Mouse Mammary Gland Development Research Articles

Hormonal regulation of miRNA during mammary gland development.

The mammary gland is a unique organ as most of its development occurs after birth through stages of proliferation, differentiation and apoptosis that are tightly regulated by circulating hormones and growth factors. Throughout development, hormonal cues induce the regulation of different pathways, ultimately leading to differential transcription and expression of genes involved in this process, but also in the activation or inhibition of post-transcriptional mechanisms of regulation. However, the role of microRNAs (miRNAs) in the different phases of mammary gland remodeling is still poorly understood. The objectives of this study were to analyze the expression of miRNA in key stages of mammary gland development in mice and to determine whether it could be associated with hormonal variation between stages. To do so, miRNAs were isolated from mouse mammary glands at stages of adulthood, pregnancy, lactation and involution, and sequenced. Results showed that 490, 473, 419, and 460 miRNAs are detected in adult, pregnant, lactating and involuting mice, respectively, most of them being common to all four groups, and 58 unique to one stage. Most genes could be divided into six clusters of expression, including two encompassing the highest number of miRNA (clusters 1 and 3) and showing opposite profiles of expression, reaching a peak at adulthood and valley at lactation, or showing the lowest expression at adulthood and peaking at lactation. GO and KEGG analyses suggest that the miRNAs differentially expressed between stages influence the expression of targets associated with mammary gland homeostasis and hormone regulation. To further understand the links between miRNA expression and hormones involved in mammary gland development, miRNAs were then sequenced in breast cells exposed to estradiol, progesterone, prolactin and oxytocin. Four, 38, 24 and 66 miRNAs were associated with progesterone, estradiol, prolactin, and oxytocin exposure, respectively. Finally, when looking at miRNAs modulated by the hormones, differentially expressed during mammary gland development, and having a pattern of expression that could be correlated with the relative levels of hormones known to be found in vivo, 16 miRNAs were identified as likely regulated by circulating hormones. Overall, our study brings a better understanding of the regulation of miRNAs throughout mammary gland development and suggests that there is a relationship between their expression and the main hormones involved in mammary gland development. Future studies will examine this role more in detail.

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM),a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM)displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB)cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

ABCD4 is associated with mammary gland development in mammals.

Mammary gland development is a critical process in mammals, crucial for their reproductive success and offspring nourishment. However, the functional roles of key candidate genes associated with teat number, including ABCD4, VRTN, PROX2, and DLST, in this developmental process remain elusive. To address this gap in knowledge, we conducted an in-depth investigation into the dynamic expression patterns, functional implications, and regulatory networks of these candidate genes during mouse mammary gland development. In this study, the spatial and temporal patterns of key genes were characterized in mammary gland development. Using time-series single-cell data, we uncovered differences in the expression of A bcd4, Vrtn, Prox2, and Dlst in cell population of the mammary gland during embryonic and adult stages, while Vrtn was not detected in any cells. We found that only overexpression and knockdown of Abcd4 could inhibit proliferation and promote apoptosis of HC11 mammary epithelial cells, whereas Prox2 and Dlst had no significant effect on these cells. Using RNA-seq and qPCR, further analysis revealed that Abcd4 can induce widespread changes in the expression levels of genes involved in mammary gland development, such as Igfbp3, Ccl5, Tlr2, and Prlr, which were primarily associated with the MAPK, JAK-STAT, and PI3K-AKT pathways by functional enrichment. These findings revealed ABCD4 as a candidate gene pivotal for regulating mammary gland development and lactation during pregnancy by influencing PRLR expression.

INO80 function is required for mouse mammary gland development, but mutation alone may be insufficient for breast cancer.

The aberrant function of ATP-dependent chromatin remodeler INO80 has been implicated in multiple types of cancers by altering chromatin architecture and gene expression; however, the underlying mechanism of the functional involvement of INO80 mutation in cancer etiology, especially in breast cancer, remains unclear. In the present study, we have performed a weighted gene co-expression network analysis (WCGNA) to investigate links between INO80 expression and breast cancer sub-classification and progression. Our analysis revealed that INO80 repression is associated with differential responsiveness of estrogen receptors (ERs) depending upon breast cancer subtype, ER networks, and increased risk of breast carcinogenesis. To determine whether INO80 loss induces breast tumors, a conditional INO80-knockout (INO80 cKO) mouse model was generated using the Cre-loxP system. Phenotypic characterization revealed that INO80 cKO led to reduced branching and length of the mammary ducts at all stages. However, the INO80 cKO mouse model had unaltered lumen morphology and failed to spontaneously induce tumorigenesis in mammary gland tissue. Therefore, our study suggests that the aberrant function of INO80 is potentially associated with breast cancer by modulating gene expression. INO80 mutation alone is insufficient for breast tumorigenesis.

Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.

Per- and Polyfluoroalkyl Substances and Breastfeeding as a Vulnerable Function: A Systematic Review of Epidemiological Studies.

Milk formation in the breast during breastfeeding is a complex hormonally regulated process, potentially sensitive to the effects of endocrine-disrupting chemical exposures. The environmental chemicals, per- and polyfluoroalkyl substances (PFAS) are known endocrine disruptors. PFAS exposure have been associated with insufficient mammary gland development in mice and reduced breastfeeding duration in humans. The aim of this review was to gather the epidemiological evidence on the association between PFAS exposure and breastfeeding duration. Using PubMed and Embase, we performed a systematic literature search (on 23 January 2023) to identify epidemiological studies examining the association between maternal PFAS exposure and breastfeeding duration. Animal studies, reviews, and non-English studies were excluded. The risk of bias was assessed using the risk of bias in non-randomized studies of exposures tool. Estimates describing the association between PFAS exposure and the duration of breastfeeding were identified, and the data were synthesized separately for each type of PFAS and for the duration of exclusive and total breastfeeding. Six studies with between 336 and 2374 participants each were identified. PFAS exposure was assessed in serum samples (five studies) or based on residential address (one study). Five out of six studies found shorter total duration of breastfeeding with higher PFAS exposure. The most consistent associations were seen for perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and perfluorononanoic acid (PFNA). The finding of a potential causal association between PFAS exposure and breastfeeding duration is in agreement with findings from experimental studies.

Embryonic Programs in Cancer and Metastasis-Insights From the Mammary Gland.

Cancer is characterized as a reversion of a differentiated cell to a primitive cell state that recapitulates, in many aspects, features of embryonic cells. This review explores the current knowledge of developmental mechanisms that are essential for embryonic mouse mammary gland development, with a particular focus on genes and signaling pathway components that are essential for the induction, morphogenesis, and lineage specification of the mammary gland. The roles of these same genes and signaling pathways in mammary gland or breast tumorigenesis and metastasis are then summarized. Strikingly, key embryonic developmental pathways are often reactivated or dysregulated during tumorigenesis and metastasis in processes such as aberrant proliferation, epithelial-to-mesenchymal transition (EMT), and stem cell potency which affects cellular lineage hierarchy. These observations are in line with findings from recent studies using lineage tracing as well as bulk- and single-cell transcriptomics that have uncovered features of embryonic cells in cancer and metastasis through the identification of cell types, cell states and characterisation of their dynamic changes. Given the many overlapping features and similarities of the molecular signatures of normal development and cancer, embryonic molecular signatures could be useful prognostic markers for cancer. In this way, the study of embryonic development will continue to complement the understanding of the mechanisms of cancer and aid in the discovery of novel therapeutic targets and strategies.

Aberrant RET expression affects normal mammary gland post-lactation transition, enhancing cancer potential.

ABSTRACTRET is a receptor tyrosine kinase with oncogenic potential in the mammary epithelium. Several receptors with oncogenic activity in the breast are known to participate in specific developmental stages. We found that RET is differentially expressed during mouse mammary gland development: RET is present in lactation and its expression dramatically decreases in involution, the period during which the lactating gland returns to a quiescent state after weaning. Based on epidemiological and pre-clinical findings, involution has been described as tumor promoting. Using the Ret/MTB doxycycline-inducible mouse transgenic system, we show that sustained expression of RET in the mammary epithelium during the post-lactation transition to involution is accompanied by alterations in tissue remodeling and an enhancement of cancer potential. Following constitutive Ret expression, we observed a significant increase in neoplastic lesions in the post-involuting versus the virgin mammary gland. Furthermore, we show that abnormal RET overexpression during lactation promotes factors that prime involution, including premature activation of Stat3 signaling and, using RNA sequencing, an acute-phase inflammatory signature. Our results demonstrate that RET overexpression negatively affects the normal post-lactation transition.

