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Although mammary gland growth and development in females is a lifelong process, it builds on isometric and allometric phases of mammary growth to establish a complex ductal network before and during puberty. Only then can other phases of branching and alveologenesis, differentiation, lactation, and involution proceed. Although the ductal network of various species differs in its histomorphology, all glands undergo a common phase of allometric growth when the mammary ducts penetrate into the supporting stromal microenvironment. Perhaps not surprisingly, different aspects of diet and nutrition can influence this allometric growth, either directly or indirectly. In this review, we outline some of the fundamental aspects of how allometric ductal growth in the mammary glands of various species is influenced by diet and nutrition and identify opportunities and questions for future investigation.

Work from numerous fields of study suggests that exposures to hormonally active chemicals during sensitive windows of development can alter mammary gland development, function, and disease risk. Stronger links between many environmental pollutants and disruptions to breast health continue to be documented in human populations, and there remain concerns that the methods utilized to identify, characterize, and prioritize these chemicals for risk assessment and risk management purposes are insufficient. There are also concerns that effects on the mammary gland have been largely ignored by regulatory agencies. Here, we provide technical guidance that is intended to enhance collection and evaluation of the mammary gland in mice and rats. We review several features of studies that should be controlled to properly evaluate the mammary gland, and then describe methods to appropriately collect the mammary gland from rodents. Furthermore, we discuss methods for preparing whole mounted mammary glands and numerous approaches that are available for the analysis of these samples. Finally, we conclude with several examples where analysis of the mammary gland revealed effects of environmental toxicants at low doses. Our work argues that the rodent mammary gland should be considered in chemical safety, hazard and risk assessments. It also suggests that improved measures of mammary gland outcomes, such as those we present in this review, should be included in the standardized methods evaluated by regulatory agencies such as the test guidelines used for identifying reproductive and developmental toxicants. • The rodent mammary gland is sensitive to effects of hormonally active agents. • Whole-mounts reveal effects of environmental pollutants on the mammary gland. • Numerous methods are available to quantify effects on rodent mammary glands. • Including the mammary gland would improve several test guidelines. [ABSTRACT FROM AUTHOR]

The mammary gland is responsive to endogenous hormones and environmental chemicals that are estrogen receptor (ER) agonists. The mouse mammary gland offers the opportunity to dissect the most sensitive windows of exposure. 17α-ethinyl estradiol (EE2) is a pharmaceutical ER agonist that often serves as a positive control for estrogen-active chemicals. Here, adult female mice were exposed to EE2 starting either at pregnancy day 7, or on lactational day 1, and exposures continued until the litters were weaned. The pups were therefore exposed during gestation + the juvenile period, or during the juvenile period alone. The morphology of the mammary gland was evaluated in both male and female offspring at two life stages: weaning (postnatal day [PND]21) and at puberty (PND32). Other hormone-sensitive outcomes evaluated included body weight, anogenital index, frequency of open vagina, and weight of the uterus. We found age- and sex-dependent effects of EE2 on these estrogen-responsive endpoints including the morphology of the mammary gland. Importantly, EE2 altered mammary gland morphology even when exposures were limited to the juvenile period. However, the number of endpoints that were affected in animals from the EE2-Juvenile-Only period were fewer, and typically of a lower magnitude, compared to those observed in the EE2-Gest-Juvenile group. Understanding the effects of environmental estrogen exposures during the juvenile period is critical because humans are exposed to estrogenic pollutants throughout life, including in early childhood., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Laura N. Vandenberg reports financial support was provided by SUDOC, LLC. Joshua P. Mogus reports financial support was provided by UMass Amherst School of Public Health & Health Sciences. Additional support was provided by the National Institutes of Health. Laura N. Vandenberg reports a relationship with Sudoc LLC that includes: consulting or advisory. LNV serves on the editorial board of Reproductive Toxicology. The other authors do not have conflicts to disclose., (Copyright © 2024. Published by Elsevier Inc.)

