Terminal Ductal Units Research Articles

Feeding an enhanced diet to Holstein heifers during the preweaning period alters steroid receptor expression and increases cellular proliferation

Preweaning diet and estradiol treatment alters mammary development. Our objectives were to study the effects of diet and estradiol on proliferation of mammary epithelial cells and expression of estrogen receptor α (ESR1) and progesterone receptors (PGR) in these cells. Thirty-six Holstein heifer calves were raised on (1) a control milk replacer fed at 0.44 kg of powder/head per day, dry matter (DM) basis (restricted, R; 20.9% crude protein, 19.8% fat, DM basis), or (2) an enhanced milk replacer fed at 1.08 kg of powder/head per day, DM basis (Enhanced, EH; 28.9% crude protein, 26.2% fat, DM basis). Milk replacer was fed for 8 wk. At weaning, a subset (n = 6/diet) of calves were euthanized and had tissue harvested. Remaining calves received estradiol implants (E2) or placebo and were euthanized at wk 10 to harvest tissue. Treatments were (1) R, (2) R + E2 (R-E2), (3) EH, and (4) EH + E2 (EH-E2). One day before euthanasia calves were given bromo-2'-deoxyuridine (BrdU; 5 mg/kg of body weight). At euthanization, mammary parenchyma was removed and fixed. Tissue sections from zone 1 (cisternal), 2 (medial), and 3 (distal) within the mammary gland were stained with hematoxylin and eosin and antibodies to measure expression of ESR1, PGR, and incorporation of BrdU. At wk 8, R-fed calves had more PGR-expressing cells in distal parenchyma; however, PGR expression intensity was greater in EH-fed calves. The proportion of cells expressing ESR1 was not affected by diet, but expression intensity (receptors per positive cell) was greater in EH-fed calves across all zones (62-81%). Overall, the percent BrdU-positive epithelial cells was 2 and 0.5 fold greater for EH-fed calves in zone 2 and 3. The proportion of labeled cells was greater in terminal ductal units than in subtending ducts, and treatment effects were more evident in terminal ductal units. At wk 10, calves treated with estradiol had 3.9-fold greater PGR expression intensity. The intensity and percent of cells expressing ESR1 was lowest in estradiol-treated calves. Overall, estradiol-treated calves had the greatest number of proliferating epithelial cells. Moreover, in zone 3, EH-E2 calves had a higher percentage of proliferating cells than in all other treatments. Results indicate both diet and estradiol administration alter proliferation rates of the mammary epithelium and that changes in expression of ESR1 and PGR are involved in enhanced mammary development. The data support our hypothesis that enhanced preweaning feeding increases the mammary tissue responsiveness to mammogenic stimulation.

Assessment of the Cancer Risk of the Fat-Grafted Breast in a Murine Model.

The results of experimental studies indicate that grafting of autologous adipose tissue may induce tumorigenesis at the recipient site, but clinical results do not support a carcinogenic effect of fat grafting to the breast. The authors assessed cancer risk following transplantation of autologous fat into murine mammary tissue. In this animal study, mammary tissues from 54 breasts of 9 female rats were either grafted with autologous subcutaneous fat, grafted with autologous omental fat, or unmanipulated. Tissues were harvested and processed for histologic and immunohistochemical analyses, and the mRNA expression levels of specific genes were determined. No atypia or changes in lobular structures were observed in lipofilled breasts compared with controls. The numbers of ductal cell layers and terminal ductal units were similar for lipofilled and control breasts. Macrophage concentrations also were similar for the 3 groups. The localization and magnitude of plasminogen activator inhibitor 1 were similar for lipofilled and unmanipulated breast tissue. The percentages of cells expressing Ki67 or estrogen receptor (ER) and the ER/Ki67 balance were similar for the 3 groups. Gene expression was not altered in lipofilled breasts, compared with controls. No theoretical risk of cancer was detected in the microenvironment of the lipofilled rat breast.

