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Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, transmembrane PCP proteins are trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in the HCC1569 breast cancer cell line, and in undifferentiated, but not differentiated, HC11 cells that serve as a model for mammary lactogenic differentiation. The loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate recruitment of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a, Ccnd1 and Csn2, larger acini and organoids, and precocious differentiation; phenotypes are rescued by overexpression of Vangl2, but not Vangl2ΔNLS. Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

Background: Although several cell culture systems have been developed to investigate the function of the mammary gland in dairy livestock, they have potential limitations, such as the loss of alveolar structure or genetic and phenotypic differences from their native counterparts. Overcoming these challenges is crucial for lactation research. Development of protocols to establish lactating organoid of livestock represents a promising goal for the future. In this study, we developed a protocol to establish a culture system for mammary organoids in dairy goats to model the mammary gland development and lactation process. Results: The organoids cultured within an extracellular matrix gel maintained a bilayer structure that closely resembled the native architecture of mammary tissue. The expansion of mammary organoids was significantly promoted by growth factors containing epidermal growth factor and fibroblast growth factor 2 whereas the proliferative index of the organoids was significantly inhibited by the treatment with WNT inhibitors. Upon stimulation with a lactogenic medium containing prolactin, the mammary organoids exhibited efficient lactation, characterized by the accumulation of lipid droplets in the lumen space. The lactation could be sustained for more than 3 weeks. Importantly, the expression patterns of genes related to fatty acid synthesis and milk proteins in lactating organoids closely mirrored those observed in mammary tissues. These observations were confirmed by data from proteomic analysis that the bulk of milk proteins was produced in the lactating organoids. Conclusion: This study is the first to establish a mammary organoid culture system modeling the mammary gland development and lactation process in ruminants. The efficient induction of lactation in ruminant mammary organoids holds promises for advancing the field of cell-based milk bio-manufacture in the food industry. [ABSTRACT FROM AUTHOR]

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo‐anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology. [ABSTRACT FROM AUTHOR]

Branching morphogenesis is a characteristic feature of many essential organs, such as the lung and kidney, and most glands, and is the net result of two tissue behaviors: branch point initiation and elongation. Each branched organ has a distinct architecture customized to its physiological function, but how patterning occurs in these ramified tubular structures is a fundamental problem of development. Here, we use quantitative 3D morphometrics, timelapse imaging, manipulation of ex vivo cultured mouse embryonic organs and mice deficient in the planar cell polarity component Vangl2 to address this question in the developing mammary gland. Our results show that the embryonic epithelial trees are highly complex in topology owing to the flexible use of two distinct modes of branch point initiation: lateral branching and tip bifurcation. This non-stereotypy was contrasted by the remarkably constant average branch frequency, indicating a ductal growth invariant, yet stochastic, propensity to branch. The probability of branching was malleable and could be tuned by manipulating the Fgf10 and Tgfβ1 pathways. Finally, our in vivo data and ex vivo time-lapse imaging suggest the involvement of tissue rearrangements in mammary branch elongation. [ABSTRACT FROM AUTHOR]

Simple Summary: Mastitis is an infection of the mammary glands that affects yaks' health and milk quality. The various bacteria found in mastitis-affected yaks were examined using a technique for reading particular genetic information. We discovered that contaminated milk had higher concentrations of bacteria than healthy milk. Our findings highlight the complexity and irregularity in the milk microbiome of yaks. Understanding these bacterial communities may aid in developing more effective mastitis control measures and enhance the health and milk quality of yaks, thereby benefiting dairy producers and yak farmers. Mastitis is an inflammation of the mammary gland that can be caused by various factors, including biological, chemical, mechanical, or physical. Microbiological culture, DNA techniques, and high-throughput next-generation sequencing have been used to identify mastitis-causing pathogens in various animal species. However, little is known about microbiota and microbiome changes linked to yak milk mastitis. This study aimed to characterize the milk microbiota of healthy and mastitis-infected yaks using full-length 16S rRNA sequencing. The results showed that the bacterial microbiota comprises 7 phyla, 9 classes, 20 orders, 39 families, 59 genera, and 72 species. Proteobacteria and Firmicutes were the predominant microbial communities, with lower abundances of Bacteroidota, Actinobacteriota, Acidobacteriota, and other minor groupings also observed. Proteobacteria dominated the clinical and subclinical mastitis groups (95.36% and 89.32%, respectively), in contrast to the healthy group (60.17%). Conversely, Firmicutes were more common in the healthy group (39.7%) than in the subclinical and clinical mastitis groups (10.49% and 2.92%, respectively). The predominant organisms found in the healthy group were Leuconostoc mesenteroides, Lactococcus piscium, Carnobacterium maltaromaticum, and Lactococcus raffinolactis. Low abundances of Staphylococcus aureus species were found in both subclinical and clinical mastitis groups, with Moraxella osloensis and Psychrobacter cibarius dominating the subclinical mastitis group and Pseudomonas fluorescens dominating the clinical mastitis group. An alpha diversity study revealed that the healthy group had a higher microbial diversity than the clinical and subclinical mastitis groups. According to beta-diversity analysis, the principal coordinate analysis identified that mastitis-infected samples significantly differed from healthy ones. The milk microbiota of healthy yaks is more varied, and specific prominent taxa within various groups can act as marker microorganisms for mastitis risk. The genera Leuconostoc and Lactococcus are promising candidates for creating probiotics. [ABSTRACT FROM AUTHOR]

Abstract Background Although several cell culture systems have been developed to investigate the function of the mammary gland in dairy livestock, they have potential limitations, such as the loss of alveolar structure or genetic and phenotypic differences from their native counterparts. Overcoming these challenges is crucial for lactation research. Development of protocols to establish lactating organoid of livestock represents a promising goal for the future. In this study, we developed a protocol to establish a culture system for mammary organoids in dairy goats to model the mammary gland development and lactation process. Results The organoids cultured within an extracellular matrix gel maintained a bilayer structure that closely resembled the native architecture of mammary tissue. The expansion of mammary organoids was significantly promoted by growth factors containing epidermal growth factor and fibroblast growth factor 2 whereas the proliferative index of the organoids was significantly inhibited by the treatment with WNT inhibitors. Upon stimulation with a lactogenic medium containing prolactin, the mammary organoids exhibited efficient lactation, characterized by the accumulation of lipid droplets in the lumen space. The lactation could be sustained for more than 3 weeks. Importantly, the expression patterns of genes related to fatty acid synthesis and milk proteins in lactating organoids closely mirrored those observed in mammary tissues. These observations were confirmed by data from proteomic analysis that the bulk of milk proteins was produced in the lactating organoids. Conclusion This study is the first to establish a mammary organoid culture system modeling the mammary gland development and lactation process in ruminants. The efficient induction of lactation in ruminant mammary organoids holds promises for advancing the field of cell-based milk bio-manufacture in the food industry.

