Mammary Gland Physiology Research Articles

Post-milking application of a Lacticaseibacillus paracasei strain impacts bovine teat microbiota while preserving the mammary gland physiology and immunity.

Bovine mastitis (BM) is a major disease in dairy industry. The current approaches- mainly antibiotic treatments- are not entirely effective and may contribute to antimicrobial resistance dissemination, rising the need for alternative treatment. The present study aims to evaluate the impact of post-milking application of Lacticaseibacillus paracasei CIRM BIA 1542 (Lp1542) on the teat skin (TS) of 20 Holstein cows in mid lactation, in order to reinforce the barrier effect of the microbiota naturally present on the teat. Treatment (Lp1542, iodine or no treatment) was applied post-milking twice a day on the 4 teats of healthy animals for 15 days. Blood and milk samples, and TS swabs were collected at day (D)1, D8, D15 and D26 before morning milking and at D15 before evening milking (D15E) to evaluate Lp1542 impact at the microbial, immune and physiological levels. Lp1542 treatment resulted in a higher lactic acid bacteria and total microbial populations on TS and in foremilk (FM) at D15(E) compared with iodine treatment. Metabarcoding analysis revealed changes in the composition of TS and FM microbiota, beyond a higher Lacticaseibacillus abundance. This included a higher abundance of Actinobacteriota, including Bifidobacterium, and a lower abundance of Pseudomonadota on TS of Lp1542 compared with iodine-treated quarters. In addition, Lp1542 treatment did not trigger any major inflammatory response in the mammary gland, except interleukin 8 production and expression which tended to be slightly higher in Lp1542-treated cows compared with the others. Finally, Lp1542 treatment had no impact on the mammary epithelium functionality (milk yield and composition) and integrity (epithelial cell exfoliation into milk and milk Na+/K+ ratio). Altogether, these results indicate that a topical treatment with Lp1542 is safe with regard to mammary gland physiology and immune system, while impacting its microbiota, inviting us to further explore its effectiveness for mastitis prevention.

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota.

Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.

A Decision Tree to Guide Human and Mouse Mammary Organoid Model Selection.

Over the past 50 years, researchers from the mammary gland field have launched a collection of distinctive 3D cell culture systems to study multiple aspects of mammary gland physiology and disease. As our knowledge about the mammary gland evolves, more sophisticated 3D cell culture systems are required to answer more and more complex questions. Nowadays, morphologically complex mammary organoids can be generated in distinct 3D settings, along with reproduction of multiple aspects of the gland microenvironment. Yet, each 3D culture protocol comes with its advantages and limitations, where some culture systems are best suited to study stemness potential, whereas others are tailored towards the study of mammary gland morphogenesis. Therefore, prior to starting a 3D mammary culture experiment, it is important to consider and select the ideal culture model to address the biological question of interest. The number and technical requirements of novel 3D cell culture methods vastly increased over the past decades, making it currently challenging and time consuming to identify the best experimental testing. In this chapter, we provide a summary of the most promising murine and human 3D organoid models that are currently used in mammary gland biology research. For each model, we will provide a brief description of the protocol and an overview of the expected morphological outcome, the advantages of the model, and the potential pitfalls, to guide the reader to the best model of choice for specific applications.

Milk proteins as a feed restriction signature indicating the metabolic adaptation of dairy cows

Milk production in dairy cows is affected by numerous factors, including diet. Feed restriction is known to have little impact on milk total protein content but its effect on the fine protein composition is still poorly documented. The objective of this study was to describe the effects of two feed restriction trials of different intensities on the milk protein composition of Holstein cows. One restriction trial was of high intensity (H: 8 mid-lactation Holstein cows) and the second of moderate intensity (M: 19 peak lactation Holstein cows). Feed restriction decreased the milk protein yield for caseins under the M trial and of all six major milk proteins under the H trial. These decreased yields lead to lower concentrations of αs1-, αs2- and β-caseins during the H trial. The milk proteome, analyzed on 32 milk samples, was affected as a function of restriction intensity. Among the 345 proteins identified eight varied under the M trial and 160 under the H trial. Ontology analyses revealed their implication in carbohydrate, lipid and protein metabolisms as well as in the immune system. These proteins reflected adaptations of the animal and mammary gland physiology to feed restriction and constituted a signature of this change.