Abstract 4558: Using reduction mammoplasty tissues from healthy young women living in USA's high breast cancer (BC) risk posing environment to open up to study the “black box” of latent stages of breast carcinogenesis; a key to develop strategies for BC

Abstract Knowledge of latent stages of breast carcinogenesis is limited to when histological changes are already present due to a lack of; a) cytochemical marker(s) needed to identify loci in histologically normal breast tissues where procarcinogenic conditions exist and; b) microscopic systems needed to geno- and phenotype cells in such loci. In 2 recent publications we presented evidence that protein adducts of 4-hydroxy-2-nonenal (4HNE), markers of a procarcinogenic state of chronic oxidative stress (OxS), might provide the needed fiducial marker. Specifically, a) we documented the presence of 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells in terminal ductal units (TDLUs) in breast tissues of healthy teenagers and young women and their association with altered expression of OxS-related mRNAs and; b) presented Fourier-transform infrared microspectroscopic evidence of differences in biomolecular profile of ME cells and associated intra- and interlobular stromal cells in 4HNE+ vs. 4HNE- TDLUs, i.e., of cells of origin of BCs and cells in their supportive stromal microenvironment. Here we present further evidence of molecular changes in breast tissues of young women (ages 17-30 y) associated with 4HNE adducts. A comparison of the transcriptome of breast tissues with many 4HNE+ vs. few 4HNE+ TDLUs (controls) by Illumina 24,000 gene platform and SABioscience 84 gene Cell Death PCR Array revealed reprogramming of many homeostatic mechanism in 4HNE+ TDLUs. Notably, a) DASL platform identified transcripts for 395 genes that differed (p = <.05) between 4HNE+ vs. 4HNE- tissues, transcripts that were assigned by GO bioinformatics tool to 21 molecular mechanism and; b) Cell Death array identified 30 genes that differed >2 fold between 4HNE+ vs. 4HNE- tissues. The findings support using 4HNE adducts to identify in histologically normal breast tissues loci harboring cells that might initiate a cancer. Our findings fit an evolutionary model of carcinogenesis and reflect adaptive responses to stressors that serve to preserve tissue integrity, e.g., by developing resistance to OxS and to apoptosis, responses however that over time can lead to the emergence of a cancer. Our recently acquired Toponome Imaging System (TIS) enables identifying such responses to chronic OxS implied by the presence of 4HNE adducts. TIS is the first high throughput fluorescence robotic microscopic system that can provide a map, a toponome, of up to 100 proteins at 40nm functional resolution in individual cells in a tissue section. In ongoing experiments we are using TIS and antibodies against up to 21 proteins to test the postulate that 4HNE+ TDLUs are enriched with cells with an apoptosis resistant phenotype, a postulate supported by our transcriptional data and predicted by the evolutionary model of carcinogenesis. Citation Format: Judith Weisz, Debra Shearer, Anna C. Salzberg. Using reduction mammoplasty tissues from healthy young women living in USA's high breast cancer (BC) risk posing environment to open up to study the “black box” of latent stages of breast carcinogenesis; a key to develop strategies for BC [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2015-4558

Infrared microspectroscopy identifies biomolecular changes associated with chronic oxidative stress in mammary epithelium and stroma of breast tissues from healthy young women

Studies of the decades-long latent stages of breast carcinogenesis have been limited to when hyperplastic lesions are already present. Investigations of earlier stages of breast cancer (BC) latency have been stymied by the lack of fiducial biomarkers needed to identify where in histologically normal tissues progression toward a BC might be taking place. Recent evidence suggests that a marker of chronic oxidative stress (OxS), protein adducts of 4-hydroxy-2-nonenal (4HNE), can meet this need. Specifically: (1) 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells were found to be prevalent in normal (reduction mammoplasty) tissues of most women (including many teenagers) studied, representative of those living in the United States’ high risk-posing environment and: (2) marked (>1.5-fold) differences were identified between tissues of healthy young women with many vs. few 4HNE+ ME cells in the relative levels of transcripts for 42 of the 84 OxS-associated genes represented in SABioscience Oxidative-Stress/Oxidative-Defense PCR array. Herein we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to identify molecular changes associated with 4HNE adducts in basal and luminal ME cells in terminal ductal units (TDLU), which are the cells of origin of BC, and associated intralobular and interlobular stroma, known contributors to carcinogenesis. Multivariate analysis-derived wavenumbers differentiated 4HNE+ and 4HNE− cells in each of the anatomical compartments. Specifically, principal component and linear discriminant analyses of mid-infrared spectra obtained from these cells revealed unambiguous, statistically highly significant differences in the “biochemical fingerprint” of 4HNE+ vs. 4HNE− luminal and basal ME cells, as well as between associated intralobular and interlobular stroma. These findings demonstrate further SR-FTIR microspectroscopy’s ability to identify molecular changes associated with altered physiological and/or pathophysiological states, in this case with a state of chronic OxS that provides a pro-carcinogenic microenvironment.

