There is currently little known about the role of extracellular matrix (ECM) in the regulation of microRNA (miRs), a family of short, non-coding RNA that repress gene expression at the post-translational level by binding to the 3’-untranslated region (3’UTR) of target mRNA.This thesis uses the mouse mammary gland (MG) to address this question by investigating whether the extracellular matrix regulates miR expression in mammary epithelial cells (MECs).miR expression profiles were generated using MECs cultured in 2D on collagen Ι and in 3D on laminin-rich basement membrane (LrBM). I identified 88 miRs that are more highly expressed in collagen cultured MECs and 8 miRs that have higher expression in MECs cultured on LrBM including miR-146b, a miR known to reduce metastases to the lung in breast cancer. The culture model used compares not only collagen to LrBM but also a stiff environment to a soft environment; raising the question of whether miR-146b is regulated by MEC interaction with ECM proteins or by cellular tension imposed by the microenvironment. Further investigation into miR-146b expression in MECs showed that its expression also increases in response to prolactin stimulation. Expression of the prolactin receptor and subsequently prolactin signalling is reduced in MECs cultured on collagen, but increases in MECs treated with blebbistatin or Y27632, which release cellular tension. However, neither drug had any affect on expression of miR-146. The ECM adhesion receptor β1-intregrin regulates MEC differentiation via cross-talk with prolactin receptor signalling. By using MECs from β1- itgfx/fx;CreER mice I identified a novel mechanism of miR regulation in which β1-intregrin signalling regulates transcription of miR-146b. This study has shows the importance of ECM in the regulation of miR expression and, whilst further investigations are still required, highlights the importance of ECM culture models in studying miR expression and function.

No description available.

The adapter protein Shc is a ubiquitously expressed Src homology 2 (SH2) domain protein implicated in the transmission of activation signals to Ras. She proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors and in v-𝘴𝘳𝘤 transformed cells and are able to transform fibroblasts and differentiate PC12 cells in a Ras-dependent fashion. To assess the transforming ability of Shc in the mouse mammary gland, I generated transgenic mice harbouring the p52ˢʰᶜ cDNA under the transcriptional control of the mouse mammary tumor virus long terminal repeat (MMTV LTR). While p52ˢʰᶜ expression was correlated with multiple enlarged terminal end buds in virgin mouse mammary glands, multiparous mice developed mammary hyperplasias and mammary carcinomas. The frequency, latency and focal nature with which these tumors arose suggests that additional events are necessary to induce malignant conversion of primary mammary epithelial cells. To directly test the role of Shc in established mammary tumor models, I have generated two strains of bigenic mice. When Shc was overexpressed with NDL 1-2, a constitutively activated form of the Neu receptor tyrosine kinase, latency of tumor onset was decreased over that of parental MMTV/NDL 1-2 mice. Polyomavirus middle T antigen (PyV MT) mutants with a functionally inactive Shc binding site (MT Y250F) are debilitated in mammary tumor formation compared to wild-type PyV MT transgenic animals. Concurrent overexpression of Shc with MT Y250F accelerated tumor kinetics and increased the propensity for metastasis to the lungs of bigenic animals. / Thesis / Master of Science (MSc)

This study was to evaluate effects of milk replacer (MR) composition on: mass and composition of mammary parenchyma (PAR) and fat pad (MFP), growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis gene expression, and putative mammary epithelial stem cells. The hypothesis was that diet during the pre-weaning period alters the development, persistence, or activity of populations of putative mammary epithelial stem cells, possibly through involvement of GH/IGF-I axis molecules. Twenty-four newborn heifers were fed one of four MR diets: CON (20% CP, 21% fat MR fed at 441 g DM/d), HPLF (28% CP, 20% fat MR fed at 951 g DM/d), HPHF (27% CP, 28% fat MR fed at 951 g DM/d), and HPHF+ (27% CP, 28% fat MR fed at 1431 g DM/d). Animals were harvested on d 65 of life and mammary tissue was subjected to biochemical, molecular, and histological examination. By design, the effects of diet were evaluated at a common chronological age, but not necessarily at the same physiological age (body weight). Results from heifers reared on CON were compared to the average results from heifers reared on the other 3 diets. The second comparison evaluated the effect of increased fat in MR when protein content and intake were the same. The final comparison evaluated the effect of increased intake of a high-fat, high-protein MR. Neither diet composition nor nutrient intake in pre-weaned heifers affected PAR weight, PAR composition, GH/IGF-I axis gene expression, or putative mammary epithelial stem cell abundance when assessed at a common chronological age. Changes in MFP size and composition were observed, but no diet effect on GH/IGF-I axis gene expression in MFP was observed. This suggests nutrition is not critical for regulating the expression of local GH/IGF-I axis components or stem cell populations in the developing heifer mammary gland. / Ph. D.