Comprehensive Proteomic Analysis Revealed a Large Number of Newly Identified Proteins in the Small Extracellular Vesicles of Milk from Late-Stage Lactating Cows.

Simple SummaryBovine milk provides excellent nutrition for infants and contains significant bioactive compounds, including small extracellular vesicles. Small extracellular vesicles are nanosized particles that have long been recognized to carry a variety of cargos, including proteins, nucleic acids, lipids, and other biomolecules. Previous research has shown that the proteomic profile in milk small extracellular vesicles differed in accordance with the lactation stages of cows, such as early- and mid-stage lactation. However, the protein profile in milk small extracellular vesicles from late-stage lactation is mostly unknown. In this study, a comprehensive proteomic analysis was performed to explore the proteins in milk small extracellular vesicles from late-stage lactating cows. The present study identified a total of 2225 proteins in milk small extracellular vesicles from late-stage lactating cows. Notably, a total of 429 proteins were newly identified after comparing with the previously published bovine milk small extracellular vesicles proteomic datasets. These findings indicate that the newly identified proteins could be helpful in better understanding the proteomic dynamics in milk small extracellular vesicles during the late-stage lactating cows.Bovine milk contains small extracellular vesicles (sEVs) that provide proteins, miRNAs, mRNAs, DNAs, and lipids to target cells and play a role in intracellular communications. Previous studies have characterized proteins in milk sEVs from early- and mid-stage lactation. However, the proteins in milk sEVs from late-stage lactation are mostly unexplored. The aim of this study was to determine the proteomic profile of milk sEVs from late-stage lactating cows. A comprehensive nanoliquid chromatography–tandem mass spectrometry (nanoLC-MS/MS) approach was carried out to reveal the proteins in milk sEVs. Additionally, bioinformatics analysis was carried out to interpret the molecular signatures of newly identified proteins in milk sEVs from three late-stage lactating cows. NanoLC-MS/MS analysis revealed a total of 2225 proteins in milk sEVs from cows. Notably, after comparing these identified proteins with previously deposited datasets of proteins in bovine milk sEVs, 429 proteins were detected as newly identified. Bioinformatic analysis indicated that these newly identified proteins in milk sEVs were engaged in a diverse range of molecular phenomena relevant to mammary gland physiology, milk production, immunity, and immune response. These findings suggest that the newly identified proteins could expand the inventory application of molecular cargos, nutritional status, and immune modulation of sEVs in milk during the late-stage lactation.