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Linear scores and measures of both udder and teat shape and size (thirteen traits) were assessed in 83 ewes of Improved Valachian breed (IV) and in crossbreds with East Friesian breed (EF 25% or EF 50%). At the same time, cistern measures and cross-section areas (fourteen traits) as well as milk yield and milkability were scanned using ultrasound technique or measured in the same individuals (130 measurements in each trait). Crossbreds with EF 50% had the largest udders in terms of depth, width, and height, as determined by either a nine-point linear score or exact measurements. Additionally, these crossbreds had teats that were more horizontally placed and the most appropriate udder attachment and shape. more horizontally and most appropriate udder attachment and shape. Ultrasound scans revealed that the areas of both the left and right udder cisterns scanned by ultrasound were the highest in EF 50% crosses. Overall cistern cross-section areas scanned from the side were 2934.6 mm2 in IV, 2932.6 mm2 in crosses of EF 25% and 3559.4 mm2 in crosses of EF 50% (P0.05). On average, the proportion of machine-stripped milk was 33.2%, and non-significant influence of genotype was shown. Furthermore, all traits under study showed non significant differences between crosses of EF 25% and 50%.

As both perimenopausal and menopausal periods are recognized critical windows of susceptibility for breast carcinogenesis, development of a physiologically relevant model has been warranted. The traditional ovariectomy model causes instant removal of the entire hormonal repertoire produced by the ovary, which does not accurately approximate human natural menopause with gradual transition. Here, we characterized the mammary glands of 4-vinylcyclohexene diepoxide (VCD)-treated animals at different time points, revealing that the model can provide the mammary glands with both perimenopausal and menopausal states. The perimenopausal gland showed moderate regression in ductal structure with no responsiveness to external hormones, while the menopausal gland showed severe regression with hypersensitivity to hormones. Leveraging the findings on the VCD model, effects of a major endocrine disruptor (polybrominated diphenyl ethers, PBDEs) on the mammary gland were examined during and after menopausal transition, with the two exposure modes; low-dose, chronic (environmental) and high-dose, subacute (experimental). All conditions of PBDE exposure did not augment or compromise the macroscopic ductal reorganization resulting from menopausal transition and/or hormonal treatments. Single-cell RNA sequencing revealed that the experimental PBDE exposure during the post-menopausal period caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts. The environmental PBDE exposure resulted in similar transcriptomic changes to a lesser extent. In summary, the VCD mouse model provides both perimenopausal and menopausal windows of susceptibility for the breast cancer research community. PBDEs, including all tested models, may affect the post-menopausal gland including impacts on the non-epithelial compartments. [ABSTRACT FROM AUTHOR]

Simple Summary: Mammary carcinomas have been diagnosed with increasing frequency in pet rabbits. Prognostic biomarkers are limited, and the only available treatment is surgical excision. Additional treatment options are needed, e.g., for animals in which metastases to internal organs preclude complete tumor removal. Human breast cancer may express the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1), that represents a prognostic biomarker and a possible therapeutic target. Since previous studies revealed similarities between human breast cancer and pet rabbit mammary carcinomas, this study investigated IDO1 immunostaining in 96 mammary carcinomas of 96 pet rabbits with an average age of 5.5 years. All rabbits with reported sex were female. Variable percentages of IDO1-positive tumor cells were detected in 90 (94%) carcinomas. Furthermore, IDO1 immunostaining was observed in the secretory epithelial cells of the adjacent non-neoplastic mammary tissue. This study provides further information on the molecular features of mammary carcinomas in rabbits. It also shows similarities in IDO1 expression between rabbit mammary carcinomas and human breast cancer. These findings can have a mutual benefit. They could lead the development of novel treatment options for rabbits with mammary carcinomas. In addition, they further support the value of rabbits with mammary carcinomas for breast cancer research. For mammary carcinomas in pet rabbits, prognostic biomarkers are poorly defined, and treatment is limited to surgical excision. Additional treatment options are needed for rabbit patients for which surgery is not a suitable option. In human breast cancer, the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) represents a prognostic biomarker and possible therapeutic target. This retrospective immunohistochemical study examined IDO1 in 96 pet rabbit mammary carcinomas with known mitotic count, hormone receptor status, and percentage of stromal tumor infiltrating lymphocytes (TILs). Tumors were obtained from 96 pet rabbits with an average of 5.5 years. All rabbits with reported sex (n = 88) were female or female-spayed. Of the carcinomas, 94% expressed IDO1, and 86% had sparse TILs consistent with cold tumors. Statistically significant correlations existed between a higher percentage of IDO1-positive tumor cells, lower mitotic counts, and increased estrogen receptor expression. The threshold for significance was IDO1 staining in >10% of tumor cells. These results lead to the assumption that IDO1 expression contributes to tumorigenesis and may represent a prognostic biomarker and possible therapeutic target also in pet rabbit mammary carcinomas. They also support the value of rabbits for breast cancer research. [ABSTRACT FROM AUTHOR]

The subareolar Sappey's plexus was studied using color lymphography and immunohistochemical methods with a panel of antibodies to podoplanin, smooth muscle actin, low molecular weight cytokeratin AE1/AE3, and GATA3 on archival material obtained during radical mastectomies and sectoral resections with lymph node dissection from 86 patients diagnosed with non-special type breast cancer. At the macro- and microscopic levels, the connection between the subareolar lymphatic plexus and the lymphatic system of the breast parenchyma has been demonstrated. In triple negative breast cancer with metastases to the axillary lymph nodes, the involvement of subareolar lymphatic plexus into lymphogenous metastasis to the lymph nodes of the axillary lymphatic collector was shown. [ABSTRACT FROM AUTHOR]

Background: Growth factor receptor‐bound 7 (Grb7) is an adaptor protein involved in signal transduction downstream of multiple receptor tyrosine kinases, including ERBB, FGFR, and PDGFR pathways. Experimental studies have implicated Grb7 in regulating cell proliferation, survival, migration, and invasion through its large repertoire of protein–protein interactions. Results: Here, we describe the generation and characterization of a Grb7 knockout mouse. These mice are viable and fertile. A lacZ knock‐in reporter was used to visualize Grb7 promoter activity patterns in adult tissues, indicating widespread Grb7 expression in glandular epithelium, the central nervous system, and other tissues. The sole defect observed in these animals was a failure of Grb7 knockout females to successfully raise pups to weaning age, a phenotype that was independent of both paternal and pup genotypes. Conclusions: These data suggest a regulatory role for Grb7 in mammary lactational physiology. Key Findings: Despite many reports implicating Grb7 in multiple signal transduction pathways, deletion of this signaling adapter protein had minimal effects on mouse development.Grb7 knockout animals were viable and fertile but knockout females were deficient in the ability to raise pups to weaning age.These data imply a requirement for Grb7 in lactational sufficiency. [ABSTRACT FROM AUTHOR]

