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Heightened energetic and nutrient demand during lactogenic differentiation of the mammary gland elicits upregulation of various stress responses to support cellular homeostasis. Here, we identify the stimulator of interferon genes (STING) as an immune supporter of the functional development of mouse mammary epithelial cells (MECs). An in vitro model of MEC differentiation revealed that STING is activated in a cGAS-independent manner to produce both type I interferons and proinflammatory cytokines in response to the accumulation of mitochondrial reactive oxygen species. Induction of STING activity was found to be dependent on the breast tumor suppressor gene single-minded 2 (SIM2). Using mouse models of lactation, we discovered that loss of STING activity results in early involution of #3 mammary glands, severely impairing lactational performance. Our data suggest that STING is required for successful functional differentiation of the mammary gland and bestows a differential lactogenic phenotype between #3 mammary glands and the traditionally explored inguinal 4|9 pair. These findings affirm unique development of mammary gland pairs that is essential to consider in future investigations into normal development and breast cancer initiation. [ABSTRACT FROM AUTHOR]

The origin of breast cancer (BC) has traditionally been a focus of medical research. It is widely acknowledged that BC originates from immortal mammary stem cells and that these stem cells participate in two division modes: symmetric cell division (SCD) and asymmetrical cell division (ACD). Although both of these modes are key to the process of breast development and their imbalance is closely associated with the onset of BC, the molecular mechanisms underlying these phenomena deserve in-depth exploration. In this review, we first outline the molecular mechanisms governing ACD/SCD and analyze the role of ACD/SCD in various stages of breast development. We describe that the changes in telomerase activity, the role of polar proteins, and the stimulation of ovarian hormones subsequently lead to two distinct consequences: breast development or carcinogenesis. Finally, gene mutations, abnormalities in polar proteins, modulation of signal-transduction pathways, and alterations in the microenvironment disrupt the balance of BC stem cell division modes and cause BC. Important regulatory factors such as mammalian Inscuteable mInsc, Numb, Eya1, PKCα, PKCθ, p53, and IL-6 also play significant roles in regulating pathways of ACD/SCD and may constitute key targets for future research on stem cell division, breast development, and tumor therapy. [ABSTRACT FROM AUTHOR]

Lauric acid (LA) has the possibility to improve milk production in dairy cows by improving mammary gland development, however, the mechanism by which it might regulate mammary gland development is unclear. The influence of LA on milk production, nutrient digestibility and the expression of proteins related to mammary gland development in dairy cows were evaluated. Forty primiparous Holstein dairy cows were divided into 4 groups in a randomized block design. Four treatments included the control (0 g/d LA per cow), low-LA (100 g/d LA per cow), medium-LA (200 g/d LA per cow), and high-LA (300 g/d LA per cow). Yields of milk, fat-corrected milk, and energy-corrected milk quadratically increased (P

Abstract Background The cell cycle of mammary stem cells must be tightly regulated to ensure normal homeostasis of the mammary gland to prevent abnormal proliferation and susceptibility to tumorigenesis. The atypical cell cycle regulator, Spy1 can override cell cycle checkpoints, including those activated by the tumour suppressor p53 which mediates mammary stem cell homeostasis. Spy1 has also been shown to promote expansion of select stem cell populations in other developmental systems. Spy1 protein is elevated during proliferative stages of mammary gland development, is found at higher levels in human breast cancers, and promotes susceptibility to mammary tumourigenesis when combined with loss of p53. We hypothesized that Spy1 cooperates with loss of p53 to increase susceptibility to tumour initiation due to changes in susceptible mammary stem cell populations during development and drives the formation of more aggressive stem like tumours. Methods Using a transgenic mouse model driving expression of Spy1 within the mammary gland, mammary development and stemness were assessed. These mice were intercrossed with p53 null mice to study the tumourigenic properties of Spy1 driven p53 null tumours, as well as global changes in signaling via RNA sequencing analysis. Results We show that elevated levels of Spy1 leads to expansion of mammary stem cells, even in the presence of p53, and an increase in mammary tumour formation. Spy1-driven tumours have an increased cancer stem cell population, decreased checkpoint signaling, and demonstrate an increase in therapy resistance. Loss of Spy1 decreases tumor onset and reduces the cancer stem cell population. Conclusions This data demonstrates the potential of Spy1 to expand mammary stem cell populations and contribute to the initiation and progression of aggressive, breast cancers with increased cancer stem cell populations.

Postpartum breast cancer (PPBC) is a unique subset of breast cancer, accounting for nearly half of the women diagnosed during their postpartum years. Mammary gland involution is widely regarded as being a key orchestrator in the initiation and progression of PPBC due to its unique wound-healing inflammatory signature. Here, we provide dialogue suggestive that lactation may also facilitate neoplastic development as a result of sterile inflammation. Immune cells are involved in all stages of postnatal mammary development. It has been proposed that the functions of these immune cells are partially directed by mammary epithelial cells (MECs) and the cytokines they produce. This suggests that a more niche area of exploration aimed at assessing activation of innate immune pathways within MECs could provide insight into immune cell contributions to the developing mammary gland. Immune cell contribution to pubertal development and mammary gland involution has been extensively studied; however, investigations into pregnancy and lactation remain limited. During pregnancy, the mammary gland undergoes dramatic expansion to prepare for lactation. As a result, MECs are susceptible to replicative stress. During lactation, mitochondria are pushed to capacity to fulfill the high energetic demands of producing milk. This replicative and metabolic stress, if unresolved, can elicit activation of innate immune pathways within differentiating MECs. In this review, we broadly discuss postnatal mammary development and current knowledge of immune cell contribution to each developmental stage, while also emphasizing a more unique area of study that will be beneficial in the discovery of novel therapeutic biomarkers of PPBC. [ABSTRACT FROM AUTHOR]

Background: The cell cycle of mammary stem cells must be tightly regulated to ensure normal homeostasis of the mammary gland to prevent abnormal proliferation and susceptibility to tumorigenesis. The atypical cell cycle regulator, Spy1 can override cell cycle checkpoints, including those activated by the tumour suppressor p53 which mediates mammary stem cell homeostasis. Spy1 has also been shown to promote expansion of select stem cell populations in other developmental systems. Spy1 protein is elevated during proliferative stages of mammary gland development, is found at higher levels in human breast cancers, and promotes susceptibility to mammary tumourigenesis when combined with loss of p53. We hypothesized that Spy1 cooperates with loss of p53 to increase susceptibility to tumour initiation due to changes in susceptible mammary stem cell populations during development and drives the formation of more aggressive stem like tumours. Methods: Using a transgenic mouse model driving expression of Spy1 within the mammary gland, mammary development and stemness were assessed. These mice were intercrossed with p53 null mice to study the tumourigenic properties of Spy1 driven p53 null tumours, as well as global changes in signaling via RNA sequencing analysis. Results: We show that elevated levels of Spy1 leads to expansion of mammary stem cells, even in the presence of p53, and an increase in mammary tumour formation. Spy1-driven tumours have an increased cancer stem cell population, decreased checkpoint signaling, and demonstrate an increase in therapy resistance. Loss of Spy1 decreases tumor onset and reduces the cancer stem cell population. Conclusions: This data demonstrates the potential of Spy1 to expand mammary stem cell populations and contribute to the initiation and progression of aggressive, breast cancers with increased cancer stem cell populations. [ABSTRACT FROM AUTHOR]

Abstract Background 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. Results 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. Conclusions These findings revealed ABCD4 as a candidate gene pivotal for regulating mammary gland development and lactation during pregnancy by influencing PRLR expression.

Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or controlmice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare. [ABSTRACT FROM AUTHOR]

This experiment was to evaluate the influence of sodium butyrate (SB) addition on milk production, ruminal fermentation, nutrient digestion, and the development and metabolism regulation of the mammary gland in dairy cows. Forty Holstein dairy cows averaging 710 ± 18.5 kg body weight, 72.8 ± 3.66 d in milk (DIM), and 41.4 ± 1.42 kg/d milk production were divided into four treatments blocked by DIM and milk production. Treatments were control group, low SB, medium SB, and high SB with 0, 100, 200 and 300 g/d of SB addition per cow, respectively. The study lasted for 105 d. Production of milk, milk protein and lactose quadratically increased (P

Simple Summary: For many decades, the cellular and molecular mechanisms that orchestrate the differentiation of epithelial subtypes in the mammary gland have been a focus of intense investigations. Despite the orderly development of epithelial lineages with specific functions, individual cells or clusters of cells can switch identities in response to stress conditions and during the onset and progression of breast cancer. This review provides a comprehensive overview of factors that promote cellular plasticity. Changes in epithelial cell identity associated with pregnancy and lactation, inflammation, tissue repair, as well as the origin and progressive development of breast cancer subtypes discussed in this review demonstrate the broad impact of cellular plasticity on normal mammary gland development and the formation of malignant tumors. Cellular plasticity is a phenomenon where cells adopt different identities during development and tissue homeostasis as a response to physiological and pathological conditions. This review provides a general introduction to processes by which cells change their identity as well as the current definition of cellular plasticity in the field of mammary gland biology. Following a synopsis of the evolving model of the hierarchical development of mammary epithelial cell lineages, we discuss changes in cell identity during normal mammary gland development with particular emphasis on the effect of the gestation cycle on the emergence of new cellular states. Next, we summarize known mechanisms that promote the plasticity of epithelial lineages in the normal mammary gland and highlight the importance of the microenvironment and extracellular matrix. A discourse of cellular reprogramming during the early stages of mammary tumorigenesis that follows focuses on the origin of basal-like breast cancers from luminal progenitors and oncogenic signaling networks that orchestrate diverse developmental trajectories of transforming epithelial cells. In addition to the epithelial-to-mesenchymal transition, we highlight events of cellular reprogramming during breast cancer progression in the context of intrinsic molecular subtype switching and the genesis of the claudin-low breast cancer subtype, which represents the far end of the spectrum of epithelial cell plasticity. In the final section, we will discuss recent advances in the design of genetically engineered models to gain insight into the dynamic processes that promote cellular plasticity during mammary gland development and tumorigenesis in vivo. [ABSTRACT FROM AUTHOR]

Abstract TAR DNA-binding protein 43 (TDP-43) is a versatile RNA/DNA-binding protein with multifaceted processes. While TDP-43 has been extensively studied in the context of degenerative diseases, recent evidence has also highlighted its crucial involvement in diverse life processes beyond neurodegeneration. Here, we mainly reviewed the function of TDP-43 in non-neurodegenerative physiological and pathological processes, including spermatogenesis, embryonic development, mammary gland development, tumor formation, and viral infection, highlighting its importance as a key regulatory factor for the maintenance of normal functions throughout life. TDP-43 exhibits diverse and sometimes opposite functionality across different cell types through various mechanisms, and its roles can shift at distinct stages within the same biological system. Consequently, TDP-43 operates in both a context-dependent and a stage-specific manner in response to a variety of internal and external stimuli. Video Abstract

Abstract Background Advanced glycation end products (AGEs) are reactive metabolites intrinsically linked with modern dietary patterns. Processed foods, and those high in sugar, protein and fat, often contain high levels of AGEs. Increased AGE levels are associated with increased breast cancer risk, however their significance has been largely overlooked due to a lack of direct cause-and-effect relationship. Methods To address this knowledge gap, FVB/n mice were fed regular, low AGE, and high AGE diets from 3 weeks of age and mammary glands harvested during puberty (7 weeks) or adulthood (12 weeks and 7 months) to determine the effects upon mammary gland development. At endpoint mammary glands were harvested and assessed histologically (n ≥ 4). Immunohistochemistry and immunofluorescence were used to assess cellular proliferation and stromal fibroblast and macrophage recruitment. The Kruskal–Wallis test were used to compare continuous outcomes among groups. Mammary epithelial cell migration and invasion in response to AGE-mediated fibroblast activation was determined in two-compartment co-culture models. In vitro experiments were performed in triplicate. The nonparametric Wilcoxon rank sum test was used to compare differences between groups. Results Histological analysis revealed the high AGE diet delayed ductal elongation, increased primary branching, as well as increased terminal end bud number and size. The high AGE diet also led to increased recruitment and proliferation of stromal cells to abnormal structures that persisted into adulthood. Atypical hyperplasia was observed in the high AGE fed mice. Ex vivo fibroblasts from mice fed dietary-AGEs retain an activated phenotype and promoted epithelial migration and invasion of non-transformed immortalized and tumor-derived mammary epithelial cells. Mechanistically, we found that the receptor for AGE (RAGE) is required for AGE-mediated increases in epithelial cell migration and invasion. Conclusions We observed a disruption in mammary gland development when mice were fed a diet high in AGEs. Further, both epithelial and stromal cell populations were impacted by the high AGE diet in the mammary gland. Educational, interventional, and pharmacological strategies to reduce AGEs associated with diet may be viewed as novel disease preventive and/or therapeutic initiatives during puberty.

TAR DNA-binding protein 43 (TDP-43) is a versatile RNA/DNA-binding protein with multifaceted processes. While TDP-43 has been extensively studied in the context of degenerative diseases, recent evidence has also highlighted its crucial involvement in diverse life processes beyond neurodegeneration. Here, we mainly reviewed the function of TDP-43 in non-neurodegenerative physiological and pathological processes, including spermatogenesis, embryonic development, mammary gland development, tumor formation, and viral infection, highlighting its importance as a key regulatory factor for the maintenance of normal functions throughout life. TDP-43 exhibits diverse and sometimes opposite functionality across different cell types through various mechanisms, and its roles can shift at distinct stages within the same biological system. Consequently, TDP-43 operates in both a context-dependent and a stage-specific manner in response to a variety of internal and external stimuli. 5_7nHgoobCHQB-MCy8-gQo Video Abstract [ABSTRACT FROM AUTHOR]

Mammary fat plays a profound role in the postnatal development of mammary glands. However, the specific types (white, brown, or beige) of adipocytes in mammary fat and their potential regulatory effects on modulating mammary gland development remain poorly understood. This study aimed to investigate the role of the browning of mammary fat on pubertal mammary gland development and explore the underlying mechanisms. Thus, the mammary gland development and the serum lipid profile were evaluated in mice treated with CL316243, a β3-adrenoceptor agonist, to induce mammary fat browning. In addition, the proliferation of HC11 cells co-cultured with brown adipocytes or treated with the altered serum lipid metabolite was determined. Our results showed that the browning of mammary fat by injection of CL316243 suppressed the pubertal development of mice mammary glands, accompanied by the significant elevation of serum dioleoylphosphocholine (DOPC). In addition, the proliferation of HC11 was repressed when co-cultured with brown adipocytes or treated with DOPC. Furthermore, DOPC suppressed the activation of the PI3K/Akt pathway, while the DOPC-inhibited HC11 proliferation was reversed by SC79, an Akt activator, suggesting the involvement of the PI3K/Akt pathway in the DOPC-inhibited proliferation of HC11. Together, the browning of mammary fat suppressed the development of the pubertal mammary gland, which was associated with the elevated serum DOPC and the inhibition of the PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

