TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Stem Cells and the Differentiation Hierarchy in Mammary Gland Development.

The mammary gland is a highly dynamic organ that undergoes profound changes within its epithelium during puberty and the reproductive cycle. These changes are fueled by dedicated stem and progenitor cells. Both short- and long-lived lineage-restricted progenitors have been identified in adult tissue as well as a small pool of multipotent mammary stem cells (MaSCs), reflecting intrinsic complexity within the epithelial hierarchy. While unipotent progenitor cells predominantly execute day-to-day homeostasis and postnatal morphogenesis during puberty and pregnancy, multipotent MaSCs have been implicated in coordinating alveologenesis and long-term ductal maintenance. Nonetheless, the multipotency of stem cells in the adult remains controversial. The advent of large-scale single-cell molecular profiling has revealed striking changes in the gene expression landscape through ontogeny and the presence of transient intermediate populations. An increasing number of lineage cell-fate determination factors and potential niche regulators have now been mapped along the hierarchy, with many implicated in breast carcinogenesis. The emerging diversity among stem and progenitor populations of the mammary epithelium is likely to underpin the heterogeneity that characterizes breast cancer.

Mutation of SOCS2 induces structural and functional changes in mammary development.

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 control mice. 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.

Immune Cell Contribution to Mammary Gland Development.

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.

ABCD4 is associated with mammary gland development in mammals.

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.

Atypical cell cycle regulation promotes mammary stem cell expansion during mammary development and tumourigenesis.

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.

Effects of nanoselenium supplementation on lactation performance, nutrient digestion and mammary gland development in dairy cows.

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.

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

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

Embryonic mammary gland development.

Embryonic mammary gland development involves the formation of mammary placodes, invagination of flask-shaped mammary buds and development of miniature bi-layered ductal trees. Currently there is a good understanding of the factors that contribute to ectodermal cell movements to create these appendages and of pathways that lead to mammary specification and commitment. Gene expression profiles of early bipotent mammary stem cells populations as well as cell surface proteins and transcription factors that promote the emergence of unipotent progenitors have been identified. Analyses of these populations has illuminated not only embryonic mammary development, but highlighted parallel processes in breast cancer. Here we provide an overview of the highly conserved pathways that shape the embryonic mammary gland. Understanding the dynamic signaling events that occur during normal mammary development holds considerable promise to advance attempts to eliminate cancer by restoring differentiative signals.

Pubertal mammary gland development is a key determinant of adult mammographic density.

Mammographic density refers to the radiological appearance of fibroglandular and adipose tissue on a mammogram of the breast. Women with relatively high mammographic density for their age and body mass index are at significantly higher risk for breast cancer. The association between mammographic density and breast cancer risk is well-established, however the molecular and cellular events that lead to the development of high mammographic density are yet to be elucidated. Puberty is a critical time for breast development, where endocrine and paracrine signalling drive development of the mammary gland epithelium, stroma, and adipose tissue. As the relative abundance of these cell types determines the radiological appearance of the adult breast, puberty should be considered as a key developmental stage in the establishment of mammographic density. Epidemiological studies have pointed to the significance of pubertal adipose tissue deposition, as well as timing of menarche and thelarche, on adult mammographic density and breast cancer risk. Activation of hypothalamic-pituitary axes during puberty combined with genetic and epigenetic molecular determinants, together with stromal fibroblasts, extracellular matrix, and immune signalling factors in the mammary gland, act in concert to drive breast development and the relative abundance of different cell types in the adult breast. Here, we discuss the key cellular and molecular mechanisms through which pubertal mammary gland development may affect adult mammographic density and cancer risk.

SOX factors as cell-state regulators in the mammary gland and breast cancer.

Emerging evidence has shown that several SOX family transcription factors are key regulators of stem/progenitor cell fates in the mammary gland. These cell-fate regulators are often upregulated in breast cancer and contribute to tumor initiation and progression. They induce lineage plasticity and the epithelial-mesenchymal transition, which promotes tumor invasion, metastasis, and therapeutic resistance. SOX factors act through modulating multiple oncogenic signaling pathways in breast cancer. In addition to the cell-autonomous functions, new evidence suggests they can shape the tumor immune microenvironment. Here, we will review the molecular and functional evidence linking SOX factors with mammary gland development and discuss how these cell-fate regulators are co-opted in breast cancer.

Pubertal exposure to dietary advanced glycation end products disrupts ductal morphogenesis and induces atypical hyperplasia in the mammary gland.

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.

Mitochondrial one-carbon metabolic enzyme MTHFD2 facilitates mammary gland development during pregnancy.

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.

Diverse roles of glucocorticoids in the ruminant mammary gland: modulation of mammary growth, milk production, and mastitis.

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.

Implications of TDP-43 in non-neuronal systems.

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.

Adipocyte-Derived Paracrine Factors Regulate the In Vitro Development of Bovine Mammary Epithelial Cells.

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.

The Characteristic Function of Blood-Derived Exosomes and Exosomal circRNAs Isolated from Dairy Cattle during the Dry Period and Mid-Lactation.

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.

Browning of Mammary Fat Suppresses Pubertal Mammary Gland Development of Mice via Elevation of Serum Phosphatidylcholine and Inhibition of PI3K/Akt Pathway.

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 ß3-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.

EEF1D facilitates milk lipid synthesis by regulation of PI3K-Akt signaling in mammals.

Mammal's milk is an abundantly foremost source of proteins, lipids, and micronutrients for human nutrition and health. Understanding the molecular mechanisms underlying synthesis of milk components provides practical benefits to improve the milk quality via systematic breeding program in mammals. Through RNAi with EEF1D in primary bovine mammary epithelial cells, we phenotypically observed aberrant formation of cytoplasmic lipid droplets and significantly decreased milk triglyceride level by 37.7%, and exploited the mechanisms by which EEF1D regulated milk lipid synthesis via insulin (PI3K-Akt), AMPK, and PPAR pathways. In the EEF1D CRISPR/Cas9 knockout mice, incompletely developed mammary glands at 9th day postpartum with small or unformed lumens, and significantly decreased triglyceride concentration in milk by 23.4% were observed, as well as the same gene expression alterations in the three pathways. For dairy cattle, we identified a critical regulatory mutation modifying EEF1D transcription activity, which interpreted 7% of the genetic variances of milk lipid yield and percentage. Our findings highlight the significance of EEF1D in mammary gland development and milk lipid synthesis in mammals.

The miR-424(322)/503 cluster orchestrates remodeling of the epithelium in the involuting mammary gland.

The mammary gland is a very dynamic organ that undergoes continuous remodeling. The critical regulators of this process are not fully understood. Here we identify the microRNA cluster miR-424(322)/503 as an important regulator of epithelial involution after pregnancy. Through the generation of a knockout mouse model, we found that regression of the secretory acini of the mammary gland was compromised in the absence of miR-424(322)/503. Mechanistically, we show that miR-424(322)/503 orchestrates cell life and death decisions by targeting BCL-2 and IGF1R (insulin growth factor-1 receptor). Furthermore, we demonstrate that the expression of this microRNA cluster is regulated by TGF-ß, a well-characterized regulator of mammary involution. Overall, our data suggest a model in which activation of the TGF-ß pathway after weaning induces the transcription of miR-424(322)/503, which in turn down-regulates the expression of key genes. Here, we unveil a previously unknown, multilayered regulation of epithelial tissue remodeling coordinated by the microRNA cluster miR-424(322)/503.