Crassocephalum rubens has several medicinal properties, making it effective against several ailments, including its local use for treating liver problems and breast cancer. The effect of crude extract of Crassocephalum rubens (CECR) against liver damage in 7, 12 - dimethylbenz[a]anthracene (DMBA)-induced mammary gland toxicity was investigated in female Wistar rats. Rats were divided into group of 5 (n = 8). Group 1 served as control. Animals in groups 2 to 4 were administered a single intraperitoneal dose of DMBA at 20 mg/kg. Groups 3 and 4 rats were thereafter treated with 250 mg/kg and 500 mg/kg b.w CECR, respectively, for 12 weeks. Rats in group 5 were given 500 mg/kg b.w. CECR only for 12 weeks. Effect of CECR on DMBA-induced anomalies was investigated by using various biochemical parameters, including liver marker enzymes, oxidative stress, and tissue histology and cell count. CECR ameliorated the hepatic damage accompanied with DMBA-induced mammary gland toxicity by mitigating the abnormal changes noted in the levels of the parameters investigated. This could be proved by a significant (p<0.05) reduction in the levels of serum transaminases, dehydrogenase (LDH), gamma-glutamyl transferase (GGT), lactate malondialdehyde (MDA) and estrogen, and a significant (p<0.05) increase in the levels of superoxide dismutase (SOD) and liver cell count when compared with DMBA-intoxicated rats. The CECR, mostly at 500 mg/kg. b.w. dose, could serve as an alternative therapy against liver inflammation and oxidative stress associated with DMBA-induced mammary gland toxicity. [ABSTRACT FROM AUTHOR]

Subcutaneous adipocytes are crucial for mammary gland epithelial development during pregnancy. Our and others' previous data have suggested that adipo-epithelial transdifferentiation could play a key role in the mammary gland alveolar development. In this study, we tested whether adipo-epithelial transdifferentiation occurs in vitro. Data show that, under appropriate co-culture conditions with mammary epithelial organoids (MEOs), mature adipocytes lose their phenotype and acquire an epithelial one. Interestingly, even in the absence of MEOs, extracellular matrix and diffusible growth factors are able to promote adipo-epithelial transdifferentiation. Gene and protein expression studies indicate that transdifferentiating adipocytes exhibit some characteristics of milk-secreting alveolar glands, including significantly higher expression of milk proteins such as whey acidic protein and β-casein. Similar data were also obtained in cultured human multipotent adipose-derived stem cell adipocytes. A miRNA sequencing experiment on the supernatant highlighted mir200c, which has a well-established role in the mesenchymal–epithelial transition, as a potential player in this phenomenon. Collectively, our data show that adipo-epithelial transdifferentiation can be reproduced in in vitro models where this phenomenon can be investigated at the molecular level. [ABSTRACT FROM AUTHOR]

Linear scores and measures of both udder and teat shape and size (thirteen traits) were assessed in 83 ewes of Improved Valachian breed (IV) and in crossbreds with East Friesian breed (EF 25% or EF 50%). At the same time, cistern measures and cross-section areas (fourteen traits) as well as milk yield and milkability were scanned using ultrasound technique or measured in the same individuals (130 measurements in each trait). Crossbreds with EF 50% had the largest udders in terms of depth, width, and height, as determined by either a nine-point linear score or exact measurements. Additionally, these crossbreds had teats that were more horizontally placed and the most appropriate udder attachment and shape. more horizontally and most appropriate udder attachment and shape. Ultrasound scans revealed that the areas of both the left and right udder cisterns scanned by ultrasound were the highest in EF 50% crosses. Overall cistern cross-section areas scanned from the side were 2934.6 mm² in IV, 2932.6 mm² in crosses of EF 25% and 3559.4 mm² in crosses of EF 50% (P<0.05). Crossbreds of EF 50% had more appropriate machine milked milk and total milk yield (311.1 and 424.7 ml) than purebred IV (231.8 and 336.1 ml; P>0.05). On average, the proportion of machine-stripped milk was 33.2%, and non-significant influence of genotype was shown. Furthermore, all traits under study showed nonsignificant differences between crosses of EF 25% and 50%. [ABSTRACT FROM AUTHOR]

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki‐67 expression, along with the Apoptotic Index (AI) determined through Caspase‐3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti‐Ki‐67 and Caspase‐3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki‐67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase‐3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland. [ABSTRACT FROM AUTHOR]

We describe here a case of canine mammary benign mixed tumor with sebaceous metaplasia in the right fifth mammary gland of an eight-year-old, intact female Poodle dog. Grossly, the mass was firm with off-white, poorly lobulated cut surfaces. Histologically, the luminal epithelial cells and myoepithelial cells proliferated with cartilage formation and focal squamous metaplasia. Moreover, a large number of nests of various sizes, which were filled with foamy cells in the center and associated with basaloid reserve-like cells in the periphery, showed sebaceous gland-like structures. Immunohistochemically, myoepithelial cells and reserve-like cells in the metaplastic sebaceous gland-like structures were CK14, α-smooth muscle actin (α-SMA) and p63 positive, suggesting a possibility that these two components may have a common cell of origin. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we looked to evaluate the variation in sensitivity, specificity, predictive values, and accuracy (Kappa index) of the microbiological diagnosis for Staphylococcus aureus in a dairy cattle herd subjected to blitz therapy to eradicate Streptococcus agalactiae. The studied dairy herd had an average of 160 lactating Holstein cows and microbiological diagnosis was carried out on all lactating cows for 5 consecutive months. After treating the animals that presented a pure culture of S. agalactiae in microbiological diagnosis in the first and second milk sample collections, isolation of S. aureus was observed in these animals in the following milk sample collections, increasing the sensitivity of the microbiological diagnosis. The results indicate that due to the release of S. agalactiae being greater than that of S. aureus, due to competition in a nutritive culture medium, it was not possible to isolate S. aureus in the first collections. However, after the elimination of S. agalactiae through intramammary antimicrobial treatment, S. aureus began to be identified in the microbiological examination as there was no longer competition between the pathogens in the nutritive culture medium. Bovine mastitis is an important and costly disease to dairy cattle. Diagnostic methods usually performed in Brazil are somatic cell counts (SCC) and milk microbiology. Low bacteria shedding in milk implies no colony growth in microbiological tests and false negative results. Streptococcus agalactiae and Staphylococcus aureus are principal pathogens of mixed mastitis. However, S. agalactiae has a higher bacterial release from the mammary gland than S. aureus, affecting microbiological sensitivity to diagnose S. aureus. This study aimed to estimate the SCC and total bacterial count (TBC) from cows according to pathogen isolated in milk and to evaluate variation in S. aureus diagnosis by a microbiological test during S. agalactiae treatment, which is called blitz therapy. Both S. agalactiae and S. aureus presented high SCC means, although S. agalactiae showed shedding of bacteria 2.3 times greater than S. aureus. Microbiological sensitivity to S. aureus increased for 5 months during S. agalactiae treatment. The prevalence of S. agalactiae fell after 5 months of therapeutic procedures. The prevalence of S. aureus increased to 39.0. The results showed that due to high sensitivity, the polymerase chain reaction (PCR) could be used at the beginning of blitz therapy with the goal of S. agalactiae eradication from the dairy herd. [ABSTRACT FROM AUTHOR]