ScRNA-seq Reveals a Role of Mammary Luminal Epithelium in Adipocyte Adaptations

Almost four decades of research suggest a dynamic role of ductal epithelial cells in adipocyte adaptation in mammary gland white adipose tissue (mgWAT), but factors that mediate such communication are not known. Here, we identify a complex intercellular crosstalk in mgWAT revealed by single-cell RNA-seq (scRNA-seq) and comprehensive data analysis suggest that epithelial luminal cells during cold exposure undergo major transcriptomic changes that lead to the expression of an array of genes that encode for secreted factors involved in adipose metabolism such as Adropin (Enho), neuregulin 4 (Nrg4), angiopoietin-like 4 (Angptl4), lipocalin 2 (Lcn2), milk fat globule-EGF factor 8 (Mfge8), Insulin-like growth factor-binding protein 1 (Igfbp1), and haptoglobin (Hp). To define the mammary epithelial secretome, we coin the phrase “mammokines”. We validated our cluster annotations and cluster-specific transcriptomics using eight different adipose scRNA-seq datasets including Tabula Muris and Tabula Muris Senis. In situ mRNA hybridization and ex vivo isolated mgWAT luminal cells show highly localized expression of mammokines in mammary ducts. Trajectory inference demonstrates that cold-exposed luminal cells have similar transcriptional profiles to lactation post-involution (PI), a phase defined by reappearance and maintenance of adipocytes in the mammary gland. Concomitantly, we found that under cold exposure female mgWAT maintains more adipogenic and less thermogenic potential than male scWAT and ex vivo removal of luminal epithelial cells from mgWAT markedly potentiates beige adipocyte differentiation. Conditioned media from isolated mammary epithelial cells treated with isoproterenol suppressed thermogenesis in differentiated beige/brown adipocytes and treatment of beige/brown differentiated adipocyte with mammokine LCN2 suppresses thermogenesis and increases adipogenesis. Finally, we find that mice lacking LCN2 show markedly higher cold-dependent thermogenesis in mgWAT than controls, and reconstitution of LCN2 in the mgWAT of LCN2 knockout mice promotes inhibition of thermogenesis. These results show a previously unknown role of mammary epithelium in adipocyte metabolism and suggest a potentially redundant evolutionary role of mammokines in maintaining mgWAT adiposity during cold exposure. Our data highlight mammary gland epithelium as a highly active metabolic cell type and mammokines could have broader implications in mammary gland physiology and lipid metabolism.

Yüksek ET-1 İfadesinin Meme Bezi Fizyolojisi Üzerine Etkileri

Objective: Alongside its role as a potent vasoconstrictor in the cardiovascular system, endothelin-1 (ET-1) is involved in numerous physiological processes. Its expression is thought to be an important function for both breastfeeding and newborns since it occurs especially during pregnancy and lactation. In this study, we projected the effects of ET-1 on mammary gland physiology employing ET-1 transgenic mice. Materials and Methods: In this study, we compared the mammary glands of ET-1 transgenic mice with the control mice in different physiological states (adolescence, pregnant, lactation and involution). Parallel sections were prepared from mammary glands and microscopic examinations were performed with hematoxylin and eosin (HE)-staining. Western blot techniques were used in the analysis of signal proteins that have important functions in mammary gland development and the metabolism (STAT5, AKT, STAT3). Genes known to play a key function in metabolic activities (WAP, beta casein, GLUT-1, SRBF-1 Claudin 8, IL-6, LIF) were analyzed employing real-time PCR techniques. Results: It was determined that ET-1 transgenic mice developed pathological conditions such as secretory damage, lactational failure and early involution, respectively. Our western blot studies showed that STAT5 activation was not affected by this process in the transgenic group, but a decrease in AKT protein level was detected. We detected STAT3 activation seen in the involution process in transgenic mice only on the 3rd day of lactation. In addition, we found that WAP was suppressed due to high ET-1 expression and that LIF protein expression increased significantly, by real-time PCR analysis. Conclusion: ET-1 expression is thought to be an important function for both lactation and newborns, especially during pregnancy and lactation. In this study, it was found that when ET-1 expression increases, it causes lactation failure in the mammary gland and directly affects the synthesis of genes known to be important in mammary physiology. Hereby, it was determined that the regulation of ET-1 plays a critical role in the development and secretory function of the mammary gland.

5-ALA Attenuates the Palmitic Acid-Induced ER Stress and Apoptosis in Bovine Mammary Epithelial Cells.