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

Mammary stroma is a prominent modulator of epithelial development, and a complex set of interactions between these tissue compartments is essential for normal development, which can be either permissive or restrictive in tumor initiation and progression. During perinatal development, exposures of mice to oxybenzone, a common UV filter, environmental pollutant and endocrine disruptor, induce alterations in mammary epithelium. Our prior research indicates that oxybenzone alters mammary epithelial structures at puberty and in adulthood. We had also previously observed changes in the expression of hormone receptors at puberty (e.g., oxybenzone induced a decrease in the number of epithelial cells positive for progesterone receptor) and in adulthood (e.g., oxybenzone induced a decrease in the number of estrogen receptor-positive epithelial cells), and increased body weight in adulthood. Here, we investigated mammary stromal changes in BALB/c animals exposed during gestation and perinatal development to 0, 30, or 3000 μg oxybenzone/kg/day. In mice exposed to 30 μg/kg/day, we observed morphological changes in adulthood (e.g., a thicker periductal stroma and adipocytes that were considerably larger). We also observed an increased number of mast cells in the mammary stroma at puberty which may represent a transient influence of oxybenzone exposure. These results provide additional evidence that even low doses of oxybenzone can disrupt hormone sensitive outcomes in the mammary gland when exposures occur during critical windows of development, and some of these effects manifest in later life.

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

Breast cancers display dynamic reprogrammed metabolic activities as cancers develop from premalignant lesions to primary tumors, and then metastasize. Numerous advances focus on how tumors develop pro-proliferative metabolic signaling that differs them from adjacent, non-transformed epithelial tissues. This leads to targetable oncogene-driven liabilities among breast cancer subtypes. Other advances demonstrate how microenvironments trigger stress-response at single-cell resolution. Microenvironmental heterogeneities give rise to cell regulatory states in cancer cell spheroids in three-dimensional cultures and at stratified terminal end buds during mammary gland morphogenesis, where stress and survival signaling juxtapose. The cell-state specificity in stress signaling networks recapture metabolic evolution during cancer progression. Understanding lineage-specific metabolic phenotypes in experimental models is useful for gaining a deeper understanding of subtype-selective breast cancer metabolism.

Highlights • The CD-1 male and female mouse mammary glands have consistent left-right asymmetry. • In males, the right mammary gland is more sensitive to estrogen than the left. • This bias in estrogen response occurs if exposures occur prenatally or peripubertally. • The asymmetric response is not due to a bias in expression of estrogen receptor. Abstract The CD-1 mouse mammary gland is sexually dimorphic, with males lacking nipples. Recent studies have revealed that the underlying epithelium in the male mammary gland is sensitive to estrogenic environmental chemicals. In ongoing investigations, we observed asymmetric morphology in the left and right male mouse mammary glands. Here, we quantified these asymmetries in the embryonic, prepubertal, pubertal and adult male mammary gland. We found that the right gland was typically larger with more branching points compared to the left gland. We next evaluated the response of the left and right glands to 17α-ethinyl estradiol (EE2) after perinatal or peripubertal exposures. We found that the right gland was more responsive to EE2 than the left at both periods of exposure. These results reveal novel aspects of male mammary gland biology and suggest that future studies should control for laterality in the evaluation of hazards associated with exposures to estrogenic chemicals. [ABSTRACT FROM AUTHOR]

The protein disulfide isomerase ERp57 (GRp58/PDIA3/1,25D3-MARRS) has been implicated in a multitude of signaling pathways throughout the entire body. Most thoroughly studied for its protein-folding role, ERp57 has also been found to have multiple binding partners, and have significant effects on cellular growth. ERp57 has been studied n the context of several neurodegenerative disorders, metabolic conditions, and can be used as a prognosis marker in certain cancers. One role, as an alternate vitamin D binding receptor, has prompted research in tissues with known vitamin D activity, such as the intestine and bone. Vitamin D has been studied in relation to mammary gland growth and development, but it is not yet known if ERp57 plays an independent role in this tissue. In this study, ERp57 was knocked out in murine mammary gland epithelial cells of 30 4-week old mice. Several markers of mammary gland growth were measured, including number of terminal end buds (TEB), ductal coverage of the fat pad, and ductal extension. It was found the knockout animals had decreased numbers of TEBs (p = 0.019), and decreased ductal extension (p = 0.018) compared to wildtype animals, with no differences in gross body weight. Immunohistochemistry analysis of mammary glands showed ERp57 localized to the apical side of alveolar branches, and on leading edges of TEBs. These results provide further evidence for ERp57 functioning separately to the VDR, and further insights into the roles of ERp57. [ABSTRACT FROM AUTHOR]