This work extensively studied the vasculature of mice mammary fat pads (BALB/c and C57BL/6) with special reference to haematogenous drainage routes. Mammary fat pads were five pairs (first cervical, second and third thoracic, fourth abdominal and fifth inguinal), bilaterally symmetrical, extending laterally and continuously with the subcutaneous fascia. The superficial cervical artery and vein primarily accomplished the blood vasculature of the first mammary fat pad, while the lateral thoracic and external thoracic arteries and veins supplied the second and third mammary fat pads. The superficial cervical vein (found parallel to the superficial cervical artery) drained into the external jugular vein. The lateral thoracic artery and external thoracic artery branched almost at the same level as the axillary artery (branch of subclavian artery), the latter being more medial in position. However, in some specimens, the branching of both arteries appeared to be at the same level, and their origins were indistinguishable. The lateral thoracic vein that was parallel to the lateral thoracic artery drained to the axillary vein close to the drainage of the external thoracic vein. The lateral thoracic, superficial caudal epigastric, iliolumbar and external thoracic arteries and veins vascularized the fourth mammary fat pad and displayed anastomosis among themselves. The iliolumbar vein (found parallel to the iliolumbar artery) drained into the inferior vena cava. The superficial caudal epigastric vein (found parallel to the superficial caudal epigastric artery (SCaEA)) drained into the femoral vein. Unlike humans, the internal thoracic artery and vein did not participate in the vasculature of mammary fat pads. The SCaEA and vein supplied blood and drained the fifth mammary fat pad. The anatomical continuity of the fourth and fifth mammary fat pads provided common drainage for both mammary fat pads. The BALB/c and C57BL/6 mice strains studied did not differ in topography and size of mammary fat pads. The vascular supply and drainage of the mammary fat pads also did not differ in the strains studied. Only minor variations could be noted in the small veins draining into the lateral thoracic vein. Lateral tributaries seen in the terminal end of the lateral thoracic vein were absent in the C57BL/6 mice. [ABSTRACT FROM AUTHOR]

Treating female canine mammary gland tumors is crucial owing to their propensity for rapid progression and metastasis, significantly impacting the overall health and well-being of dogs. Mitoquinone (MitoQ), an antioxidant, has shown promise in inhibiting the migration, invasion, and clonogenicity of human breast cancer cells. Thus, we investigated MitoQ's potential anticancer properties against canine mammary gland tumor cells, CMT-U27 and CF41.Mg. MitoQ markedly suppressed the proliferation and migration of both CMT-U27 and CF41.Mg cells and induced apoptotic cell death in a dose-dependent manner. Furthermore, treatment with MitoQ led to increased levels of pro-apoptotic proteins, including cleaved-caspase3, BAX, and phospho-p53. Cell cycle analysis revealed that MitoQ hindered cell progression in the G1 and S phases in CMT-U27 and CF41.Mg cells. These findings were supported using western blot analysis, demonstrating elevated levels of cleaved caspase-3, a hallmark of apoptosis, and decreased expression of cyclin-dependent kinase (CDK) 2 and cyclin D4, pivotal regulators of the cell cycle. In conclusion, MitoQ exhibits in vitro antitumor effects by inducing apoptosis and arresting the cell cycle in canine mammary gland tumors, suggesting its potential as a preventive or therapeutic agent against canine mammary cancer. [ABSTRACT FROM AUTHOR]

ABSTRACT: A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency.

Abstract Background Macrophages residing in milk are vital during intramammary infections. This study sought to develop a method enabling the investigation of macrophage responses to pathogens. Streptococcus uberis is the predominant cause of bovine mastitis UK-wide and its pathogenesis is unusual compared to other intramammary pathogens. Previous studies utilise macrophage cell lines, isolated bovine blood derived monocytes, or macrophages from raw milk through complex or inconsistent strategies such as fluorescence activated cell sorting (FACS), centrifugation and selective adherence, and CD14 antibody-microbeads. The centrifuge steps required in the initial stages often damage cells. Thus, the aim of this study was to develop a reliable, reproducible, and cost-effective method for isolating mammary macrophages from milk in a way that allows their culture, challenge with bacteria, and measurement of their response ex-vivo. Results This method achieves an average yield of 1.27 × 107 cells per litre of milk. Whole milk with somatic cell range of 45–65 cells/µL produced excellent yields, with efficient isolations accomplished with up to 150 cells/µL. This strategy uses milk diluted in PAE buffer to enable low-speed centrifugation steps followed by seeding on tissue-culture-treated plastic. Seeding 1,000,000 milk-extracted cells onto tissue culture plates was sufficient to obtain 50,000 macrophage. Isolated macrophage remained responsive to challenge, with the highest concentration of IL-1β measured by ELISA at 20 h after challenge with S. uberis. In this model, the optimal multiplicity of infection was found to be 50:1 bacteria:macrophage. No difference in IL-1β production was found between macrophages challenged with live or heat-killed S. uberis. Standardisation of the production of IL-1β to that obtained following macrophage stimulation with LPS allowed for comparisons between preparations. Conclusions A cost-effective method, utilising low-speed centrifugation followed by adherence to plastic, was established to isolate bovine mammary macrophages from raw milk. This method was shown to be appropriate for bacterial challenge, therefore providing a cost-effective, ex-vivo, and non-invasive model of macrophage-pathogen interactions. The optimal multiplicity of infection for S. uberis challenge was demonstrated and a method for standardisation against LPS described which removes sample variation. This robust method enables, reproducible and reliable interrogation of critical pathogen-host interactions which occur in the mammary gland.

Abstract In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased β-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

Mammals depend on the secretion of milk to rear their offspring, which exposes the organ in charge of the function, the mammary gland (MG), to bacterial threat. The essential driving force that conditions the interactions of bacteria with the MG is the abundant secretion of milk, a nutritious fluid which endows the common mastitis-causing pathogens with a doubling time of less than 30 min. From this angle, mammals rely on a potential bacterial bioreactor for the survival of their offspring. The MG is lined with a two-layered epithelium devoid of protective mucus. This means that the mammary epithelium is exposed directly to bacteria once they have passed through the opening lactiferous canal. To cope with the threat, the MG resorts to neutrophilic inflammation to check bacterial proliferation in its lumen and at its epithelial lining. Promptness of neutrophil recruitment is a necessity, which requires a low threshold of activation on the part of the mammary epithelium. Constrained by natural selection, the MG has evolved an innate and adaptive immunity intolerant to bacteria regardless of their level of virulence. The evolutionary issue has been to find a compromise between the deleterious tissue-damaging side effects of inflammation and the maintenance of the secretory function indispensable for the offspring’s survival. It appears that the MG relies mainly on neutrophilic inflammation for its protection and is regulated by type 3 immunity. Advances in knowledge of type 3 immunity in the MG will be necessary to induce immune protection adapted to the physiology of this peculiar organ.

