial cells and that it is required for EGF-mediated progression past the G(1)-S checkpoint in these cells.

Requirement for IGF-I in Epidermal Growth Factor-Mediated Cell Cycle Progression of Mammary Epithelial Cells

Induction of cyclin proteins is required for progression of cells through the G1-S and G2-M cell cycle checkpoints and is a primary mechanism by which mitogens regulate cell cycle progression. IGF-I and the epidermal growth factor (EGF)-related ligands are mitogens for mammary epithelial cells in vitro and are essential for growth of the mammary epithelium during development. We report here that IGF-I in combination with EGF or TGFα is synergistic in promoting DNA synthesis in mammary epithelial cells in the intact mammary gland cultured in vitro. We further investigated the role of IGF-I and EGF in cyclin expression and cell cycle progression in the mammary gland and demonstrate that IGF-I and EGF induce expression of early G1 cyclins. However, we show that IGF-I, but not EGF, induces late G1 and G2 cyclins and is required for mammary epithelial cells to overcome the G1-S checkpoint. These data demonstrate that IGF-I is essential for cell cycle progression in mammary epithelial cells and that it is required for EGF-mediated progression past the G1-S checkpoint in these cells.

Prolactin receptor expression in the epithelia and stroma of the rat mammary gland.

The importance of prolactin (PRL) in regulating growth and differentiation of the mammary gland is well known. However, it is not well established whether PRL acts solely on the mammary epithelia or if it can also directly affect the mammary stroma. To determine where PRL could exert its effects within the mammary gland, we investigated the levels of expression and the localization of the PRL receptor (PRLR) in the epithelia and stroma of the rat mammary gland at different physiological stages. For these studies, we isolated parenchymal-free 'cleared' fat pads and intact mammary glands from virgin, 18-day-pregnant and 6-day-lactating rats. In addition, intact mammary tissues were enzymatically digested to obtain epithelial cells, free of stroma. The mammary tissues, intact gland, stroma and isolated epithelia, were then used for immunocytochemistry, protein extraction and isolation of total RNA. PRLR protein was detected in tissues using specific polyclonal antisera (PRLR-l) by immunocytochemistry and Western blot analysis. Messenger RNA for PRLR was measured by ribonuclease protection assay. Immunocytochemistry and Western blots with the PRLR-1 antisera detected PRLR in wild-type rat and mouse tissues, whereas the receptor protein was absent in tissues from PRLR gene-deficient mice. PRLR was found to be present both in the epithelia and stroma of mammary glands from virgin, pregnant and lactating rats, as determined by immunocytochemistry and Western blotting. Western blots revealed the predominance of three bands migrating at 88, 90 and 92 kDa in each of the rat mammary samples. These represent the long form of the PRLR. During pregnancy and lactation, PRLR protein increased in the epithelial compartment of the mammary gland but did not change within the stromal compartment at any physiological stage examined. We also found PRLR mRNA in both the epithelia and stroma of the mammary gland. Again, the stroma contained lower levels of PRLR mRNA compared with the epithelia at all physiological stages examined. Also, the PRLR mRNA levels within the stroma did not change significantly during pregnancy or lactation, whereas PRLR mRNA within the epithelia increased twofold during pregnancy and fourfold during lactation when compared with virgin rats. We conclude from this study that PRLR is expressed both in the stromal and epithelial compartment of the mammary gland. This finding suggests PRL may have a direct affect on the mammary stroma and by that route affect mammary gland development.

Preparation of an epithelium-free mammary fat pad and subsequent mammogenesis in ewes.

The objective of this study was to develop a surgical procedure for the preparation of an epithelium-free mammary fat pad (cleared mammary fat pad; CFP) in ewes. At 7 to 10 d of age, ewe lambs (n = 43, mean BW 9.2 +/- .2 kg at 14 d) were sedated and one mammary gland was locally anesthetized. An incision circumscribing the base of the teat was made and blunt dissection was performed through the extraneous mammary fat pad tissue to enable the parenchyma and teat to be wholly removed. Failure to completely remove the epithelium enabled it to regenerate and grow into the mammary fat pad. Mean diameter of the parenchymal rudiment at 7 to 10 d of age was 8.9 +/- .5 mm (range of 5 to 16 mm). The excision site was closed with wound clips and recovered lambs returned to their dams. The contralateral mammary gland remained intact, allowing it to undergo normal development. Live weight gain was unaffected by this procedure. Ewes were subsequently slaughtered in groups at various stages of prepuberty, puberty, gestation, and lactation. Of 39 operated glands recovered, only one demonstrated epithelial outgrowth within the CFP. Parenchyma within the contralateral, intact gland underwent phases of rapid growth in prepuberty, puberty, and late gestation and was capable of milk synthesis after steroid induction or parturition. Change in weight of the CFP paralleled that of the intact mammary gland to 100 d of pregnancy. Sham CFP surgery was performed on four additional ewes wherein the parenchyma was completely excised and immediately replaced. Sham-operated epithelium populated the mammary fat pad and synthesized milk that could be expressed from the teat. A CFP in sheep will be a useful model for future investigations into the local growth regulatory mechanisms associated with ruminant mammogenesis.

Ontogeny and epithelial–stromal interactions regulate IGF expression in the ovine mammary gland

Although the insulin-like growth factors (IGF-I and -II) have been implicated in the stimulation of mammogenesis, little is known of their regulation in the mammary gland. In this study we removed epithelial tissue from one of the two mammary glands of 1-week-old ewe lambs and examined IGF-I and -II mRNA expression during postnatal development in both the intact mammary gland and in the gland cleared of epithelial tissue. Expression of IGF-I mRNA was highest at 6 and 10 weeks of age, coincident with the prepubertal phase of rapid mammary growth, then declined and remained low until expression increased during late pregnancy. IGF-I mRNA was more abundant in the mammary fat pad adjacent to parenchyma (MFP) than in the contralateral fat pad that had been surgically cleared of epithelium (CFP). The level of IGF-II mRNA in parenchyma was highest at 1–23 weeks of age due to an increase in the abundance of specific mRNAs. Expression was lower in the fat pads, with generally higher levels in the intact MFP than the CFP, and in these tissues IGF-II expression was shown to increase with age between 6 and 23 weeks. We also investigated the influence of the ovary and estrogen on the expression of IGFs. While IGF-I mRNA abundance was unaffected by ovariectomy, exogenous estrogen resulted in higher levels of expression in the MFP of ovariectomized ewes and tended to increase its level in the parenchyma of intact ewes. Ovariectomy increased IGF-II mRNA within mammary parenchyma whereas estrogen suppressed levels in both the parenchyma and MFP. These findings demonstrate that IGF-I and -II mRNAs are expressed locally within the developing ovine mammary gland and are regulated by stage of ontogeny, ovarian hormones, and epithelial–stromal interaction.