The conservation of mammary gland physiology by maintaining the maximum number of mammary epithelial cells (MECs) is of the utmost importance for the optimum amount of milk production. In a state of negative energy balance, palmitic acid (PA) reduces the number of bovine MECs. However, there is no effective strategy against PA-induced apoptosis of MECs. In the present study, 5-aminolevulinic acid (5-ALA) was established as a remedial agent against PA-induced apoptosis of MAC-T cells (an established line of bovine MECs). In PA-treated cells, the apoptosis-related genes BCL2 and BAX were down- and upregulated, respectively. The elevated expression of major genes of the unfolded protein response (UPR), such as CHOP, a proapoptotic marker (C/EBP homologous protein), reduced the viability of PA-treated MAC-T cells. In contrast, 5-ALA pretreatment increased and decreased BCL2 and BAX expression, respectively. Moreover, cleaved caspase-3 protein expression was significantly reduced in the 5-ALA-pretreated group in comparison with the PA group. The downregulation of major UPR-related genes, including CHOP, extended the viability of MAC-T cells pretreated with 5-ALA and also reduced the enhanced intensity of the PA-induced expression of phospho-protein kinase R-like ER kinase. Moreover, the enhanced expression of HO-1 (antioxidant gene heme oxygenase) by 5-ALA reduced PA-induced oxidative stress (OxS). HO-1 is not only protective against OxS but also effective against ER stress. Collectively, these findings offer new insights into the protective effects of 5-ALA against PA-induced apoptosis of bovine MECs.

Abstract PR05: Genome-wide screening identifies novel YAP modulators

Abstract The transcriptional coactivator YAP is the key downstream effector of the Hippo pathway, which is a central regulator of organ size, stem cell/progenitor cell expansion, and tissue regeneration. Diverse studies have shown the importance of a precise regulation of this pathway and its key downstream regulator YAP for tissue homeostasis. To achieve new insights in the Hippo-independent transcriptional regulation of YAP in breast cancer, we set out to identify new YAP modulators using a genome-wide CRISPR screen and Perturb-Seq. We identified the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation, using a genome-wide CRISPR screen. TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. Moreover, TRPS1 antagonizes activating YAP/TEAD complexes by recruiting corepressor complexes to joint sites. Loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter-enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer, suggesting that restrained YAP activity favors tumor growth. Combined, we uncover TRPS1 as an epigenetic regulator of YAP activity in breast cancer. Additionally, we developed a Trps1-deficient mouse line that we will use to test the in vivo role of Trps1 in mammary gland physiology and breast cancer. Furthermore, we combined CRISPR-based gene perturbation with droplet-based single-cell RNA sequencing (Perturb-Seq), to perform a second large-scale analysis on potential YAP modulators. To this end, we used a CRISPR-library containing 158 previously from us in a Bio-ID assay identified YAP interactors. Together, we uncover several novel YAP modulators, contributing to a better understanding of the Hippo-independent transcriptional regulation of YAP. This abstract is also being presented as Poster B40. Citation Format: Dana Elster, Marie Tollot, Karin Schlegelmilch, Alessandro Ori, Andreas Rosenwald, Erik Sahai, Björn von Eyss. Genome-wide screening identifies novel YAP modulators [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR05.

Shifts in the Holstein dairy cow milk fat globule membrane proteome that occur during the first week of lactation are affected by parity