Pseudoangiomatous stromal hyperplasia (PASH) is a benign interstitial hyperplasia of the breast that usually occurs in premenopausal or perimenopausal women. It is usually characterized by localized lesions or clear boundary masses, and diffuse double breast enlargement is rare. PASH is considered a hormone-dependent disease that is commonly progesterone related. There are no imaging characteristics, and both benign and suspicious malignant signs can be seen. The definitive diagnosis of PASH depends on a pathological diagnosis, and it is necessary to be vigilant in distinguishing between benign and malignant tumors with similar breast histopathology. Here, we report the case of a 23-year-old multipara patient with bilateral diffuse pseudoangiomatous stromal hyperplasia of the breast during pregnancy who presented with macromastia and reviewed the literature to further understand the clinical features, pathological diagnosis, differential diagnosis, treatment and prognosis of pseudoangiomatous stromal hyperplasia of the breast. [ABSTRACT FROM AUTHOR]

A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency. [Display omitted] [ABSTRACT FROM AUTHOR]

The mammary gland is a target organ for hormones in the hypothalamus, pituitary gland, thyroid gland, and ovaries. In the postnatal period of animal ontogenesis, breast growth, and development are determined by the estrous cycle and pregnancy. One of the most pressing problems in morphology, mammology, and reproductology of animals is the study of the structure of the breast at the macro and microscopic levels. However, modern studies on the morphology of animals' breasts are mainly devoted to the features of pathomorphological changes in the breast during carcinogenesis. The work aims to conduct a scientific review of the study of normal breast morphology in domestic carnivores. Electronic publication searches have been conducted in the Web of Science, Scopus, PubMed, and Google Scholar databases over the past 20 years. The principles of objectivity and a comprehensive attitude to studying the chosen problem are used. The authors of the review article review the current scientific literature and summarize current knowledge on the features of dog and cat breast morphology. The anatomical structure of the breast of animals is presented with an emphasis on its topography. According to the description of the microscopic structure of the breast, its main structural components are characterized by secretory parts (alveoli and tubes) and intraparticle excretory ducts. Anatomical characteristics of the nerves, blood, and lymphatic vessels of the mammary glands of dogs and cats are presented. Morphological changes in the mammary gland of dogs and cats at different stages of the estrus cycle and during pregnancy are described; in particular, the mammary gland is at rest (inactive) during proestrus and estrus. According to diestrus, due to an increase in progesterone levels, successive morphological changes occur, in particular, stromal and ductal proliferation; early development of lobules with branching ducts and proliferation of alveoli; the dominance of glandular tissue with large lobules containing secretory material; early regression, an increase in interparticle connective tissue, eosinophilic protein secretion in dilated ducts and alveoli. Complete involution of the alveoli and tubes of the breast is recorded at the end of the anestrus. The described anatomical and microscopic features of the structure of the mammary glands of dogs and cats are relevant for the comparative morphology of carnivores at different stages of the sexual cycle and during pregnancy. Data on blood supply, lymphatic drainage, and innervation of the breast are essential for its functional characteristics in normal and pathological conditions, particularly for developing a reasonable model of breast extirpation in malignant tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: In the dairy industry, bovine mastitis poses significant challenges due to production losses and treatment costs. Understanding innate immunity in bovine milk could be crucial for fighting intra-mammary infections. We conducted cytofluorimetric analyses to assess bacterial killing activity in bovine skim milk against Gram-positive (S. aureus) and Gram-negative (E. coli) pathogens. We found that milk exhibited higher bacterial killing activity against S. aureus compared to E. coli, and those results correlated with N-acetyl-β-D-glucosaminidase (NAGase) levels. Both S. aureus killing and NAGase activity decreased with lactation duration. Milk samples from autochthonous Modenese cows displayed stronger killing activity compared to samples from cosmopolitan breed Holstein Friesian cows. Our findings suggest that skim milk is endued with distinct control mechanisms against different pathogens in the mammary gland, and provide insights into innate immune responses and potential strategies for managing mastitis in the dairy industry, which could lead to improved production efficiency and reduced treatment costs. In the dairy industry, bovine mastitis represents a major concern due to substantial production losses and costs related to therapies and early culling. The mechanisms of susceptibility and effective response to intra-mammary infections are still poorly understood. Therefore, we investigated innate immunity in acellular bovine skim milk through cytofluorimetric analyses of bacterial killing activity against both Gram-positive and Gram-negative pathogens. Freshly cultured E. coli and S. aureus strains were incubated with colostrum and milk samples at different lactation time points from two groups of cows, purportedly representing mastitis-resistant and mastitis-susceptible breeds; bacterial cells were analyzed for vitality by flow cytometry following incorporation of vital dyes. N-acetyl-β-D-glucosaminidase (NAGase) activity was also investigated in milk and colostrum samples. Our findings revealed that colostrum and milk bacterial killing activity was greater against S. aureus compared to E. coli., with this activity correlated with milk NAGase levels. Furthermore, both killing of S. aureus and NAGase activity were negatively correlated to the elapsed time of lactation. Interestingly, samples from the allegedly mastitis-resistant breed displayed higher bacterial killing and NAGase activities. Our study suggests that diverse control mechanisms are exerted against Gram-positive and Gram-negative pathogens in the mammary glands of cows, probably beyond those already described in the literature. [ABSTRACT FROM AUTHOR]

Mammary gland tumors are commonly observed in older, intact female dogs, while male dogs are rarely affected. The incidence of mammary gland tumors in dogs is significantly higher than in other domestic species, accounting for about 50% of tumor cases in dogs. A 7-year-old intact German Shepherd bitch was presented at the Veterinary Teaching Hospital, University of Ibadan, with swollen mammary glands and a history of conception failure, despite a successful mating a few months prior. Samples were collected using the fine needle aspirate technique from the swollen inguinal mammary glands and popliteal lymph nodes for cytological examination, which revealed confirmatory evidence suggestive of lymphoma. This article describes the mammary gland tumor in an adult intact bitch and her subsequent euthanasia due to tumor metastasis. [ABSTRACT FROM AUTHOR]

Genomic imprinting is an epigenetically-regulated process of central importance in mammalian development and evolution. It involves multiple levels of regulation, with spatio-temporal heterogeneity, leading to the contextdependent and parent-of-origin specific expression of a small fraction of the genome. Genomic imprinting studies have therefore been essential to increase basic knowledge in functional genomics, evolution biology and developmental biology, as well as with regard to potential clinical and agrigenomic perspectives. Here we offer an overview on the contribution of livestock research, which features attractive resources in several respects, for better understanding genomic imprinting and its functional impacts. Given the related broad implications and complexity, we promote the use of such resources for studying genomic imprinting in a holistic and integrative view. We hope this mini-review will draw attention to the relevance of livestock genomic imprinting studies and stimulate research in this area. [ABSTRACT FROM AUTHOR]