Background: Advanced glycation end products (AGEs) are reactive metabolites intrinsically linked with modern dietary patterns. Processed foods, and those high in sugar, protein and fat, often contain high levels of AGEs. Increased AGE levels are associated with increased breast cancer risk, however their significance has been largely overlooked due to a lack of direct cause-and-effect relationship. Methods: To address this knowledge gap, FVB/n mice were fed regular, low AGE, and high AGE diets from 3 weeks of age and mammary glands harvested during puberty (7 weeks) or adulthood (12 weeks and 7 months) to determine the effects upon mammary gland development. At endpoint mammary glands were harvested and assessed histologically (n ≥ 4). Immunohistochemistry and immunofluorescence were used to assess cellular proliferation and stromal fibroblast and macrophage recruitment. The Kruskal–Wallis test were used to compare continuous outcomes among groups. Mammary epithelial cell migration and invasion in response to AGE-mediated fibroblast activation was determined in two-compartment co-culture models. In vitro experiments were performed in triplicate. The nonparametric Wilcoxon rank sum test was used to compare differences between groups. Results: Histological analysis revealed the high AGE diet delayed ductal elongation, increased primary branching, as well as increased terminal end bud number and size. The high AGE diet also led to increased recruitment and proliferation of stromal cells to abnormal structures that persisted into adulthood. Atypical hyperplasia was observed in the high AGE fed mice. Ex vivo fibroblasts from mice fed dietary-AGEs retain an activated phenotype and promoted epithelial migration and invasion of non-transformed immortalized and tumor-derived mammary epithelial cells. Mechanistically, we found that the receptor for AGE (RAGE) is required for AGE-mediated increases in epithelial cell migration and invasion. Conclusions: We observed a disruption in mammary gland development when mice were fed a diet high in AGEs. Further, both epithelial and stromal cell populations were impacted by the high AGE diet in the mammary gland. Educational, interventional, and pharmacological strategies to reduce AGEs associated with diet may be viewed as novel disease preventive and/or therapeutic initiatives during puberty. [ABSTRACT FROM AUTHOR]

Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis. [Display omitted] • MTHFD2 is expressed in both luminal and basal cell layers within the mammary glands. • Mice with a specific knockout of Mthfd2 in mammary epithelial cells are generated. • The mutant mice display a delay in pregnancy-induced mammary gland development. [ABSTRACT FROM AUTHOR]

The mammary gland is composed of epithelial tissue forming ducts and lobules, and the stroma, composed of adipocytes, connective tissue, and other cell types. The stromal microenvironment regulates mammary gland development by paracrine and cell–cell interactions. In the present study, primary cultures of bovine mammary epithelial cells (bMEC) and bovine adipose-derived stem cells (bASC) subjected to adipogenic differentiation were used to investigate the influence of paracrine factors secreted by preadipocytes and adipocytes on bMEC development. Four types of conditioned media (CM) were collected from undifferentiated preadipocytes (preA) and adipocytes on days: 8, 12, 14 of differentiation. Next, bMEC were cultured for 24 h in CM and cell viability, apoptosis, migratory activity, ability to form spheroids on Matrigel, and secretory activity (alpha S1-casein concentration) were evaluated. CM derived from fully differentiated adipocytes (12 d and 14 d) significantly decreased the number of apoptotic cells in bMEC population and increased the size of spheroids formed by bMEC on Matrigel. CM collected from preadipocytes significantly enhanced bMEC's migration, and stimulated bMEC to produce alpha S1-casein, but only in the presence of prolactin. These results confirm that preadipocytes and adipocytes are important components of the stroma, providing paracrine factors that actively regulate the development of bovine mammary epithelium. [ABSTRACT FROM AUTHOR]

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.

S100 proteins have emerged as key regulators in the mammary gland and have been implicated in breast cancer development and metastasis. This review provides a comprehensive overview of the roles of S100 proteins in mammary gland regulation and their impact on breast cancer progression. The mammary gland, a complex organ involved in lactation and tissue homeostasis, undergoes dynamic changes during different physiological stages. S100 proteins play crucial roles in mammary gland development, differentiation, and function, participating in cellular processes such as proliferation, migration, and apoptosis. However, dysregulation of S100 proteins can contribute to breast cancer initiation and metastasis. These proteins are involved in angiogenesis, invasion, migration, and epithelial-mesenchymal transition, promoting aggressive behavior in breast cancer cells. Understanding the intricate mechanisms by which S100 proteins exert their effects in the mammary gland and breast cancer is crucial for the development of targeted therapies and identification of diagnostic and prognostic biomarkers. Further research in this field will provide valuable insights and potential advancements in breast cancer management. This review highlights the significance of unraveling the role of S100 proteins in mammary gland regulation and their impact on breast cancer metastasis.

Exosomes are key mediators of intercellular communication. They are secreted by most cells and contain a cargo of protein-coding genes, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), which modulate recipient cell behavior. Herein, we collected blood samples from Holstein cows at days 30 (mid-lactation) and 250 (dry period) of pregnancy. Prolactin, follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone levels showed an obvious increase during D250. We then extracted exosomes from bovine blood samples and found that their sizes generally ranged from 100 to 200 nm. Further, Western blotting validated that they contained CD9, CD63, and TSG101, but not calnexin. Blood-derived exosomes significantly promoted the proliferation of mammary epithelial cells, particularly from D250. This change was accompanied by increased expression levels of proliferation marker proteins PCNA, cyclin D, and cyclin E, as detected by EdU assay, cell counting kit-8 assay, and flow cytometric cell cycle analysis. Moreover, we treated mammary epithelial cells with blood-derived exosomes that were isolated from the D30 and D250 periods. And RNA-seq of two groups of cells led to the identification of 839 differentially expressed genes that were significantly enriched in KEGG signaling pathways associated with apoptosis, cell cycle and proliferation. In bovine blood-derived exosomes, we found 12,747 protein-coding genes, 31,181 lncRNAs, 9374 transcripts of uncertain coding potential (TUCP) candidates, and 460 circRNAs, and 32 protein-coding genes, 806 lncRNAs, 515 TUCP candidates, and 45 circRNAs that were differentially expressed between the D30 and D250 groups. We selected six highly expressed and four differentially expressed circRNAs to verify their head-to-tail splicing using PCR and Sanger sequencing. To summarize, our findings improve our understanding of the key roles of blood-derived exosomes and the characterization of exosomal circRNAs in mammary gland development. [ABSTRACT FROM AUTHOR]

The mammary gland is a highly dynamic tissue that undergoes repeated cycles of growth and involution during pregnancy and menstruation. It is also the site from which breast cancers emerge. Organoids provide an in vitro model that preserves several of the cellular, structural, and microenvironmental features that dictate mammary gland function in vivo and have greatly advanced our understanding of glandular biology. Their tractability for genetic manipulation, live imaging, and high throughput screening have facilitated investigation into the mechanisms of glandular morphogenesis, structural maintenance, tumor progression, and invasion. Opportunities remain to enhance cellular and structural complexity of mammary organoid models, including incorporating additional cell types and hormone signaling.