A matrisome RNA signature from early-pregnancy mouse mammary fibroblasts predicts distant metastasis-free breast cancer survival in humans

BackgroundDuring pregnancy, the mouse mammary ductal epithelium branches and grows into the surrounding stroma, requiring extensive extracellular matrix (ECM) and tissue remodelling. It therefore shows parallels to cancer invasion. We hypothesised that similar molecular mechanisms may be utilised in both processes, and that assessment of the stromal changes during pregnancy-associated branching may depict the stromal involvement during human breast cancer progression.MethodsImmunohistochemistry (IHC) was employed to assess the alterations within the mouse mammary gland extracellular matrix during early pregnancy when lateral branching of the primary ductal epithelium is initiated. Primary mouse mammary fibroblasts from three-day pregnant and age-matched non-pregnant control mice, respectively, were 3D co-cultured with mammary epithelial cells to assess differences in their abilities to induce branching morphogenesis in vitro. Transcriptome analysis was performed to identify the underlying molecular changes. A signature of the human orthologues of the differentially expressed matrisome RNAs was analysed by Kaplan–Meier and multi-variate analysis in two large breast cancer RNA datasets (Gene expression-based Outcome for Breast cancer Online (GOBO) und Kaplan–Meier Plotter), respectively, to test for similarities in expression between early-pregnancy mouse mammary gland development and breast cancer progression.ResultsThe ECM surrounding the primary ductal network showed significant differences in collagen and basement membrane protein distribution early during pregnancy. Pregnancy-associated fibroblasts (PAFs) significantly enhanced branching initiation compared to age-matched control fibroblast. A combined signature of 64 differentially expressed RNAs, encoding matrisome proteins, was a strong prognostic indicator of distant metastasis-free survival (DMFS) independent of other clinical parameters. The prognostic power could be significantly strengthened by using only a subset of 18 RNAs (LogRank P ≤ 1.00e−13; Hazard ratio (HR) = 2.42 (1.8–3.26); p = 5.61e−09). The prognostic power was confirmed in a second breast cancer dataset, as well as in datasets from ovarian and lung cancer patients.ConclusionsOur results describe for the first time the early stromal changes that accompany pregnancy-associated branching morphogenesis in mice, specify the early pregnancy-associated molecular alterations in mouse mammary fibroblasts, and identify a matrisome signature as a strong prognostic indicator of human breast cancer progression, with particular strength in oestrogen receptor (ER)-negative breast cancers.

Expression of Grainyhead-like 2 in the Process of Ductal Development of Mouse Mammary Gland.

Grainyhead-like 2 (Grhl2) is a transcription factor regulating cell adhesion genes. Grhl2 acts as an epithelial-mesenchymal transition suppressor, and it is a proto-oncogene involved in estrogen-stimulated breast cancer proliferation. However, its expression during ovarian hormone-dependent mammary ductal development remains obscure. We here examined Grhl2 expression in the mammary gland of normal and steroid-replaced ovariectomized mice. Grhl2 protein signals were detected in both the mammary luminal epithelial and myoepithelial nuclei. The ratio and density of Grhl2-positive nuclei increased after the onset of puberty and progressed with age, whereas Grhl2-negative epithelial cells were detected in mature ducts. Claudin 3, claudin 4, claudin 7, and E-cadherin gene expression in the mammary gland was upregulated, and their expression was highly correlated with Grhl2 gene expression. Furthermore, Grhl2 mRNA expression and ductal lumen width were significantly increased by the combined treatment of estrogen and progesterone compared with estrogen alone. These results suggest that Grhl2 expressed in the luminal epithelial and myoepithelial cells from the early phase of ductal development, controlling the expression of cell adhesion molecules to establish functional ducts.

Progesterone receptor membrane component 1 is required for mammary gland development†.

The physiological functions of progesterone (P4) in female reproductive organs including the mammary glands are mediated via the progesterone receptor (PR), but not all P4 functions can be explained by PR-mediated signaling. Progesterone receptor membrane component 1 (PGRMC1), a potential mediator of P4 actions, plays an important role in the ovary and uterus in maintaining female fertility and pregnancy, but its function in mammary glands has not been elucidated. This study investigated the role of PGRMC1 in mouse mammary gland development. Unlike in the uterus, exogenous estrogen (E2) and/or P4 did not alter PGRMC1 expression in the mammary gland, and Pgrmc1-knockout (KO) mice displayed reduced ductal elongation and side branching in response to hormone treatment. During pregnancy, PGRMC1 was expressed within both the luminal and basal epithelium and gradually increased with gestation and decreased rapidly after parturition. Moreover, although lactogenic capacity was normal after parturition, Pgrmc1 KO resulted in defective mammary gland development from puberty until midpregnancy, while the expression of PR and its target genes was not significantly different between wild-type and Pgrmc1-KO mammary gland. These data suggest that PGRMC1 is essential for mammary gland development during puberty and pregnancy in a PR-independent manner.

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

SummaryThe link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.