Background: Macrophages residing in milk are vital during intramammary infections. This study sought to develop a method enabling the investigation of macrophage responses to pathogens. Streptococcus uberis is the predominant cause of bovine mastitis UK-wide and its pathogenesis is unusual compared to other intramammary pathogens. Previous studies utilise macrophage cell lines, isolated bovine blood derived monocytes, or macrophages from raw milk through complex or inconsistent strategies such as fluorescence activated cell sorting (FACS), centrifugation and selective adherence, and CD14 antibody-microbeads. The centrifuge steps required in the initial stages often damage cells. Thus, the aim of this study was to develop a reliable, reproducible, and cost-effective method for isolating mammary macrophages from milk in a way that allows their culture, challenge with bacteria, and measurement of their response ex-vivo. Results: This method achieves an average yield of 1.27 × 107 cells per litre of milk. Whole milk with somatic cell range of 45–65 cells/µL produced excellent yields, with efficient isolations accomplished with up to 150 cells/µL. This strategy uses milk diluted in PAE buffer to enable low-speed centrifugation steps followed by seeding on tissue-culture-treated plastic. Seeding 1,000,000 milk-extracted cells onto tissue culture plates was sufficient to obtain 50,000 macrophage. Isolated macrophage remained responsive to challenge, with the highest concentration of IL-1β measured by ELISA at 20 h after challenge with S. uberis. In this model, the optimal multiplicity of infection was found to be 50:1 bacteria:macrophage. No difference in IL-1β production was found between macrophages challenged with live or heat-killed S. uberis. Standardisation of the production of IL-1β to that obtained following macrophage stimulation with LPS allowed for comparisons between preparations. Conclusions: A cost-effective method, utilising low-speed centrifugation followed by adherence to plastic, was established to isolate bovine mammary macrophages from raw milk. This method was shown to be appropriate for bacterial challenge, therefore providing a cost-effective, ex-vivo, and non-invasive model of macrophage-pathogen interactions. The optimal multiplicity of infection for S. uberis challenge was demonstrated and a method for standardisation against LPS described which removes sample variation. This robust method enables, reproducible and reliable interrogation of critical pathogen-host interactions which occur in the mammary gland. [ABSTRACT FROM AUTHOR]

Context: The hypothesis of interdependency among cows' mammary glands has been shown, and quarters adjacent to glands with clinical mastitis may have their composition and health status impaired. Aims: The main focus of this study was to compare foremilk composition (fat, total protein, lactose, solids non-fat (SNF), and chloride) and health status (somatic cell count (SCC), differential leukocytes) of healthy mammary glands adjacent to a gland previously treated (on average, 24 days after treatment, varying from 20 to 39 days) for clinical mastitis with foremilk samples of healthy mammary glands of healthy cows. Our previous study compared these traits (foremilk composition and health status) between healthy mammary glands adjacent to a mastitis-infected quarter and healthy mammary glands from healthy cows (no clinical mastitis) immediately after the identification of mastitis on case quarters. Methods: The study was designed as a prospective case–control study and the enrolled cows (cases and controls) were matched by days in milk and parity. Case cows (n = 50) were defined as cows that previously had a single quarter infected with mild or moderate clinical mastitis, and which were treated according to an on-farm protocol 1 day after mastitis identification. Control cows (n = 50) were defined as cows that had not suffered clinical mastitis in the current lactation. Foremilk samples from each quarter of case and control cows were collected so as to assess concentrations of fat, total protein, lactose, SCC, and SNF, conduct microbiological analyses, and determine the concentration of chloride and differential leukocyte counts. Multivariate linear regression and logistic regression analyses were performed to assess possible associations of milk-composition and health-status traits between quarter types (case or control cows). Key results: On average, 24 days after the treatment of the case quarter began, milk composition (fat, lactose, SNF, and chloride) and some health status (lymphocyte and macrophage counts) of adjacent quarters returned to similar levels as in control quarters, while total protein, SCC, and neutrophils remained greater for adjacent quarters. No effect of quarter position or microbiological results of enrolled quarters were identified. Conclusions: Our results suggested that at least 20 days (average of 24 days) after an episode of clinical mastitis in a neighbouring gland, adjacent quarters were still recovering from this mastitis episode, and we conclude that interdependency of quarters is a physiological systemic two-way immune response route, and each particular milk component has a different behaviour after clinical mastitis. Implications: Our results suggest that clinical mastitis is far more costly for producers and industry than has been previously reported. Bovine mastitis is the disease that causes the most economical losses to dairy farmers, including veterinary expenses, milk disposal, loss of production and reduction of animal pregnancies. The milk composition of the mammary quarter affected by mastitis is not the only one compromised, because the milk quality of the entire udder is also affected. A period of 20 days is not enough for animals to fully recover from a case of udder inflammation. Mastitis is more costly to producers and industry than has been previously reported. Bovine mastitis is the disease that causes the most economical losses to dairy farmers, including veterinary expenses, milk disposal, loss of production and reduction of animal pregnancies. The milk composition of mammary quarter affected by mastitis is not the only one compromised, as the milk quality of entire udder is also affected. A period of 20 days is not enough for animals to fully recover from a case of udder inflammation. Mastitis is more costly to producers and industry than has been previously reported. [ABSTRACT FROM AUTHOR]

The three-dimensional (3D) structure of the ductal epithelium and the surrounding extracellular matrix (ECM) are integral aspects of the breast tissue, and they have important roles during mammary gland development, function and malignancy. However, the architecture of the branched mammary epithelial network is poorly recapitulated in the current in vitro models. 3D bioprinting is an emerging approach to improve tissue-mimicry in cell culture. Here, we developed and optimized a protocol for 3D bioprinting of normal and cancerous mammary epithelial cells into a branched Y-shape to study the role of cell positioning in the regulation of cell proliferation and invasion. Non-cancerous cells formed continuous 3D cell networks with several organotypic features, whereas the ductal carcinoma in situ (DCIS) –like cancer cells exhibited aberrant basal polarization and defective formation of the basement membrane (BM). Quantitative analysis over time demonstrated that both normal and cancerous cells proliferate more at the branch tips compared to the trunk region of the 3D-bioprinted cultures, and particularly at the tip further away from the branch point. The location-specific rate of proliferation was independent of TGFβ signaling but invasion of the DCIS-like breast cancer cells was reduced upon the inhibition of TGFβ. Thus, our data demonstrate that the 3D-bioprinted cells can sense their position in the branched network of cells and proliferate at the tips, thus recapitulating this feature of mammary epithelial branching morphogenesis. In all, our results demonstrate the capacity of the developed 3D bioprinting method for quantitative analysis of the relationships between tissue structure and cell behavior in breast morphogenesis and cancer. [ABSTRACT FROM AUTHOR]