The molecular events underlying the lactation curve of dairy animals have long been a biological conundrum for mammary biologists, and a cause of a considerable loss of income for dairy farmers. To better understand the regulatory mechanisms and developmental processes of the mammary gland during the lactation cycle, whole-genome transcriptome profiles of bovine mammary tissues at five different lactation stages were investigated using the RNA-Seq technique. The differentially expressed genes were identified by comparing different lactation stage combinations. Gene expression changes were validated using quantitative reverse-transcription polymerase chain reaction. Some differentially expressed genes were found to locate in the known quantitative trait loci regions that are associated with multiple milk production traits. Proteins of potentially important genes were confirmed to be expressed in the mammary epithelium of dairy cows. An integrated analysis of differential gene expression indicated that internal development regularities underlying the shape of the milk production curve. First, the mammary gland is enriched in similar activities during lactation onset and involution, but in opposite regulatory directions. Second, the lactation stages before and after the milk peak shared similar biological processes that were mainly involved in extracellular matrix remodelling. The discoveries of the study provide information necessary to improve milk production.

Objective Water buffalo, an important domestic animal in tropical and subtropical regions, play an important role in agricultural economy. It is an important source for milk, meat, horns, skin, and draft power, especially its rich milk that is the great source of cream, butter, yogurt, and many cheeses. In recent years, long noncoding RNAs (lncRNAs) have been reported to play pivotal roles in many biological processes. Previous studies for the mammary gland development of water buffalo mainly focus on protein coding genes. However, lncRNAs of water buffalo remain poorly understood, and the regulation relationship between mammary gland development/milk production traits and lncRNA expression is also unclear. Methods Here, we sequenced 22 samples of the milk somatic cells from three lactation stages and integrated the current annotation and identified 7,962 lncRNA genes. Results By comparing the lncRNA genes of the water buffalo in the early, peak, and late different lactation stages, we found that lncRNA gene lnc-bbug14207 displayed significantly different expression between early and late lactation stages. And lnc-bbug14207 may regulate neighboring milk fat globule-EGF factor 8 (MFG-E8) and hyaluronan and proteoglycan link protein 3 (HAPLN3) protein coding genes, which are vital for mammary gland development. Conclusion This study provides the first genome-wide identification of water buffalo lncRNAs and unveils the potential lncRNAs that impact 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. [ABSTRACT FROM AUTHOR]

Background: Alcohol exposure increases the risk of breast cancer. Alcohol consumption by adolescents is a serious social and public health issue. This study investigated the impact of adolescent alcohol consumption on mammary tumorigenesis and progression and compared it to that of adult alcohol exposure in animal models. Methods: Female adolescent (5 weeks) and adult (8 weeks) MMTV‐Wnt1 mice were exposed to alcohol either chronically or acutely. For chronic alcohol exposure, animals were fed a liquid diet containing 6.7% ethanol for 23 weeks. For acute exposure, animals were treated with ethanol (2.5 g/kg, 25% w/v) via intraperitoneal (IP) injection for 15 days. Results: In control animals, the tumor latency was 18.5 to 22 weeks. Both chronic and acute alcohol exposure in adolescent mice significantly shortened the tumor latency to 9.5 and 8.4 weeks, respectively. However, adult‐initiated alcohol exposure had little effect on the tumor latency. Both adolescent‐ and adult‐initiated alcohol exposure significantly increased lung metastasis. Adolescent‐initiated alcohol exposure but not adult‐initiated alcohol exposure increased the breast cancer stem cell population. Adolescent‐initiated alcohol exposure significantly altered the proliferation of mammary epithelial cells, ductal growth, and the formation of terminal end buds in the mammary glands. Adolescent‐initiated alcohol exposure but not adult‐initiated alcohol exposure increased estradiol levels in the blood. Acute adolescent alcohol exposure also significantly increased blood progesterone levels. Furthermore, adolescent‐initiated alcohol exposure activated PAK1 and p38γ MAPK, critical regulators of mammary tumorigenesis and aggressiveness, respectively, while adult‐initiated alcohol exposure activated only p38γ MAPK. In addition, both adolescent and adult alcohol exposure significantly decreased the levels of a prognostic marker miR200b. Conclusions: Adolescent‐initiated alcohol exposure enhanced both tumorigenesis and aggressiveness of mammary tumors, while adult‐initiated alcohol exposure mainly promoted tumor metastasis. Thus, adolescent mice were more sensitive than adult mice in response to alcohol‐induced tumor promotion. [ABSTRACT FROM AUTHOR]

As endocrine hormones, glucocorticoids (GCs) play a pivotal role in numerous physiological processes, including mammary growth and lactation, circulatory metabolism, and responses to external stimuli. In the dairy industry, milk production from cows or goats is important for newborns and economic benefits. However, the milk yields from ruminant animals are always affected by the extent of mammary development, mammary disease, stress, or changes in metabolism. Thus, it is necessary to clarify how GCs changes in ruminants affect ruminant mammary gland function and mammary disease. This review summarizes the findings identifying that GCs modulate mammary gland development before lactation, but the stress-induced excessive release of GCs leads to milk production loss. In addition, the manner of GCs release may change under different concentrations of metabolites or during mastitis or inflammatory challenge. Nevertheless, exogenous GCs administration to animals may alleviate the clinical symptoms of mastitis. This review demonstrates that GCs offer a fascinating contribution to both physiologic and pathogenic conditions of the mammary gland in ruminant animals. Characterizing and understanding these changes or functions of endogenous and exogenous GCs in animals will be crucial for developing more endocrine regulators and therapies for improving milk production in ruminants. [ABSTRACT FROM AUTHOR]

Genome-wide association studies of breast cancer susceptibility have revealed risk-associated genetic variants and nominated candidate genes; however, the identification of causal variants and genes is often undetermined by genome-wide association studies. Comparative genomics, utilizing Rattus norvegicus strains differing in susceptibility to mammary tumor development, is a complimentary approach to identify breast cancer susceptibility genes. Mammary carcinoma susceptibility 3 (Mcs3) is a Copenhagen (COP/NHsd) allele that confers resistance to mammary carcinomas when introgressed into a mammary carcinoma susceptible Wistar Furth (WF/NHsd) genome. Here, Mcs3 was positionally mapped to a 7.2-Mb region of RNO1 spanning rs8149408 to rs107402736 (chr1:143700228-150929594, build 6.0/rn6) using WF.COP congenic strains and 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis. Male and female WF.COP-Mcs3 rats had significantly lower body mass compared to the Wistar Furth strain. The effect on female body mass was observed only when females were raised in the absence of males indicating a socioenvironmental interaction. Furthermore, female WF.COP-Mcs3 rats, raised in the absence of males, did not develop enhanced lobuloalveolar morphologies compared to those observed in the Wistar Furth strain. Human 15q25.1-25.2 was determined to be orthologous to rat Mcs3 (chr15:80005820-82285404 and chr15:83134545-84130720, build GRCh38/hg38). A public database search of 15q25.1-25.2 revealed genome-wide significant and nominally significant associations for body mass traits and breast cancer risk. These results support the existence of a breast cancer risk-associated allele at human 15q25.1-25.2 and warrant ultrafine mapping of rat Mcs3 and human 15q25.1-25.2 to discover novel causal genes and variants. [ABSTRACT FROM AUTHOR]