Expression of antigen tf and galectin-3 in fibroadenoma

BackgroundFibroadenomas are benign human breast tumors, characterized by proliferation of epithelial and stromal components of the terminal ductal unit. They may grow, regress or remain unchanged, as the hormonal environment of the patient changes. Expression of antigen TF in mucin or mucin-type glycoproteins and of galectin-3 seems to contribute to proliferation and transformations events; their expression has been reported in ductal breast cancer and in aggressive tumors.FindingsLectin histochemistry, immunohistochemistry, and immunofluorescence were used to examine the expression and distribution of antigen TF and galectin-3. We used lectins from Arachis hypogaea, Artocarpus integrifolia, and Amaranthus lecuocarpus to evaluate TF expression and a monoclonal antibody to evaluate galectin-3 expression. We used paraffin-embedded blocks from 10 breast tissues diagnosed with fibroadenoma and as control 10 healthy tissue samples. Histochemical and immunofluorescence analysis showed positive expression of galectin-3 in fibroadenoma tissue, mainly in stroma, weak interaction in ducts was observed; whereas, in healthy tissue samples the staining was also weak in ducts. Lectins from A. leucocarpus and A. integrifolia specificaly recognized ducts in healthy breast samples, whereas the lectin from A. hypogaea recognized ducts and stroma. In fibroadenoma tissue, the lectins from A. integrifolia, A. Hypogaea, and A. leucocarpus recognized mainly ducts.ConclusionsOur results suggest that expression of antigen TF and galectin-3 seems to participate in fibroadenoma development.

Expression of keratin 19, Na-K-Cl cotransporter and estrogen receptor alpha in developing mammary glands of ewes.

Mammary gland remodelling is strictly related to intracellular signals and stem cell biology. Among the best candidates to identify the nature and development of mammary cells are cytokeratin 19 (CK19), the Na-K-Cl cotransporter (NKCC1) and receptor of estrogen alpha (ERα). In this study, we analyzed the expression of these genes in ewe mammary glands from prepubertal stage to involution. Using Real time PCR we showed that NKCC1 transcription was significantly down regulated during lactation and at involution in comparison to the expression measured in the prepubertal group. No significant differences were found in CK19 expression, whereas ERα transcription was significantly down regulated before lambing, during lactation and at involution. In situ hybridization analysis confirmed quantitative data and localized the CK 19 transcript at basal and luminal compartment of terminal ductal unit (TDU) of prepubertal mammary glands. NKCC1 expression was also present in lactating glands and ERα in connective tissue surrounding TDU. The characterization and identification of mammary developmental markers in the tissue of dairy animals is necessary to gain knowledge in mammary gland biology.

O-GLYCOSYLATION EXPRESSION IN FIBROADENOMA

Fibroadenomas are human benign breast tumors characterized by proliferation of epithelial and stroma cells of the terminal ductal unit. Expression of O-glycans seems to contribute to the proliferation and transformation events. With this in mind, we evaluated the expression of glycans in fibroadenoma tissue through immunohistochemistry with antibodies against mucin epitopes (Anti CA15-3 and MUC1), as well as with lectins specific for glycans linked to proteins or lipids, and we compared findings with healthy breast specimens. Our results show positive expression of CA15-3 and MUC1 in fibroadenoma tissue, mainly in duct and stroma cells, whereas, in normal samples, staining was observed in duct cells. The lectin from Glycine max recognized equally well duct and stroma cells; this was the only lectin showing co-localization with anti-CA15-3 in healthy and tumor tissues. Dolichos biflorus, Artocarpus integrifolia, and Griffonia simplicifolia lectins recognized duct cells in control healthy tissues as well as in fibroadenoma tissue. The lectin from Amaranthus leucocarpus recognized only duct cells in control samples, whereas, in fibroadenoma tissue, it recognized duct and some stromal cells, suggesting that O-glycans-type mucin linked to proteins and mucin participate in the development of fibroadenomas.