The production of milk by dairy cows far exceeds the nutritional needs of the calf and is vital for the economical use of dairy cattle. High milk yield is a unique production trait that can be effectively enhanced through traditional selection methods. The process of lactation in cows serves as an excellent model for studying the biological aspects of lactation with the aim of exploring the mechanistic base of this complex trait at the cellular level. In this study, we analyzed the milk transcriptome at the single-cell level by conducting scRNA-seq analysis on milk samples from two Holstein Friesian cows at mid-lactation (75 and 93 days) using the 10× Chromium platform. Cells were pelleted and fat was removed from milk by centrifugation. The cell suspension from each cow was loaded on separate channels, resulting in the recovery of 9313 and 14,544 cells. Library samples were loaded onto two lanes of the NovaSeq 6000 (Illumina) instrument. After filtering at the cell and gene levels, a total of 7988 and 13,973 cells remained, respectively. We were able to reconstruct different cell types (milk-producing cells, progenitor cells, macrophages, monocytes, dendritic cells, T cells, B cells, mast cells, and neutrophils) in bovine milk. Our findings provide a valuable resource for identifying regulatory elements associated with various functions of the mammary gland such as lactation, tissue renewal, native immunity, protein and fat synthesis, and hormonal response. [ABSTRACT FROM AUTHOR]

In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased β-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis. [ABSTRACT FROM AUTHOR]

During bovine mastitis, immune responses include the release of cytokines and the recruitment of leukocytes, resulting in profound structural and functional changes in the mammary gland. Our aims were to delineate systemic and local cytokine responses and to quantify histological changes in the mammary tissue of lactating cows after acute intramammary lipopolysaccharide (LPS) challenge. Ten multiparous dairy cows were paired to either treatment (TRT) or control (CON) groups. For TRT cows, one side of the udder was randomly assigned to receive treatment with LPS (50 µg in 10 mL of saline, TL) into both the front and rear quarters; the contralateral quarters received saline (10 mL). Udder-halves of CON cows were similarly assigned randomly to receive either saline (10 mL, CS) or no infusion (untreated). Temporal changes in the concentrations of 15 cytokines in the blood (0, 3, 6, 12, and 24 h relative to the LPS infusion) and in mammary tissue (0, 3, and 12 h) were determined, as were concomitant changes in mammary histology. The cytokines IL-6, IL-10, MCP-1, and MIP-1β showed a systemic response as their concentrations were significantly different in the plasma of TRT cows as compared with CON cows after LPS challenge. The cytokines IL-1α, IL-1β, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1β, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1β, and VEGF-A showed systemic changes in the nonchallenged mammary glands adjacent to LPS-infused glands. Endotoxin challenge evoked changes in the histology of mammary tissue that included a 5.2- and 7.2-fold increases in the number of neutrophils in alveolar lumens at 3 h and 12 h, respectively. In summary, LPS challenge induced specific local and systemic responses in cytokine induction and elicited neutrophil infiltration in bovine mammary tissue. [ABSTRACT FROM AUTHOR]

An adverse perinatal environment can increase long-term cancer risk, although the precise nature of associated perinatal triggers remain unknown. Sleep apnea is a common condition during pregnancy, characterized by recurrent cessations in breathing during sleep, and the potential consequences of sleep apnea during pregnancy as it relates to breast cancer risk in offspring have not been explored. To model sleep apnea, Sprague-Dawley dams were exposed during gestation to nightly intermittent hypoxia (GIH) or normoxia (GNx), and the mammary glands of female offspring were examined. GIH offspring demonstrated increased epithelial stem and progenitor cell populations, which are associated with diminished transforming growth factor beta (TGFβ) activity. Elevations in adipose tissue stem cells in the mammary gland were also identified in GIH offspring. In aging females, mammary tumors formed in GIH offspring. These tumors displayed a dramatic increase in stroma compared to tumors from GNx offspring, as well as distinct patterns of expression of stem cell-related pathways. Together, these results suggest that exposure to sleep apnea during pregnancy leads to lasting changes in the mammary glands of female offspring. Increased stem and progenitor cell populations as a result of GIH exposure could enhance long-term breast cancer risk, as well as alter the clinical behavior of resulting breast tumors. [ABSTRACT FROM AUTHOR]

MiRNAs play crucial roles in a broad spectrum of biological processes, both physiological and pathological. Different reports implicate miR-205 in the control of breast stem cell properties. Differential miR-205 expression has been observed in different stages of mammary gland development and maturation. However, a functional role in this process has not been clearly demonstrated. We generated an miR-205 knockout in the FVB/N mouse strain, which is viable and characterized by enhanced mammary gland development. Indeed, mammary glands of miR-205−/− female mice at different ages (1.5 and 5.5 months) show increased outgrowth and branching. This evidence is consistent with our previously reported data demonstrating the direct miR-205-mediated targeting of HER3, a master regulator of mammary gland development, and the oncosuppressive activity of this microRNA in different types of breast cancer. [ABSTRACT FROM AUTHOR]

The mammary glands are dynamic tissues affected by pregnancy-related hormones during the pregnancy-lactation cycle. Collagen production and its dynamics are essential to the remodeling of the mammary glands. Alterations of the mammary microenvironment and stromal cells during the pregnancy-lactation cycle are important for understanding the physiology of the mammary glands and the development of breast tumors. In this study, we performed an evaluation of collagen dynamics in the mammary fat pad during the pregnancy-lactation cycle. Reanalysis of single-cell RNA-sequencing (scRNA-Seq) data showed the ectopic collagen expression in the immune cells and cell-cell interactions for collagens with single-cell resolution. The scRNA-Seq data showed that type I and type III collagen were produced not only by stromal fibroblasts but also by lymphoid and myeloid cell types in the pregnancy phase. Furthermore, the total cell-cell interaction score for collagen interactions was dramatically increased in the pregnancy tissue. The data presented in this study provide evidence that immune cells contribute, at least in part, to mammary collagen dynamics. Our findings suggest that immune cells, including lymphoid and myeloid cells, might be supportive members of the extracellular matrix orchestration in the pregnancy-lactation cycle of the mammary glands. NEW & NOTEWORTHY Our study evaluated mammary gland collagen dynamics during the pregnancy-lactation cycle using single-cell RNA-sequencing data. We found ectopic collagen expression in immune cells and an increase in collagen interactions during pregnancy. Type I and type III collagen were produced by lymphoid, myeloid, and stromal fibroblast cells during pregnancy. These findings suggest that immune cells, including lymphoid and myeloid cells, play a crucial role in supporting the extracellular matrix in mammary glands during pregnancy-lactation cycles. [ABSTRACT FROM AUTHOR]

Introduction: Macrophages are innate immune cells that are associated with extensive phenotypic and functional plasticity and contribute to normal development, tissue homeostasis, and diseases such as cancer. In this review, we discuss the heterogeneity of tissue resident macrophages in the normal mammary gland and tumor‐associated macrophages in breast cancer. Tissue resident macrophages are required for mammary gland development, where they have been implicated in promoting extracellular matrix remodeling, apoptotic clearance, and cellular crosstalk. In the context of cancer, tumor‐associated macrophages are key drivers of growth and metastasis via their ability to promote matrix remodeling, angiogenesis, lymphangiogenesis, and immunosuppression. Method: We identified and summarized studies in Pubmed that describe the phenotypic and functional heterogeneity of macrophages and the implications of targeting individual subsets, specifically in the context of mammary gland development and breast cancer. We also identified and summarized recent studies using single‐cell RNA sequencing to identify and describe macrophage subsets in human breast cancer samples. Results: Advances in single‐cell RNA sequencing technologies have yielded nuances in macrophage heterogeneity, with numerous macrophage subsets identified in both the normal mammary gland and breast cancer tissue. Macrophage subsets contribute to mammary gland development and breast cancer progression in differing ways, and emerging studies highlight a role for spatial localization in modulating their phenotype and function. Conclusion: Understanding macrophage heterogeneity and the unique functions of each subset in both normal mammary gland development and breast cancer progression may lead to more promising targets for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