Postnatal wheel running mitigates endocrine disruption of mammary gland development in mice

Postnatal wheel running mitigates endocrine disruption of mammary gland development in mice

DC-SCRIPT deficiency delays mouse mammary gland development and branching morphogenesis

Mammary glands are unique organs in which major adaptive changes occur in morphogenesis and development after birth. Breast cancer is the most common cancer and a major cause of mortality in females worldwide. We have previously identified the loss of expression of the transcription regulator DC-SCRIPT (Zfp366) as a prominent prognostic event in estrogen receptor positive breast cancer patients. DC-SCRIPT affects multiple transcriptional events in breast cancer cells, including estrogen and progesterone receptor-mediated transcription, and promotes CDKN2B-related cell cycle arrest. As loss of DC-SCRIPT expression appears an early event in breast cancer development, we here investigated the role of DC-SCRIPT in mammary gland development using wild-type and DC-SCRIPT knockout mice. Mice lacking DC-SCRIPT exhibited severe breeding problems and showed significant growth delay relative to littermate wild-type mice. Subsequent analysis revealed that DC-SCRIPT was expressed in mouse mammary epithelium and that DC-SCRIPT deficiency delayed mammary gland morphogenesis in vivo. Finally, analysis of 3D mammary gland organoid cultures confirmed that loss of DC-SCRIPT dramatically delayed mammary organoid branching in vitro. The study shows for the first time that DC-SCRIPT deficiency delays mammary gland morphogenesis in vivo and in vitro. These data define DC-SCRIPT as a novel modulator of mammary gland development.

Bisphenol S alters development of the male mouse mammary gland and sensitizes it to a peripubertal estrogen challenge

Humans are exposed to estrogenic chemicals in food and food packaging, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS; 4,4′-sulfonyldiphenol), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. Here, we quantified the effects of perinatal exposures to BPS on the male mouse mammary gland. In our first study, pregnant CD-1 mice were orally exposed to BPS (2 or 200 μg/kg/day) starting on pregnancy day 9 through lactation day 20, and male mammary glands were evaluated on embryonic day 16, prior to puberty, and in early adulthood. We observed modest changes in tissue organization in the fetal gland, and significant increases in growth of the gland induced by developmental BPS exposure in adulthood. In our second study, pregnant CD-1 mice were orally exposed to BPS (2, 200 or 2000 μg/kg/day) starting on pregnancy day 9 through lactational day 2. After weaning, the male pups were administered either oil (vehicle) or an estrogen challenge (1 μg ethinyl estradiol/kg/day) for ten days starting prior to puberty. After the 10-day estrogen challenge, we examined hormone-sensitive outcomes including anogenital index (AGI), weight of the seminal vesicles, and morphological parameters of the mammary gland. Although AGI and seminal vesicle weight were not affected by BPS, we observed dose-specific effects on the response of male mammary glands to the peripubertal estrogen challenge. Because male mammary glands are structurally less developed compared to females, they may provide a simple model tissue to evaluate the effects of putative xenoestrogens.

Sfrp3 modulates stromal\u2013epithelial crosstalk during mammary gland development by regulating Wnt levels

Mammary stroma is essential for epithelial morphogenesis and development. Indeed, postnatal mammary gland (MG) development is controlled locally by the repetitive and bi-directional cross-talk between the epithelial and the stromal compartment. However, the signalling pathways involved in stromal–epithelial communication are not entirely understood. Here, we identify Sfrp3 as a mediator of the stromal–epithelial communication that is required for normal mouse MG development. Using Drosophila wing imaginal disc, we demonstrate that Sfrp3 functions as an extracellular transporter of Wnts that facilitates their diffusion, and thus, their levels in the boundaries of different compartments. Indeed, loss of Sfrp3 in mice leads to an increase of ductal invasion and branching mirroring an early pregnancy state. Finally, we observe that loss of Sfrp3 predisposes for invasive breast cancer. Altogether, our study shows that Sfrp3 controls MG morphogenesis by modulating the stromal-epithelial cross-talk during pubertal development.

OR26-2 Phosphorylation of Estrogen Receptor Alpha is Required for Mammary Gland Development and MCF-7 Breast Tumor Response to Estradiol and Tamoxifen