The molecular events underlying the lactation curve of dairy animals have long been a biological conundrum for mammary biologists, and a cause of a considerable loss of income for dairy farmers. To better understand the regulatory mechanisms and developmental processes of the mammary gland during the lactation cycle, whole-genome transcriptome profiles of bovine mammary tissues at five different lactation stages were investigated using the RNA-Seq technique. The differentially expressed genes were identified by comparing different lactation stage combinations. Gene expression changes were validated using quantitative reverse-transcription polymerase chain reaction. Some differentially expressed genes were found to locate in the known quantitative trait loci regions that are associated with multiple milk production traits. Proteins of potentially important genes were confirmed to be expressed in the mammary epithelium of dairy cows. An integrated analysis of differential gene expression indicated that internal development regularities underlying the shape of the milk production curve. First, the mammary gland is enriched in similar activities during lactation onset and involution, but in opposite regulatory directions. Second, the lactation stages before and after the milk peak shared similar biological processes that were mainly involved in extracellular matrix remodelling. The discoveries of the study provide information necessary to improve milk production. [ABSTRACT FROM AUTHOR]

Abstract Breast Cancer (BrC) is a common malignancy with genetically diverse subtypes. There is evidence that specific BrC subtypes originate from particular normal mammary cell populations. However, the cell populations that give rise to most BrC subtypes are unidentified. Several human breast scRNAseq datasets are available. In this research, we utilized a robust human scRNAseq dataset to identify population-specific marker genes and then identified the expression of these marker genes in specific BrC subtypes. In humans, several BrC subtypes, HER2-enriched, basal-like, and triple-negative (TN), are more common in women who have had children. This observation suggests that cell populations that originate during pregnancy give rise to these BrCs. The current human datasets have few normal parous samples, so we supplemented this research with mouse datasets, which contain mammary cells from various developmental stages. This research identified two novel normal breast cell populations that may be the origin of the basal-like and HER2-overexpressing subtypes, respectively. A stem cell-like population, SC, that expresses gestation-specific genes has similar gene expression patterns to basal-like BrCs. A novel luminal progenitor cell population and HER2-overexpressing BrCs are marked by S100A7, S100A8, and S100A9 expression. We bolstered our findings by examining SC gene expression in TN BrC scRNAseq datasets and S100A7-A9 gene expression in BrC cell lines. We discovered that several potential cancer stem cell populations highly express most of the SC genes in TN BrCs and confirmed S100A8 and A9 overexpression in a HER2-overexpressing BrC cell line. In summary, normal SC and the novel luminal progenitor cell population likely give rise to basal-like and HER2-overexpressing BrCs, respectively. Characterizing these normal cell populations may facilitate a better understanding of specific BrCs subtypes.

Adult mammary stem cells (aMaSCs) are vital to tissue expansion and remodeling during the process of postnatal mammary development. The protein C receptor (Procr) is one of the well-identified surface markers of multipotent aMaSCs. However, an understanding of the regulatory mechanisms governing Procr's protein stability remains incomplete. In this study, we identified Glycoprotein m6a (Gpm6a) as a critical protein for aMaSC activity modulation by using the Gpm6a knockout mouse model. Interestingly, we determined that Gpm6a depletion results in a reduction of Procr protein stability. Mechanistically, Gpm6a regulates Procr protein stability by mediating the formation of lipid rafts, a process requiring Zdhhc1 and Zdhhc2 to palmitate Gpm6a at Cys17,18 and Cys246 sites. Our findings highlight an important mechanism involving Zdhhc1- and Zdhhc2-mediated Gpm6a palmitoylation for the regulation of Procr stability, aMaSC activity, and postnatal mammary development. [Display omitted] • Loss of Gpm6a hinders mammary gland development and reduces the activity of aMaSCs • Gpm6a deficiency disrupts Procr protein stability by inhibiting the formation of lipid rafts • Palmitoylation of Gpm6a, mediated by Zdhhc1 and Zdhhc2, is crucial for the aMaSC activity Chen et al. demonstrate that Gpm6a deficiency reduces the activity of adult MaSCs. Gpm6a regulates the stability of the Procr protein by mediating the formation of lipid rafts. The palmitoylation of Gpm6a, facilitated by Zdhhc1 and Zdhhc2, is essential for Procr protein stability and adult MaSC activity. [ABSTRACT FROM AUTHOR]

Tributyltin (TBT) is an endocrine-disrupting chemical (EDC) related to reproductive dysfunctions. However, few studies have investigated the effects of TBT exposure on mammary gland development. Thus, we assessed whether subacute TBT exposure causes irregularities in mammary gland development. We administered TBT (100 and 1,000 ng/kg/day for 30 days) to female rats from postnatal day (PND) 25 to PND 55, and mammary gland development, morphology, inflammation, collagen deposition, and protein expression were evaluated. Abnormal mammary gland development was observed in both TBT groups. Specifically, TBT exposure reduced the number of terminal end buds (TEBs), type 1 (AB1) alveolar buds, and type 2 (AB2) alveolar buds. An increase in the lobule and differentiation (DF) 2 score was found in the mammary glands of TBT rats. TBT exposure increased mammary gland blood vessels, mast cell numbers, and collagen deposition. Additionally, both TBT rats exhibited intraductal hyperplasia and TEB-like structures. An increase in estrogen receptor alpha (ERα), progesterone receptor (PR), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) - positive cells was observed in the mammary glands of TBT rats. A strong negative correlation was observed between CYP19A1- positive cells and TEB number. In addition, CYP19A1 - positive cells were positively correlated with mammary gland TEB-like structure, ductal hyperplasia, inflammation, and collagen deposition. Thus, these data suggest that TBT exposure impairs mammary gland development through the modulation of CYP19A1 signaling pathways in female rats. [Display omitted] • Tributyltin (TBT) impairs mammary gland development. • TBT leads to intraductal hyperplasia and TEB-like structures. • TBT leads to mammary gland ductal and stromal abnormalities. • TBT changes ductal and stromal ERα and PR expression. • TBT changes mammary gland development via mainly CYP19A1 modulation. [ABSTRACT FROM AUTHOR]

Mammary fat plays a profound role in the postnatal development of mammary glands. However, the specific types (white, brown, or beige) of adipocytes in mammary fat and their potential regulatory effects on modulating mammary gland development remain poorly understood. This study aimed to investigate the role of the browning of mammary fat on pubertal mammary gland development and explore the underlying mechanisms. Thus, the mammary gland development and the serum lipid profile were evaluated in mice treated with CL316243, a β3-adrenoceptor agonist, to induce mammary fat browning. In addition, the proliferation of HC11 cells co-cultured with brown adipocytes or treated with the altered serum lipid metabolite was determined. Our results showed that the browning of mammary fat by injection of CL316243 suppressed the pubertal development of mice mammary glands, accompanied by the significant elevation of serum dioleoylphosphocholine (DOPC). In addition, the proliferation of HC11 was repressed when co-cultured with brown adipocytes or treated with DOPC. Furthermore, DOPC suppressed the activation of the PI3K/Akt pathway, while the DOPC-inhibited HC11 proliferation was reversed by SC79, an Akt activator, suggesting the involvement of the PI3K/Akt pathway in the DOPC-inhibited proliferation of HC11. Together, the browning of mammary fat suppressed the development of the pubertal mammary gland, which was associated with the elevated serum DOPC and the inhibition of the PI3K/Akt pathway.