We now live in an era where metabolic data are increasingly accessible and available. Analysis of this data can be done using novel techniques (e.g., machine learning and artificial intelligence) but this does not alleviate scientists to use “human intelligence”. The objective of this paper is to combine the information of a large database of biochemical reactions with a method and tool to make nutritional biochemistry more accessible to nutritionists. A script was developed to extract information from a database with more than 16 000 biochemical reactions so that it can be used for “biochemical bookkeeping”. A system of more than 300 meta-reactions (i.e., the outcome reaction of a series of connected individual reactions) was constructed covering a wide range of metabolic pathways for macro- and micronutrients. Meta-reactions were constructed by identifying metabolic nodes, which are inputs or outputs of a metabolic system or that serve as connection points between meta-reactions. Complete metabolic pathways can be constructed by combining and balancing the meta-reactions using a simple Excel tool. To illustrate the use of meta-reactions and the tool in the teaching of nutritional biochemistry, examples are given to illustrate how much ATP can be synthesized from glucose, either directly or indirectly (i.e., via storage and mobilization or via transfer of intermediate metabolites between tissues and generations). To illustrate how meta-reactions and the tool can be used in research, nutrient balance data of the mammary gland of a dairy cow were used to construct a plausible pathway of nutrient metabolism of the whole mammary gland. The balance data included 34 metabolites taken up or exported by the mammary gland and 39 meta-reactions were used to construct a metabolic pathway that accounted for the uptake and output of metabolites. The results highlighted the importance of the synthesis of proline from arginine and the concomitant synthesis of urea by the mammary gland. It also raised the question of whether the availability of metabolic pathways or glucose uptake would be the more limiting factor for the synthesis of NADPH required for fatty acid synthesis. The availability of an open database with biochemical reactions, the concept of meta-reactions, and the provision of a tool allow users to construct metabolic pathways, which helps acquiring a more comprehensive and integrated view of metabolism and may raise issues that may be difficult to identify otherwise.

Objective: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. Methods: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active Ucp1 expression in adult mice. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. Results: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. However, Ucp1-CreERT2 showed no or only partial activation in these tissues of adult mice, indicating the potential for low or transient expression of endogenous Ucp1. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. Conclusions: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

Abstract Background Obesity is a risk factor for breast cancer, and women with obesity that develop breast cancer have a worsened prognosis. Within the mammary gland, obesity causes chronic, macrophage-driven inflammation and adipose tissue fibrosis. Weight loss is a recommended intervention to resolve obesity, but the impact of weight loss on the mammary gland microenvironment and in tumors has not been well identified. Methods To examine the effects of weight loss following obesity, mice were fed a high-fat diet for 16 weeks to induce obesity, then switched to a low-fat diet for 6 weeks. We examined changes in immune cells, including fibrocytes, which are myeloid lineage cells that have attributes of both macrophages and myofibroblasts, and collagen deposition within the mammary glands of non-tumor-bearing mice and within the tumors of mice that were transplanted with estrogen receptor alpha positive TC2 tumor cells. Results In formerly obese mice, we observed reduced numbers of crown-like structures and fibrocytes in mammary glands, while collagen deposition was not resolved with weight loss. Following transplant of TC2 tumor cells into the mammary glands of lean, obese, and formerly obese mice, diminished collagen deposition and cancer-associated fibroblasts were observed in tumors from formerly obese mice compared to obese mice. Within tumors of obese mice, increased myeloid-derived suppressor cells and diminished CD8+ T cells were identified, while the microenvironment of tumors of formerly obese mice were more similar to tumors from lean mice. When TC2 tumor cells were mixed with CD11b+CD34+ myeloid progenitor cells, which are the cells of origin for fibrocytes, and transplanted into mammary glands of lean and obese mice, collagen deposition within the tumors of both lean and obese was significantly greater than when tumor cells were mixed with CD11b+CD34− monocytes or total CD45+ immune cells. Conclusions Overall, these studies demonstrate that weight loss resolved some of the microenvironmental conditions within the mammary gland that may contribute to tumor progression. Additionally, fibrocytes may contribute to early collagen deposition in mammary tumors of obese mice leading to the growth of desmoplastic tumors.

The mammary glands are the main site of milk synthesis and secretion, and its conditions such as mastitis are the common cause of decreased milk production. For this reason, knowledge of the anatomy of the mammary gland in its various stages is esirable, in order to understand the necessary elements of the physiology, pathology and management of various mammary diseases. In this bibliographic report, the main elements of anatomy (internal and external structure), morphology, histology and cytology of the mammary gland are presented, as well as the cellular elements of milk synthesis and secretion.

Bovine mastitis, a bout of inflammation of the mammary gland in high-yield dairy cows, frequently brings on by bacterial infections. One of the biggest illnesses that harm the dairy sector financially is mastitis, which lowers milk production and quality. Taking into consideration the impact of mastitis on milk production and dairy industry, research studies are already underway, but till now no concrete solution is there. It is assuming that molecular level understanding behind the mastitis can boost the process to control the mastitis at some extent. Thus, the goal of this scientific study was to identify immunogenic genes and their functional characterization. Along with that study also attempts to explore the possibilities for the use of immunogenic proteins as therapeutic targets to control mastitis. For the purpose cDNA microarray data of bovine mammary epithelial cells after the in vitro stimulation with Escherichia coli was considered. This investigation proceeds to study immunological responses on mammary epithelial (BME) cells. Using network based approach the investigation pinpointed 25 essential key genes for E. coli mastitis and their contribution related to the immune response. Through molecular docking, study also reported 33 herbal compounds with considerable affinity against toll-like receptor (TLR) and bovine granulocyte-colony stimulating factor proteins which are critical for mastitis.

The assessment of the prevalence of Subclinical Mastitis (SCM) in dairy farms is essential to evaluate the health status of mammary gland. The aim of the study was to assess the prevalence of SCM in dual-purpose livestock systems in Arauca, Colombia, through the analysis the values found on-farm diagnostic California Mastitis Test (CMT) and Electric Conductivity (EC) test. Milk samples were taken from the individual mammary quarters of 481 cows. The cow level prevalence, per affected individual mammary quarters prevalence and the mammary quarter level prevalence were determined according to the values obtained for each test using two methods of analysis. An ANOVA was performed to determine the difference between prevalences. Cohen’s kappa coefficient was used for intertest agreement, and an analysis of sensitivity and specificity. The cow level prevalence was similar between the tests (CMT = 31.4%; EC = 29.7%). The quarter-level prevalence was lower with EC (11.4% vs 14.3%) (p