Most mammary development is postnatal. Mammary growth that occurs before puberty is diminutive in amount but consequential for future milk production, especially in dairy heifers. With advanced knowledge on fundamental aspects that govern prepubertal mammary development, scientists and farmers alike can ensure that heifers perform their best once they become cows. The Hippo pathway has been identified as an evolutionarily conserved pathway that regulates organ size in many animal species; it might contribute to mammary growth in dairy heifers. This pathway is mediated by yes-associated protein (YAP) and through downstream gene transcription activation, results in cell proliferation. Because YAP has never been identified in bovine mammary tissue, questions examined in this body of work mainly focused on the abundance and localization of YAP in mammary tissue of prepubertal heifers. The first trial investigated effects of in vivo estradiol administration on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. While YAP was present in nuclei and cytoplasm of both cell types, it was also discovered that estrogen did not influence YAP abundance or location. The second research trial focused on determining the effects of in vivo estradiol blockade on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. Similar to the first experiment, results indicate that YAP abundance and localization was not influenced by estrogen blockade. Despite not being responsive to in vivo estradiol administration (experiment 1) or estradiol blockade (experiment 2) under the conditions of our experiments, YAP was present in nearly all mammary epithelial cells and myoepithelial cells of the 21 total prepubertal heifers examined. Its presence hints at an underlying biological function but that function was not ascertained here. It will be up to the next researcher to deduce what YAP contributes to mammary growth in prepubertal dairy heifers. / MSLFS

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Post-natal mammary gland development is a complex process in which epithelial proliferation and branching of lactiferous ducts is followed by extensive formation of lobuloalveolar units that produce milk. Classical nuclear factor-kappa B (NF-κB) p65/p50 transcription factors are dynamically induced in the mammary gland during pregnancy, and inhibitor of NF-κB-alpha (IκB-α) deficiency leads to hyperplasia of the mammary epithelium. To further elucidate the role of NF-κB factors in mammary development, we examined NF-κB subunit expression in the mammary glands of transgenic mice expressing the IκB-α S32/36A super-repressor (SR) protein under control of the mouse mammary tumor virus (MMTV)-long terminal repeat promoter, in which mammary gland development is transiently delayed, but not completely blocked. Developmental recovery correlated with induction of RelB/p52 NF-κB complexes, which failed to interact with an IκB-α fusion protein and potently induced cyclin D1 and c-myc promoter activities. Activation of IκB-α kinase alpha (IKKα) and NF-κB inducing kinase (NIK) was detected by day 5.5, and were hypothesized to be responsible for the induction of ReIB/p52. In support of this hypothesis, we found that constitutively active IKKα induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mammary tumors induced by high-dose 7,12-dimethylbenz(a)anthracene (DMBA) treatment in wild type FVB/N mice, displayed increased RelB/p52 binding activity. These results implicate activation of RelB/p52 complexes by the alternative NF-κB signaling pathway in branching of lateral ducts and alveolar development during mammary gland development, and in mammary carcinogenesis. / 2031-01-01