As endocrine hormones, glucocorticoids (GCs) play a pivotal role in numerous physiological processes, including mammary growth and lactation, circulatory metabolism, and responses to external stimuli. In the dairy industry, milk production from cows or goats is important for newborns and economic benefits. However, the milk yields from ruminant animals are always affected by the extent of mammary development, mammary disease, stress, or changes in metabolism. Thus, it is necessary to clarify how GCs changes in ruminants affect ruminant mammary gland function and mammary disease. This review summarizes the findings identifying that GCs modulate mammary gland development before lactation, but the stress-induced excessive release of GCs leads to milk production loss. In addition, the manner of GCs release may change under different concentrations of metabolites or during mastitis or inflammatory challenge. Nevertheless, exogenous GCs administration to animals may alleviate the clinical symptoms of mastitis. This review demonstrates that GCs offer a fascinating contribution to both physiologic and pathogenic conditions of the mammary gland in ruminant animals. Characterizing and understanding these changes or functions of endogenous and exogenous GCs in animals will be crucial for developing more endocrine regulators and therapies for improving milk production in ruminants.

Lysine is one of the essential amino acids. The effect of lysine on milk protein and milk fat anabolism has been reported, but the effect on mammary glands development has not been studied in detail. The normal development of the mammary glands at puberty is crucial to lactation of mammals. In this study, to explore the effect of lysine on mammary glands development, we fed different concentrations of lysine (0.025%, 0.05%, 0.1%) to pubertal mice and found that the addition of 0.1% lysine to drinking water significantly promoted mammary glands development. Furthermore, we treated mMECs (mouse mammary epithelial cells) with different concentrations of lysine (0, 0.2, 0.4, 0.6, 0.8 and 1 mM) to explore the underlying mechanism, and found that lysine promoted the proliferation of mMECs and development of mammary glands through PI3K/AKT/mTOR signalling pathway in pubertal mice. Overall, the results of this study revealed that lysine activated the PI3K/AKT/mTOR signal axis, elevated protein concentrations of cell proliferation markers, such as PCNA, Cyclin D1 and D3, and enhanced the proliferation of mMECs, finally promoted the murine mammary glands development at puberty. [ABSTRACT FROM AUTHOR]

Amino acids have been shown to affect the development of mammary gland (MG). However, it is unclear whether L‐arginine promotes the development of pubertal MG. Therefore, our study aims to explore the effect of L‐arginine on the development of MG in pubertal mice. To investigate its internal mechanism of action, we will use mouse mammary epithelial cells (mMECs) line. Whole‐mount staining showed that L‐arginine can promote the extension of MG duct. In vitro, 0.4 mM L‐arginine could activate the G protein‐coupled receptor family C, group 6, subtype A (GPRC6A)/phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway and increase the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4EBP1) to promote the synthesis of cell cycle regulatory protein D1 (Cyclin D1), leading to the dissociation of the retinoblastoma tumour suppressor protein (Rb)‐E2F1 transcription factor (E2F1) complex in mMECs and releasing E2F1 to promote cell proliferation. Furthermore, GPRC6A was knocked down or inhibition of the PI3K/AKT/mTOR signalling pathway with corresponding inhibitors completely abolished the arginine‐induced promotion of mMECs proliferation. In vivo, it was further confirmed that 0.1% L‐arginine can activate the PI3K/AKT/mTOR signalling pathway in the MG of pubertal mice. These results were able to indicate that L‐arginine stimulates the development of MG in pubertal mice through the GPRC6A/PI3K/AKT/mTOR signalling pathway. [ABSTRACT FROM AUTHOR]

The mammary gland is a unique organ with the ability to undergo repeated cyclic changes throughout the life of mammals. Among domesticated livestock species, ruminants (cattle and buffalo) constitute a distinct class of livestock species that are known milk producers. Cattle and buffalo contribute to 51 and 13% of the total milk supply in the world, respectively. They also play an essential role in the development of the economy for farming communities by providing milk, meat, and draft power. The development of the ruminant mammary gland is highly dynamic and multiphase in nature. There are six developmental stages: embryonic, prepubertal, pubertal, pregnancy, lactation, and involution. There has been substantial advancement in our understanding of the development of the mammary gland in both mouse and human models. Until now, there has not been a thorough investigation into the molecular processes that underlie the various stages of cow udder development. The current review sheds light on the morphological and molecular changes that occur during various developmental phases in diverse species, with a particular focus on the cow udder. It aims to explain the physiological differences between cattle and non-ruminant mammalian species such as humans, mice, and monkeys. Understanding the developmental biology of the mammary gland in molecular detail, as well as species-specific variations, will facilitate the researchers working in this area in further studies on cellular proliferation, differentiation, apoptosis, organogenesis, and carcinogenesis. Additionally, in-depth knowledge of the mammary gland will promote its use as a model organ for research work and promote enhanced milk yield in livestock animals without affecting their health and welfare. [ABSTRACT FROM AUTHOR]

Our hypothesis was that maternal nutrient restriction would negatively impact the endocrine and metabolic status of the pregnant cow, therefore influencing the mammary gland in preparation for lactation. We further hypothesized that earlier timing of realimentation could prevent negative impacts of nutrient restriction. The objectives were to investigate the influence of nutrient restriction and realimentation during early to late gestation on endocrine profile, umbilical hemodynamics, and mammary gland development and hemodynamics in pregnant beef cows. In Experiment 1, on d 30 of pregnancy cows (initial BW = 667.5 ± 13.4 kg, BCS = 6.2 ± 0.1) were randomly assigned to one of 3 treatments: 1) 100% NRC requirements from d 30 to 254 of gestation (CCC; n = 6); 2) 60% NRC from d 30 to 85, thereafter being re-alimented to 100% NRC to d 254 (RCC; n = 5); 3) or receive 60% NRC from d 30 to 140, thereafter being re-alimented to 100% NRC to d 254 (RRC; n = 6). Cows were returned to a common outdoor facility for calving thereafter and were fed ad libitum. In Experiment 2, on d 30 of pregnancy, cows (initial BW = 620.5 ± 11.3 kg, BCS = 5.1 ± 0.1) were randomly assigned to dietary treatments including: control (CON; 100% NRC; n = 18) and nutrient restriction (RES; 60% NRC; n = 30). On d 85 of pregnancy, cows were either slaughtered (CON, n = 6 and RES, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12) treatments, or were realimented to control (RC; n = 11). On d 140 of pregnancy, cows were either slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5), or were realimented to control (RRC, n = 6). On d 254 of pregnancy, all remaining cows were slaughtered (CCC, n = 6; RCC, n = 5; RRC, n = 6). Mammary hemodynamics and endocrine profile were measured. Serum urea nitrogen, NEFA, as well as fetal parameters were measured in Experiment 1; whereas in Experiment 2, mammary gland development was recorded. In Experiment 1, RRC cows had lower dry matter intake (P = 0.001) and consequently lower BW change (P = 0.06). However, maternal nutrition did not alter mammary hemodynamics, hormonal patterns, and fetal characteristics (P > 0.11). In Experiment 2, CCC cows had increased (P = 0.02) mammary gland blood flow ipsilateral to the gravid horn as well as greater (P = 0.02) mammary gland fat on d 254. Nevertheless, plane of nutrition did not alter hormonal concentrations nor mammary gland characteristics (P > 0.15). These data indicate that nutrient restriction did not alter mammary hemodynamics nor endocrine profile throughout gestation. • Nutritional plane in pregnancy beef cows does not alter maternal hormonal patterns or mammary gland vascularity. • Nutrient restriction during early to mid-pregnancy did not alter mammary hemodynamics. • Mammary gland fat near parturition is decreased when cows experience a restriction during early or early to mid-gestation. [ABSTRACT FROM AUTHOR]