BackgroundThe milk fat globule membrane (MFGM) proteomes of colostrum and transition milk are rich sources of proteins that are likely important for neonatal calf health. In addition, characterization of these proteomes could also yield valuable information regarding mammary gland physiology of the early postpartum lactating cow. The objectives of this research were to characterize the MFGM proteomes of colostrum and transition milk through sample collections at four timepoints postpartum, including the first milking (M1, colostrum), second milking (M2, transition milk), fourth milking (M4, transition milk), and fourteenth milking (M14, mature milk), and compare these proteomes between multiparous (MP; n = 10) and primiparous (PP; n = 10) Holstein dairy cows. Isolated MFGM proteins were labeled using Tandem Mass tagging and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein identification was completed using MASCOT and Sequest in Proteome Discoverer 2.2. The scaled abundance values were analyzed using PROC MIXED in SAS to determine the effects of milking (MIL), parity (PAR), and MIL × PAR. The adaptive false-discovery rate (FDR)-adjusted P values were determined using PROC MULTTEST. Protein characterization and bioinformatic analysis were completed using a combination of PANTHER, Blast, and Uniprot.ResultsA total of 104 common proteins were identified in each of the MFGM samples. Statistical analysis revealed that 70.2% of identified proteins were affected by MIL. Of these, 78.1% were lower in M14 compared with M1, including immune-related proteins lactotransferrin, lactadherin and hemopexin. Parity affected 44.2% of proteins. Of the proteins affected by PAR, 84.8% were higher in MP cows compared with PP cows, including apolipoprotein E and histones 2A, 2B, 3, and 4 b. Butyrophilin subfamily 1 member 1A and annexin 5 were higher in samples from PP cows. Milking × parity affected 32.7% of identified proteins, including lactotransferrin, gelsolin, vitamin D binding protein, and S100 proteins.ConclusionsThis research supports previous findings that the Holstein MFGM proteome changes rapidly during the first week of lactation. In addition, this research identifies the impact of parity on the colostrum and transition milk MFGM proteomes, which may be important for milk-fed calf health or for the identification of protein biomarkers for mammary functionality.

Characterization of microRNA profiles in the mammary gland tissue of dairy goats at the late lactation, dry period and late gestation stages.

MicroRNAs (miRNAs) play an important role in regulating mammary gland development and lactation. We previously analyzed miRNA expression profiles in Laoshan dairy goat mammary glands at the early (20 d postpartum), peak (90 d postpartum) and late lactation (210 d postpartum) stages. To further enrich and clarify the miRNA expression profiles during the lactation physiological cycle, we sequenced miRNAs in the mammary gland tissues of Laoshan dairy goats at three newly selected stages: the late lactation (240 d postpartum), dry period (300 d postpartum) and late gestation (140 d after mating) stages. We obtained 4038 miRNAs and 385 important miRNA families, including mir-10, let-7 and mir-9. We also identified 754 differentially expressed miRNAs in the mammary gland tissue at the 3 different stages and 6 groups of miRNA clusters that had unique expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that GO terms such as mammary gland development (GO:0030879) and mammary gland morphogenesis (GO:0060443) and important signaling pathways, including the insulin signaling pathway (chx04910), hippo signaling pathway (chx04390) and estrogen signaling pathway (chx04915), were enriched. We screened miRNAs and potential target genes that may be involved in the regulation of lactation, mammary gland growth and differentiation, cell apoptosis, and substance transport and synthesis and detected the expression patterns of important genes at the three stages. These miRNAs and critical target genes may be important factors for mammary gland development and lactation regulation and potentially valuable molecular markers, which may provide a theoretical reference for further investigation of mammary gland physiology.

Genome\u2011wide integrated analysis demonstrates widespread functions of lncRNAs in mammary gland development and lactation in dairy goats

BackgroundThe mammary gland is a unique organ for milk synthesis, secretion and storage, and it undergoes cyclical processes of development, differentiation, lactation and degeneration. At different developmental periods, the biological processes governing mammary gland physiology and internal environmental homeostasis depend on a complex network of genes and regulatory factors. Emerging evidence indicates that lncRNAs have arbitrarily critical functions in regulating gene expression in many organisms; however, the systematic characteristics, expression, and regulatory roles of lncRNAs in the mammary gland tissues of dairy goats have not been determined.ResultIn the present study, we profiled long noncoding RNA (lncRNA) expression in the mammary gland tissues of Laoshan dairy goats (Capra hircus) from different lactation periods at the whole-genome level, to identify, characterize and explore the regulatory functions of lncRNAs. A total of 37,249 transcripts were obtained, of which 2381 lncRNAs and 37,249 mRNAs were identified, 22,488 transcripts, including 800 noncoding transcripts and 21,688 coding transcripts, differed significantly (p ≤ 0.01) among the different lactation stages. The results of lncRNA-RNA interaction analysis showed that six known lncRNAs belonging to four families were identified as the precursors of 67 known microRNAs; 1478 and 573 mRNAs were predicted as hypothetical cis-regulation elements and antisense mRNAs, respectively. GO annotation and KEGG analysis indicated that the coexpressed mRNAs were largely enriched in biological processes related to such activities as metabolism, immune activation, and stress,., and most genes were involved in pathways related to such phenomena as inflammation, cancer, signal transduction, and metabolism.ConclusionsOur results clearly indicated that lncRNAs involved in responses to stimuli, multiorganism processes, development, reproductive processes and growth, are closely related to mammary gland development and lactation.