Prepartum, multiparous ewes were randomly assigned to experimental groups and sacrificed at 50(n=5), SO(n=4),115(n=5), and 140(n=4) days of gestation. Serum harvested the week prior to slaughter was assayed for progesterone (PG), prolactin (PRL) and growth hormone (GH) concentrations. Mammary tissue obtained at slaughter was assayed for receptor concentrations of progesterone (PGr), prolactin (PRLr) and insulin (Ir). Quantitative biochemical, histological and autoradiographical analyses were used to measure mammary gland growth and indicated no significant glandular growth occurs prior to 8O days of gestation. However, a major phase of parenchymal growth occurred between 8O and 115 days which coincided with significant increases in PG, PGr and PRLr concentrations. Parenchymal growth continued further into late pregnancy. GH and Ir concentrations did not change significantly during pregnancy and were not strongly correlated to growth measurements. These results suggest that mammary gland growth is receptor-mediated and direct or indirect regulation of PGr and PRLr is primarily responsible for the observed growth phenomenon. / Master of Science

Bacterial mastitis is a significant problem for the dairy industry. A vaccine against mastitis pathogens could potentially target dendritic cells (DC). While there has been some research describing bovine DC populations in-vitro, little is known about DC in mammary tissue. In this study, immunohistofluorescence was used to identify and localize bovine mammary DC. DC were found in alveoli, in epithelia, and in interalveolar tissue. Fluorescence-activated cell sorting (FACS) was used to characterize mammary DC as expressing CD11c, MHC-II, CD205, CD11b, and CD8α. FACS allowed us to distinguish DC (CD14lo) from macrophages (CD14hi). Two DC subsets, CD11a-, CD11alo, were evident in the mammary gland while an additional CD11ahi population was identified in the supramammary lymph node. After phagocytosis of bacterial components such as lipopolysaccharide (LPS), DC undergo a maturation process, in which they upregulate homing receptors, such as CCR7, and antigen presentation markers, including MHCII and CD80. A primary cell culture model was used to evaluate changes in transcription of CD80 and CCR7 after LPS stimulation. Cell cultures contained digested and Ficoll separated mammary tissue or supramammary lymph node tissue. While the presence of CCR7 and CD80 was confirmed, CD80 and CCR7 transcripts were not upregulated after LPS stimulation. Further, CD11c, CD14, MHCII, CD11b, CD11a, and CD205 protein levels, as assessed by FACS, were similar in LPS stimulated cultures and unstimulated controls. Overall, these studies provide a better understanding of mammary gland immunology, while potentially aiding in the development of novel DC based vaccines. / Master of Science

Previous research has shown that dairy cows require a dry period of at least 40 d for maximal milk yield in the subsequent lactation. Reducing the dry period requirement could prove beneficial to animal health and dairy profitability if subsequent milk yield was not reduced. Studies were conducted to evaluate the effects of continuous milking (CM) and hormonal treatments on milk yield, mammary epithelial cell (MEC) turnover during late gestation and early lactation, and mammary gene expression in dairy cows. A commercial trial using primiparous and multiparous cows demonstrated equal milk yields in bST-supplemented, CM and 60-d dry (CTL) multiparous cows, but lower milk yields in bST-supplemented CM, primiparous cows treated with bST. Subsequent experiments evaluated mammary development requirements during the dry period in primiparous cows and methods of rescuing milk yield. MEC growth was lower in CM glands during most of late gestation. Maintenance of lactation in CM glands resulted in a marked reduction in the MEC turnover process that occurs in the early dry period. In the last 20 d of gestation, MEC growth remained reduced in CM glands. By the last week of gestation, MEC growth was 50% less in CM tissue vs. CTL tissue. MEC apoptosis was unaffected by CM during the last 20 d of gestation, but a premature decrease in early lactation apoptosis occurred in CM glands at 7 d postpartum. Mammary gene expression demonstrated bax and insulin-like growth factor binding protein 5 are involved in apoptosis and cyclin D1, CCAT/enhancer binding protein-β, and bcl2 are involved in mammary development. Ultrastructure of CM tissue revealed large populations of resting or involuting alveoli by d 20 postpartum, whereas CTL glands had a homogenous population of secretory alveoli. Collectively, these data suggest that a 40-53% reduction in milk yield in CM glands is caused by reductions in MEC renewal and reduced secretory capacity. Treatments (bST, prostaglandin E2) to stimulate milk synthesis or MEC growth in CM primiparous glands were unsuccessful. In conclusion, primiparous cows continue to require a 60-d dry period, but multiparous cows are good candidates for short dry periods, and potentially no dry period.

No description available.