The mammary gland is one of the most regenerative organs in the body, with the majority of development occurring postnatally and in the adult mammal. Formation of the ductal tree is orchestrated by a specialized structure called the terminal end bud (TEB). The TEB is responsible for the production of mature cell types leading to the elongation of the subtending duct. The TEB is also the regulatory control point for basement membrane deposition, branching, angiogenesis, and pattern formation. While the hormonal control of TEB growth is well characterized, the local regulatory factors are less well understood. Recent studies of pubertal outgrowth and ductal elongation have yielded surprising details in regards to ongoing processes in the TEB. Here we summarize the current understanding of TEB biology, discuss areas of future study, and discuss the use of the TEB as a model for the study of breast cancer. [ABSTRACT FROM AUTHOR]

The inverted teat defect is the most common disorder of the mammary complex in pigs. It is characterized by the failure of teats to protrude from the udder surface, preventing normal milk flow and thus limiting the rearing capacity and increasing the risk of mastitis. The inverted teat defect is a liability trait with a complex mode of inheritance. We previously identified QTL for inverted teats. As a complementary approach that integrates map-based efforts to identify candidate genes for the inverted teat defect with function-driven expression analysis, application-specific microarrays were constructed that cover 1525 transcripts mapping in QTL regions on pig chromosomes 2, 3, 4, 6 and 11. About 950 transcripts were expressed in epithelial and mesenchymal teat tissue. The expression of three categories of teats was compared: normal teats of both non-affected and affected animals and inverted teats of affected animals. In epithelium and mesenchyme, 62 and 24 genes respectively were significantly differentially expressed ( DE). The majority of biofunctions to which a significant number of DE genes were assigned are related to the following: (1) cell maintenance, proliferation, differentiation and replacement; (2) organismal, organ and tissue development; or (3) genetic information and nucleic acid processing. Moreover, the DE genes belong almost exclusively to canonical pathways related to signaling rather than metabolic pathways. This is in line with findings obtained by genome-wide catalogue microarrays. This study adds another piece to the puzzle of the etiology of inverted teats by indicating that causal genetic variation leading to the disorder is likely among the genes encoding for members of the signaling cascades of growth factors. [ABSTRACT FROM AUTHOR]

Mammary stroma is a prominent modulator of epithelial development, and a complex set of interactions between these tissue compartments is essential for normal development, which can be either permissive or restrictive in tumor initiation and progression. During perinatal development, exposures of mice to oxybenzone, a common UV filter, environmental pollutant and endocrine disruptor, induce alterations in mammary epithelium. Our prior research indicates that oxybenzone alters mammary epithelial structures at puberty and in adulthood. We had also previously observed changes in the expression of hormone receptors at puberty (e.g., oxybenzone induced a decrease in the number of epithelial cells positive for progesterone receptor) and in adulthood (e.g., oxybenzone induced a decrease in the number of estrogen receptor-positive epithelial cells), and increased body weight in adulthood. Here, we investigated mammary stromal changes in BALB/c animals exposed during gestation and perinatal development to 0, 30, or 3000 μg oxybenzone/kg/day. In mice exposed to 30 μg/kg/day, we observed morphological changes in adulthood (e.g., a thicker periductal stroma and adipocytes that were considerably larger). We also observed an increased number of mast cells in the mammary stroma at puberty which may represent a transient influence of oxybenzone exposure. These results provide additional evidence that even low doses of oxybenzone can disrupt hormone sensitive outcomes in the mammary gland when exposures occur during critical windows of development, and some of these effects manifest in later life., Competing Interests: LV has received funding from the Cornell Douglas foundation and Paul G. Allen Foundation. She has been reimbursed for travel expenses by numerous organizations including SweTox, Israel Environment Fund, the Mexican Endocrine Society, Advancing Green Chemistry, ShiftCon, US EPA, CropLife America, BeautyCounter, and many universities, to speak about endocrine disrupting chemicals. LV is a paid scientific advisor to Sudoc LLC. KM, JB and SS have no conflicts to disclose., (Copyright © 2022 Matouskova, Bugos, Schneider and Vandenberg.)