Super-enhancers are large clusters of enhancers critical for cell-type-specific development. In a previous study, 440 mammary-specific super-enhancers, highly enriched for an active enhancer mark H3K27ac; a mediator MED1; and the mammary-enriched transcription factors ELF5, NFIB, STAT5A, and GR, were identified in the genome of the mammary epithelium of lactating mice. However, the triggering mechanism for mammary-specific super-enhancers and the molecular interactions between key transcription factors have not been clearly elucidated. In this study, we investigated in vivo protein–protein interactions between major transcription factors that activate mammary-specific super-enhancers. In mammary epithelial cells, ELF5 strongly interacted with NFIB while weakly interacting with STAT5A, and it showed modest interactions with MED1 and GR, a pattern unlike that in non-mammary cells. We further investigated the role of key transcription factors in the initial activation of the mammary-specific Wap super-enhancer, using CRISPR-Cas9 genome editing to introduce single or combined mutations at transcription factor binding sites in the pioneer enhancer of the Wap super-enhancer in mice. ELF5 and STAT5A played key roles in igniting Wap super-enhancer activity, but an intact transcription factor complex was required for the full function of the super-enhancer. Our study demonstrates that mammary-enriched transcription factors within a protein complex interact with different intensities and synergize to activate the Wap super-enhancer. These findings provide an important framework for understanding the regulation of cell-type-specific development. [ABSTRACT FROM AUTHOR]

The objective of this experiment was to evaluate the influence of nanoselenium (NANO-Se) addition on milk production, milk fatty acid synthesis, the development and metabolism regulation of mammary gland in dairy cows. Forty-eight Holstein dairy cows averaging 720 ± 16.8 kg of body weight, 66.9 ± 3.84 d in milk (dry matter intake [DIM]) and 35.2 ± 1.66 kg/d of milk production were divided into four treatments blocked by DIM and milk yields. Treatments were control group, low-Se (LSe), medium-Se (MSe) and high-Se (HSe) with 0, 0.1, 0.2 and 0.3 mg Se, respectively, from NANO-Se per kg dietary dry matter (DM). Production of energy- and fat-corrected milk (FCM) and milk fat quadratically increased ( p  < 0.05), while milk lactose yields linearly increased ( p  < 0.05) with increasing NANO-Se addition. The proportion of saturated fatty acids (SFAs) linearly decreased ( p  < 0.05), while proportions of monounsaturated fatty acids (MUFAs) linearly increased and polyunsaturated fatty acids (PUFAs) quadratically increased. The digestibility of dietary DM, organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) quadratically increased ( p  < 0.05). Ruminal pH quadratically decreased ( p  < 0.01), while total VFA linearly increased ( p  < 0.05) with increasing NANO-Se addition. The acetic to propionic ratio decreased ( p  < 0.05) linearly due to the unaltered acetic molar percentage and a quadratical increase in propionic molar percentage. The activity of CMCase, xylanase, cellobiase and pectinase increased linearly ( p  < 0.05) following NANO-Se addition. The activity of α-amylase increased linearly ( p  < 0.01) with an increase in NANO-Se dosage. Blood glucose, total protein, estradiol, prolactin, IGF-1 and Se linearly increased ( p  < 0.05), while urea nitrogen concentration quadratically decreased ( p  = 0.04). Moreover, the addition of Se at 0.3 mg/kg from NANO-Se promoted ( p  < 0.05) mRNA and protein expression of PPARγ, SREBP1, ACACA, FASN, SCD, CCNA2, CCND1, PCNA, Bcl-2 and the ratios of p-ACACA/ACACA and BCL2/BAX4 , but decreased ( p  < 0.05) mRNA and protein expressions of Bax, Caspase-3 and Caspase-9. The results suggest that milk production and milk fat synthesis increased by NANO-Se addition by stimulating rumen fermentation, nutrients digestion, gene and protein expressions concerned with milk fat synthesis and mammary gland development.

The mammary gland is composed of epithelial tissue forming ducts and lobules, and the stroma, composed of adipocytes, connective tissue, and other cell types. The stromal microenvironment regulates mammary gland development by paracrine and cell–cell interactions. In the present study, primary cultures of bovine mammary epithelial cells (bMEC) and bovine adipose-derived stem cells (bASC) subjected to adipogenic differentiation were used to investigate the influence of paracrine factors secreted by preadipocytes and adipocytes on bMEC development. Four types of conditioned media (CM) were collected from undifferentiated preadipocytes (preA) and adipocytes on days: 8, 12, 14 of differentiation. Next, bMEC were cultured for 24 h in CM and cell viability, apoptosis, migratory activity, ability to form spheroids on Matrigel, and secretory activity (alpha S1-casein concentration) were evaluated. CM derived from fully differentiated adipocytes (12 d and 14 d) significantly decreased the number of apoptotic cells in bMEC population and increased the size of spheroids formed by bMEC on Matrigel. CM collected from preadipocytes significantly enhanced bMEC’s migration, and stimulated bMEC to produce alpha S1-casein, but only in the presence of prolactin. These results confirm that preadipocytes and adipocytes are important components of the stroma, providing paracrine factors that actively regulate the development of bovine mammary epithelium.

Exosomes are key mediators of intercellular communication. They are secreted by most cells and contain a cargo of protein-coding genes, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), which modulate recipient cell behavior. Herein, we collected blood samples from Holstein cows at days 30 (mid-lactation) and 250 (dry period) of pregnancy. Prolactin, follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone levels showed an obvious increase during D250. We then extracted exosomes from bovine blood samples and found that their sizes generally ranged from 100 to 200 nm. Further, Western blotting validated that they contained CD9, CD63, and TSG101, but not calnexin. Blood-derived exosomes significantly promoted the proliferation of mammary epithelial cells, particularly from D250. This change was accompanied by increased expression levels of proliferation marker proteins PCNA, cyclin D, and cyclin E, as detected by EdU assay, cell counting kit-8 assay, and flow cytometric cell cycle analysis. Moreover, we treated mammary epithelial cells with blood-derived exosomes that were isolated from the D30 and D250 periods. And RNA-seq of two groups of cells led to the identification of 839 differentially expressed genes that were significantly enriched in KEGG signaling pathways associated with apoptosis, cell cycle and proliferation. In bovine blood-derived exosomes, we found 12,747 protein-coding genes, 31,181 lncRNAs, 9374 transcripts of uncertain coding potential (TUCP) candidates, and 460 circRNAs, and 32 protein-coding genes, 806 lncRNAs, 515 TUCP candidates, and 45 circRNAs that were differentially expressed between the D30 and D250 groups. We selected six highly expressed and four differentially expressed circRNAs to verify their head-to-tail splicing using PCR and Sanger sequencing. To summarize, our findings improve our understanding of the key roles of blood-derived exosomes and the characterization of exosomal circRNAs in mammary gland development.