Host Microbe Interactions in the Lactating Mammary Gland.

The bacteria present in human milk constitute the human milk microbiome (hMM). Both the older culture-based work and the more recent studies using molecular detection of bacterial DNA have reached similar conclusions: the hMM mostly consists of commensal staphylococci such as Staphylococcus epidermidis, and streptococci. The prevalence of other bacterial groups such lactobacilli varies widely, while the abundance and prevalence of bifidobacteria is generally low. Recently, the hMM became accepted as a part of a physiologically normal state with suggested potential health benefits. Most research on the hMM has focused on its composition and potential effect on the breastfed infant. A major role as a microbiome inoculum for the infant gut has been proposed, but remains to be clearly demonstrated. Herein, we also discuss the emerging connection between the hMM and mammary gland physiology and lactation. Similarities between the mammary gland and mucosal interfaces are considerable, and in particular mucosal-like immune attributes of mammary gland. The potential role of hMM-host interactions in the mammary gland in maternal health is explored with a primary focus on lactational mastitis.

Somatic cell count in sheep milk

The review reports recent knowledge on the role and significance of somatic cells in sheep mammary gland physiology. Somatic cell count represents an index of animal health status and it is considered an elective standard for the milk quality detection. However, there is still the need to deepen the knowledge on this complex parameter and to define a cut-off value of SCC in sheep milk in order to guarantee safety and quality of sheep milk and to sustain the development of dairy sheep productive system. Quantitative limits and detection methods for somatic cell count in sheep milk production are discussed in relation to qualitative, hygienic and technological aspects. Furthermore somatic cell level and qualitative patterns in sheep milk are discussed in relation to milk cytokines, endogenous proteolytic enzymes and pathogenic bacteria; this new approach to milk quality evaluation is reported to highlight possible early and reliable indicators able to predict and ensure quality and safety of sheep milk and dairy products. Finally, the role of mammary epithelial cells in the defence of the mammary gland represents a new research field, which still needs to be explored in sheep milk.

Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Maternal diet during lactation affects offspring metabolic health throughout life. Prolactin (PRL) is present in high quantities in maternal milk; however, the effects of milk PRL on the offspring remain poorly characterized. In this study, we evaluated whether feeding a high-fat diet (HFD) to rats during lactation alters PRL, both in the mother's serum and in milk, and whether this factor contributes to HFD-induced metabolic dysfunction in the offspring. Maternal HFD resulted in decreased PRL levels in milk (but not in serum), reduced mammary gland (MG) PRL receptor expression, and altered MG structure and function. Offspring from HFD-fed dams had increased body weight and adiposity, and developed fatty liver, hyperinsulinemia, and insulin resistance at weaning. Increasing PRL levels in the HFD-fed mothers by subcutaneous osmotic minipumps releasing PRL normalized MG function and PRL levels in milk. Moreover, PRL treatment in HFD-fed mothers, or directly in their pups via oral PRL administration, increased liver STAT5 phosphorylation, reduced visceral adiposity, ameliorated fatty liver, and improved insulin sensitivity in offspring. Our results show that HFD impairs PRL actions during lactation to negatively affect MG physiology and directly impair offspring metabolism.-De los Ríos, E. A., Ruiz-Herrera, X., Tinoco-Pantoja, V., López-Barrera, F., Martínez de la Escalera, G., Clapp, C., Macotela, Y. Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Transcriptome analysis of the mammary gland from GH transgenic goats during involution