Effects of Feeding Prepubertal Heifers a High-Energy Diet for Three, Six, or Twelve Weeks on Mammary Growth and Composition

The experimental objective was to determine the effects of feeding prepubertal dairy heifers a high-energy diet for 3, 6, or 12wk on mammary growth and composition. Holstein heifers (age = 11wk; body weight = 107±1kg) were assigned to 1 of 4 treatments (n=16/ treatment). The treatment period lasted 12wk and treatments were H0 (low-energy diet fed for 12 wk, with no weeks on the high-energy diet); H3 (low-energy diet fed for 9wk, followed by the high-energy diet for 3 wk); H6 (low-energy diet fed for 6wk, followed by the high-energy diet for 6wk); and H12 (high-energy diet for all 12wk). The low- and high-energy diets were formulated to achieve 0.6 and 1.2kg of average daily gain, respectively. Heifers were slaughtered at 23wk of age and mammary tissue was collected. A longer duration of feeding the high-energy diet increased total mass of the mammary gland, extraparenchymal fat, and intraparenchymal fat, but did not alter the mass of fat-free parenchymal tissue. When adjusted for carcass weight to reflect differences in physical maturity, the mass of fat-free parenchymal tissue decreased in a linear fashion with a longer duration on the high-energy diet. Total masses of mammary parenchymal DNA and RNA were not different. However, after adjustment for carcass weight, the masses of DNA and RNA decreased as heifers were fed the high-energy diet for a longer duration. The percentages of epithelium, stroma, and lumen, the number of epithelial structures, and the developmental scores of mammary parenchymal tissue were not different among treatments. However, the percentage of proliferating epithelial cells in the terminal ductal units, as indicated by Ki-67 labeling, decreased as heifers were fed the high-energy diet for a longer duration. We concluded that feeding prepubertal heifers a high-energy diet for a longer duration resulted in a linear decrease in both the percentage of mammary epithelial cells that were proliferating and in the mass of fat-free mammary parenchyma per unit of carcass. High-energy feeding hastens puberty and, in this study, decreased mammary epithelial cell proliferation in areas of active ductal expansion. These data are consistent with the idea that feeding heifers a high-energy diet will reduce mammary parenchymal mass at puberty.

Histologia mamária após uso de esteróides sexuais - estudo em ratas

To evaluate the association of histologic changes in the breasts of female rats undergoing therapy with sex steroids. An experimental study was conducted of 40 castrated female non-pubertal rats, 20 had given birth (Group B) and 20 had no offspring (Group A). After four weeks, these rats were randomly allocated to subgroups: A1, A2, A3, A4, A5 and B1, B2, B3, B4, B5. Steroids were given to subgroups as follows: A1 and B1--estradiol benzoate; A2 and B2--medroxyprogesterone acetate; A3 and B3--estradiol benzoate and medroxyprogesterone acetate; A4 and B4--tibolone; A5 and B5--placebo. After 10 weeks of treatment, animals were sacrificed and their mammary glands were analyzed. Histologic parameters evaluated were: epithelial cell proliferation, epithelial cells with secretory activity; and cell atypia in terminal duct units and buds or terminal alveoli. The association between microscopic analysis and diverse therapeutic regimens were analyzed by calculating the odds ratio and its respective 95% confidence interval. Histologic changes were observed in 29 rats: moderate hyperplasia (52.5%), hyperplastic alveolar nodule (42.5%), epithelial atypia (35%), mild hyperplasia (32.5%), secretory activity (20%) and severe hyperplasia (5%). In rats with no offspring when compared to the control, 1.3 times more hyperplastic alveolar nodules were found in the group treated with estradiol, the same was true for moderate hyperplasia in the rats that received medroxyprogesterone acetate, hyperplastic alveolar nodules and epithelial atypia in the group treated with estradiol plus medroxyprogesterone acetate. In the rats with offspring 1.3 times more secretory activity was found with estradiol. Epithelial hyperplasia and epithelial atypia with no proliferation are strongly associated to combined therapy with estradiol plus medroxyprogesterone acetate, mainly in the rats without offspring.