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

Brominated flame retardants (BFR) are molecules added to consumer products to reduce fire hazards. They were banned in North America and Europe because of their persistence and biomagnification. However, BFR are still released in the environment due to continued use of products manufactured before restriction, and from waste and recycling processes of those products. As a result, they remain sources of chronic environmental and human exposure worldwide. BFR are well-characterized endocrine disruptors. They have been associated with a wide range of alterations in endocrine and reproductive systems both in humans and experimental models in vivo and in vitro. Paradoxically, the effects of BFR on mammary glands, whose development and carcinogenesis are mainly under hormonal dependency are poorly known. There is increasing weight of evidence that exposure to endocrine disruptors promotes breast cancer, especially if the exposure occurs during sensitivity windows. For the mammary gland, these windows include the perinatal life, puberty, and pregnancy, as important remodeling of the organ happens during those periods. The peak of exposure to BFRs happened during late 1990s and beginning of 2000s in most countries. Women who were pregnant at that time are reaching menopause while their daughters are 20–30 years old. It is thus important to better understand the effects of BFRs on mammary gland development and breast cancer to determine whether these women are more at risk. Thus, this review aims to propose a comprehensive review of data reporting the effects of exposure to BFR on female endocrine and reproductive systems, with a particular focus on mammary gland development and of a potential increased risk of breast cancer. [ABSTRACT FROM AUTHOR]

Branching morphogenesis is the process that gives rise to branched structures in several organs, such as the lung, the kidney, and the mammary gland. Although morphologically well described, the exact mechanisms driving branch elongation and bifurcation are still poorly understood. Signaling cues from the stroma and extracellular matrix have an important role in driving branching morphogenesis. Organoid models derived from primary mammary epithelial cells have emerged as a powerful tool to gain insight into branching morphogenesis of the mammary gland. However, current available mammary organoid culture protocols result in morphologically simple structures which do not resemble the complex branched structure of the in vivo mammary gland. Supplementation of growth factors to mammary organoids cultured in basement membrane extract or collagen I were shown to induce bud formation and elongation but are not sufficient to drive true branching events. Here, we present an improved culture approach based on 3D primary mammary epithelial cell culture to develop branched organoids with a complex morphology. By alternating the addition of fibroblast growth factor 2 and epidermal growth factor to mammary organoids cultured in a basement membrane extract matrix enriched with collagen type I fibers, we obtain complex mammary organoid structures with primary, secondary, and tertiary branches over a period of 15–20 days. Mammary organoid structures grow >1 mm in size and show an elongated and branched shape which resembles in vivo mammary gland morphology. This novel branched mammary organoid model offers many possibilities to study the mechanisms of branching in the developing mammary gland. [ABSTRACT FROM AUTHOR]

Summary: Real-time in vivo imaging provides an essential window into the spatiotemporal cellular events contributing to tissue development and pathology. By coupling longitudinal intravital imaging with genetic lineage tracing, here we capture the earliest cellular events arising in response to active Wnt/β-catenin signaling and the ensuing impact on the organization and differentiation of the mammary epithelium. This enables us to interrogate how Wnt/β-catenin regulates the dynamics of distinct subpopulations of mammary epithelial cells in vivo and in real time. We show that β-catenin stabilization, when targeted to either the mammary luminal or basal epithelial lineage, leads to cellular rearrangements that precipitate the formation of hyperplastic lesions that undergo squamous transdifferentiation. These results enhance our understanding of the earliest stages of hyperplastic lesion formation in vivo and reveal that, in mammary neoplastic development, β-catenin activation dictates a hair follicle/epidermal differentiation program independently of the targeted cell of origin.

Branching morphogenesis is the process that gives rise to branched structures in several organs, such as the lung, the kidney, and the mammary gland. Although morphologically well described, the exact mechanisms driving branch elongation and bifurcation are still poorly understood. Signaling cues from the stroma and extracellular matrix have an important role in driving branching morphogenesis. Organoid models derived from primary mammary epithelial cells have emerged as a powerful tool to gain insight into branching morphogenesis of the mammary gland. However, current available mammary organoid culture protocols result in morphologically simple structures which do not resemble the complex branched structure of the in vivo mammary gland. Supplementation of growth factors to mammary organoids cultured in basement membrane extract or collagen I were shown to induce bud formation and elongation but are not sufficient to drive true branching events. Here, we present an improved culture approach based on 3D primary mammary epithelial cell culture to develop branched organoids with a complex morphology. By alternating the addition of fibroblast growth factor 2 and epidermal growth factor to mammary organoids cultured in a basement membrane extract matrix enriched with collagen type I fibers, we obtain complex mammary organoid structures with primary, secondary, and tertiary branches over a period of 15–20 days. Mammary organoid structures grow >1 mm in size and show an elongated and branched shape which resembles in vivo mammary gland morphology. This novel branched mammary organoid model offers many possibilities to study the mechanisms of branching in the developing mammary gland.

Lauric acid (LA) has the possibility to improve milk production in dairy cows by improving mammary gland development, however, the mechanism by which it might regulate mammary gland development is unclear. The influence of LA on milk production, nutrient digestibility and the expression of proteins related to mammary gland development in dairy cows were evaluated. Forty primiparous Holstein dairy cows were divided into 4 groups in a randomized block design. Four treatments included the control (0 g/d LA per cow), low-LA (100 g/d LA per cow), medium-LA (200 g/d LA per cow), and high-LA (300 g/d LA per cow). Yields of milk, fat-corrected milk, and energy-corrected milk quadratically increased ( P  < 0.05), and yield and content of milk fat linearly increased ( P  < 0.05) with LA supplementation. Percentages of C12:0, C18:1 and C20:1 fatty acids in milk fat linearly increased ( P  < 0.05), but that of C16:0 fatty acid linearly decreased ( P  = 0.046). Supplementation of LA led to a linear and quadratical increase ( P  < 0.05) in digestibility of dry matter, organic matter, neutral detergent fibre and acid detergent fibre, and ruminal total volatile fatty acid concentration but a linear reduction ( P  = 0.018) in the ratio of acetate to propionate. The enzymatic activities of ruminal pectinase, xylanase, and α-amylase, and populations of total bacteria and anaerobic fungi increased linearly ( P  < 0.05), while populations of total protozoa and methanogens decreased linearly ( P  < 0.05) with increased LA addition. Following LA addition, blood glucose, triglyceride, estradiol, prolactin, and insulin-like growth factor 1 concentrations increased linearly ( P  < 0.05) and albumin and total protein concentrations increased quadratically ( P  < 0.05). Moreover, addition of 200 g/d LA promoted ( P  < 0.05) the expression of protein involved in mammary gland development and fatty acids synthesis. These results suggested that LA addition enhanced milk production and fatty acids synthesis by stimulating nutrient digestion, the expression of proteins associated with milk fat synthesis and mammary gland development., (© 2024 The Authors.)