Mammary glands are organs for milk production in female mammals. Growth hormone (GH) is known to affect the growth and development of the mammary gland, as well as to increase milk production in dairy goats. This study performed a comprehensive expression profiling of genes expressed in the mammary gland of early involution GH transgenic (n=4) and non-transgenic goats (n=4) by RNA sequencing. RNA was extracted from mammary gland tissues collected at day 3 of involution. Gene expression analysis was conducted by Illumina RNA sequencing and sequence reads were assembled and analyzed using TopHat. FPKM (fragments per kilobase of exon per million) values were analyzed for differentially expressed genes using the Cufflinks package. Gene ontology analysis of differentially expressed genes was categorized using agriGO, while KEGG pathway analysis was performed with the online KEGG automatic annotation server. Our results revealed that 75% of NCBI goat annotated genes were expressed during early involution. A total of 18,323 genes were expressed during early involution in GH transgenic goats, compared with 18,196 expressed genes during early involution of non-transgenic goats. In these expressed genes, the majority (17,589) were ubiquitously expressed in GH transgenic and non-transgenic goats. However, there were 745 differentially expressed genes, 421 of which were upregulated and 324 were downregulated in GH transgenic goats. GO and KEGG pathway analysis showed that these genes were involved in mammary gland physiology, including cell adhesion molecules, ECM-receptor interaction, Jak-STAT signaling pathway, and fat metabolism. Our results demonstrated that the GH receptor was strongly affected in GH transgenic goats, which may activate the IGF-1/Stat3 signaling pathway. Overall, our study provided a global view of the transcriptome during involution of GH transgenic and non-transgenic goats, which increases our understanding of the biology of involution in the goat.

Obesity alters gene expression for GH/IGF-I axis in mouse mammary fat pads: differential role of cortistatin and somatostatin.

Locally produced growth hormone (GH) and IGF-I are key factors in the regulation of mammary gland (MG) development and may be important in breast cancer development/progression. Somatostatin (SST) and cortistatin (CORT) regulate GH/IGF-I axis at various levels, but their role in regulating GH/IGF-I in MGs remains unknown. Since obesity alters the expression of these systems in different tissues and is associated to MG (patho) physiology, we sought to investigate the role of SST/CORT in regulating GH/IGF-I system in the MGs of lean and obese mice. Therefore, we analyzed GH/IGF-I as well as SST/CORT and ghrelin systems expression in the mammary fat pads (MFPs) of SST- or CORT-knockout (KO) mice and their respective littermate-controls fed a low-fat (LF) or a high-fat (HF) diet for 16wks. Our results demonstrate that the majority of the components of GH/IGF-I, SST/CORT and ghrelin systems are locally expressed in mouse MFP. Expression of elements of the GH/IGF-I axis was significantly increased in MFPs of HF-fed control mice while lack of endogenous SST partially suppressed, and lack of CORT completely blunted, the up-regulation observed in obese WT-controls. Since SST/CORT are known to exert an inhibitory role on the GH/IGFI axis, the increase in SST/CORT-receptor sst2 expression in MFPs of HF-fed CORT- and SST-KOs together with an elevation on circulating SST in CORT-KOs could explain the differences observed. These results offer new information on the factors (GH/IGF-I axis) involved in the endocrine/metabolic dysregulation of MFPs in obesity, and suggest that CORT is not a mere SST sibling in regulating MG physiology.

IGFBP-2 and -5: important regulators of normal and neoplastic mammary gland physiology.