Serine-167 (S167) is a major phosphorylation site in estrogen receptor alpha (ERα) and a predictor for endocrine therapy response in breast cancer. We previously showed using stable transfection that mutation of S167 to alanine (S167A) impacted MCF-7 breast cancer cell growth and morphology. Using CRISPR, we evaluated S167A mutation in the endogenous ERα gene in MCF-7 breast cancer cells and xenograft tumors. In vitro, MCF-7 cells with wildtype ERα (WT) exhibited >60% increase in growth after 4 days incubation with 10-9 M 17-β estradiol (E2), and >300% decrease in growth after 4 days incubation with 10-8 M 4-hydroxy-tamoxifen (TAM) as assessed by MTS assay. In marked contrast, S167A cells showed resistance to both E2 growth stimulation and TAM growth arrest. WT cells exhibited >300% increase in migration after 24 hr E2 as assessed by Boyden Chamber assay. S167A cells exhibited >3 fold increase in basal migration compared to WT cells but were insensitive to E2 stimulation of migration. RNAseq was performed on WT and S167A cells incubated with E2 or vehicle (6 hr). Sequencing data was assessed using FastQC and sequenced libraries mapped to the human genome (UCSC hg38) using STAR RNAseq aligner. RNASeq data was normalized using TMM. Differential expression analysis by edgeR revealed significant differences in gene expression between WT and S167A cells. Vehicle-treated S167A cells exhibited 623 differentially regulated genes compared to WT cells. Notably, S167A cells exhibited marked E2 resistance with only 9 genes regulated by E2 in S167A cells compare to 93 genes in WT cells. In tumor xenograft experiments using ovariectomized nude mice, similar to WT tumors the S167A tumors did not grow in the absence of implanted 17 beta-estradiol (0.72 mg/pellet; 60 day release) demonstrating dependency of S167A tumors on E2 for tumor establishment and growth. However, unlike WT tumors that exhibited tumor regression with E2 + TAM (5 mg/pellet; 60 day release) treatment (week 4, P<0.05), S167A tumors were resistant to TAM and exhibited continued growth similar to E2 alone treatment. Serine-212 (S216 in mouse) is a phosphorylation site located between the two zinc fingers in the DNA binding domain and is highly conserved in the nuclear receptor superfamily. To understand the importance of ERα phosphorylation for mammary gland development, ERα knock-in (KI) mice (Esr1S216A) that express the ERα S216A mutation were evaluated. Mammary glands were whole-mounted with carmine-alum from 5 week-old pubertal female ERα wildtype (WT) and KI mice (n=3). KI mice exhibited a significant decrease in ductal elongation (P<0.05), number of terminal end buds (P<0.01) and branching morphogenesis (P<0.01) compared to WT mice. Collectively, these results demonstrate that S216 phosphorylation is critical for mouse mammary gland development and S167 phosphorylation is required for MCF-7 breast tumor response to estradiol and tamoxifen.

Defects of the endoplasmic reticulum and changes to lipid droplet size in mammary epithelial cells due to miR-30b-5p overexpression are correlated to a reduction in Atlastin 2 expression

During lactation, mammary epithelial cells secrete fat in the form of milk fat globules that originate from intracellular lipid droplets. These droplets may form de novo from the endoplasmic reticulum or be derived from existing lipid droplets; they then either grow because enzymes of triacylglycerol synthesis relocate from the reticulum to their surface, or due to fusion and fission with other droplets. The overexpression of miR-30b-5p in the developing mouse mammary gland impairs lactation, which includes an increase in lipid droplet size. This study was performed to understand the origin of this defect affecting lipid droplets observed in transgenic mice.Electron microscopy analyses revealed a fragmented and discontinued tubular network of endoplasmic reticulum in the mammary epithelial cells of transgenic mice. The milk fatty acid composition was modified, with lower levels of medium-chain saturated fatty acids and a proportional increase in long-chain monounsaturated fatty acids in transgenic versus wild-type mice. Further, investigations of microRNA targets revealed a significant downregulation of ATLASTIN 2 (a GTPase described as playing a key role in lipid droplet formation) due to miR-30b-5p overexpression.Our results suggest that the increase in lipid droplet size observed in the mammary epithelial cells of transgenic mice might result from changes to lipid droplet formation and secretion because of direct modifications to Atl2 expression and indirect changes to endoplasmic reticulum morphology resulting from the overexpression of miR-30b-5p.

Asymmetric development of the male mouse mammary gland and its response to a prenatal or postnatal estrogen challenge

The CD-1 mouse mammary gland is sexually dimorphic, with males lacking nipples. Recent studies have revealed that the underlying epithelium in the male mammary gland is sensitive to estrogenic environmental chemicals. In ongoing investigations, we observed asymmetric morphology in the left and right male mouse mammary glands. Here, we quantified these asymmetries in the embryonic, prepubertal, pubertal and adult male mammary gland. We found that the right gland was typically larger with more branching points compared to the left gland. We next evaluated the response of the left and right glands to 17α-ethinyl estradiol (EE2) after perinatal or peripubertal exposures. We found that the right gland was more responsive to EE2 than the left at both periods of exposure. These results reveal novel aspects of male mammary gland biology and suggest that future studies should control for laterality in the evaluation of hazards associated with exposures to estrogenic chemicals.