The insulin-like growth factor (IGF) axis plays an important role in mammary gland physiology. In addition, dysregulation of this molecular axis may have a causal role in the aetiology and development of breast cancer (BC). This report discusses the IGF axis in normal and neoplastic mammary gland with special reference to IGF binding proteins (IGFBPs) -2 and -5. We describe how these high affinity binders of IGF-1 and IGF-2 may regulate local actions of growth factors in an autocrine and/or paracrine manner and how they also have IGF-independent effects in mammary gland. We discuss clinical studies which investigate both the prognostic value of IGFBP-2 and -5 expression in BC and possible involvement of these genes in the development of resistance to adjuvant endocrine therapies.

Androgen receptor inactivation resulted in acceleration in pubertal mammary gland growth, upregulation of ERα expression, and Wnt/β-catenin signaling in female mice.

The androgen receptor (AR) is widely expressed in mammary cells of female mammals including humans and mice, indicating a possible role for AR-mediated androgen actions in breast development, function, and pathology, although the specific mechanisms remain unclear. To elucidate the mechanisms of androgen action in mammary gland physiology and development, we used AR-knockout (AR(Δex3)KO) female mice with a universally expressed, transcriptionally inactive AR protein harboring an in-frame deletion of its second zinc finger. Although in sexually mature wild-type (WT) and AR(ex3Δ)KO females, the mammary epithelial growth was fully extended to the edge of the fat pad, during puberty, AR(ex3Δ)KO females exhibit significantly accelerated mammary ductal growth and an increased number of terminal end buds compared with WT females. Accelerated AR(ex3Δ)KO female mammary growth was associated with significantly increased mammary epithelial ERα expression and activated Wnt/β-catenin signaling as shown by increased Wnt4 expression and accumulation of nuclear β-catenin. These findings are consistent with increased mammary estrogen exposure although ovarian estradiol content was unchanged compared with WT females. Furthermore, treatment with the potent pure androgen DHT markedly reduced ductal extension and terminal end bud numbers in WT but not in AR(Δex3)KO females, further supporting the concept that AR-mediated, androgen-induced suppression of murine mammary growth is a physiological characteristic of puberty. In summary, our findings reveal an inhibitory role of AR-mediated androgen actions in pubertal mammary gland development by reducing epithelial cell proliferation and could be mediated by regulation of Wnt/β-catenin signaling.

The impact of vitamin D in breast cancer: genomics, pathways, metabolism

Nuclear receptors exert profound effects on mammary gland physiology and have complex roles in the etiology of breast cancer. In addition to receptors for classic steroid hormones such as estrogen and progesterone, the nuclear vitamin D receptor (VDR) interacts with its ligand 1α,25(OH)2D3 to modulate the normal mammary epithelial cell genome and subsequent phenotype. Observational studies suggest that vitamin D deficiency is common in breast cancer patients and that low vitamin D status enhances the risk for disease development or progression. Genomic profiling has characterized many 1α,25(OH)2D3 responsive targets in normal mammary cells and in breast cancers, providing insight into the molecular actions of 1α,25(OH)2D3 and the VDR in regulation of cell cycle, apoptosis, and differentiation. New areas of emphasis include regulation of tumor metabolism and innate immune responses. However, the role of VDR in individual cell types (i.e., epithelial, adipose, fibroblast, endothelial, immune) of normal and tumor tissues remains to be clarified. Furthermore, the mechanisms by which VDR integrates signaling between diverse cell types and controls soluble signals and paracrine pathways in the tissue/tumor microenvironment remain to be defined. Model systems of carcinogenesis have provided evidence that both VDR expression and 1α,25(OH)2D3 actions change with transformation but clinical data regarding vitamin D responsiveness of established tumors is limited and inconclusive. Because breast cancer is heterogeneous, analysis of VDR actions in specific molecular subtypes of the disease may help to clarify the conflicting data. The expanded use of genomic, proteomic and metabolomic approaches on a diverse array of in vitro and in vivo model systems is clearly warranted to comprehensively understand the network of vitamin D regulated pathways in the context of breast cancer.