Abstract: The stromal microenvironment regulates mammary gland branching morphogenesis. We have observed that mast cells are present in the mammary gland throughout its postnatal development and, in particular, are found around the terminal end buds and ductal epithelium of the pubertal gland. Mast cells contribute to allergy, inflammatory diseases, and cancer development but have not been implicated in normal development. Genetic and pharmacological disruption of mast cell function in the mammary gland revealed that mast cells are involved in rapid proliferation and normal duct branching during puberty, and this effect is independent of macrophage recruitment, which also regulates mammary gland development. For mast cells to exert their effects on normal morphogenesis required activation of their serine proteases and degranulation. Our observations reveal a novel role for mast cells during normal pubertal development in the mammary gland. [Copyright &y& Elsevier]

Findings in BRCA1 mutation carriers suggest that physical activity, particularly during childhood, may be linked to a reduced risk of developing breast cancer. We investigated whether physical activity at puberty alters the expression of BRCA1 and two other tumor suppressor genes—p53 and estrogen receptor (ER)-β—in rats. In addition, the effects on ER-α expression, mammary proliferation and functional epithelial differentiation were investigated as markers of altered mammary cancer risk in rats exposed to regular physical activity at puberty. Female Sprague Dawley rat pups were randomized to voluntary exercise, sham-exercise control and non-manipulated control groups. Treadmill training (20–25 m/min, 15% grade, 30 min/day, 5 days/week) started on postnatal day 14 and continued through day 32. Third thoracic mammary glands ( n = 5 per group and age) were obtained at days 32, 48 and 100 and assessed for changes in morphology through wholemounts, and at 100 days cell proliferation by using Ki67 staining, protein levels of ER-α and ER-β by immunohistochemistry, and mRNA expression levels of BRCA1, p53, ER-α and ER-β by real-time PCR. Mammary glands of rats exposed to exercise during puberty contained fewer terminal end buds (TEBs) and a higher number of differentiated alveolar buds and lobules than the sham controls. However, cell proliferation was not significantly altered among the groups. ER-α protein levels were significantly reduced, while ER-β levels were increased in the mammary ducts and lobular epithelial structures of 100-day old rays which were voluntarily exercised at puberty, compared to sham controls. ER-β, BRCA1 and p53 mRNA levels were significantly higher in the mammary glands of 100-day-old exercised versus sham control rats. Pubertal physical activity reduced mammary epithelial targets for neoplastic transformation through epithelial differentiation and it also up-regulated tumor suppressor genes BRCA1, p53 and ER-β, and reduced ER-α/ER-β ratio in the mammary gland. It remains to be determined whether the up-regulation of BRCA1, and perhaps p53, explains the protective effect of childhood physical activity against breast cancer in women who carry a germline mutation in one of the BRCA1 alleles. [ABSTRACT FROM AUTHOR]

Various doses of diethylstilbestrol (DES) were administered to rats once at birth. Thereafter, at 50 days after birth, the rats in all groups were given 10 mg 7, 12-dimethylbenz[a]anthracene (DMBA) and undergone necropsy at 300 days after birth. The incidence of mammary carcinomas (MCs) were 50, 54, 91, 39, 19% at 175 days after birth, and 77, 87, 100, 85, 75% at necropsy in the 0, 0.1, 1, 10, 100 μg groups, respectively. The incidence of rats without corpus luteum were 0, 0, 0, 30, 100% at 50 days after birth, and 0, 40, 53, 93, 100% at necropsy in the 0, 0.1, 1, 10, 100 μg groups, respectively. Observation of the whole mount specimens showed a higher number of terminal end buds (TEBs) in the 1 μ g group and a lower number in the 100 μg group compared with the control at 50 days after birth. It suggested that the administration of a relatively low dose (1 μg) of DES during neonatal period may increase TEBs, thus resulting in a stimulatory effect on the initiation of MCs. [ABSTRACT FROM AUTHOR]

Abstract: Postnatal development of the mammary gland is determined by reciprocal interactions between the ductal epithelia and adjacent stroma. Alx4 is a mesenchymally restricted homeodomain transcription factor expressed in a number of developing tissues, including skin appendages such as hair follicles, whiskers and teeth. We show here that Alx4 is expressed in a subset of ERα-expressing mammary stromal cells adjacent to terminal end buds and alveoli during puberty and pregnancy, respectively. Alx4 expression is induced in mammary stromal cells at the onset of puberty and can be induced in prepubescent mice by administration of 17β-estradiol. In order to determine the role of Alx4 during mammary gland development, we characterized mammary gland development of mice homozygous for the null allele of Alx4, lst D . Mammary glands from animals lacking Alx4 activity exhibit profound alterations in ductal morphogenesis. Overall development is delayed, ducts being grossly distorted in size and structure. Terminal end buds are also disoriented, displaying aberrant architecture during bifurcation. Despite the developmental delay, the ductal network typically reaches the limits of the fat pad. However, during puberty and in the adult virgin mice, the frequency and density of branch points is significantly reduced. We show further that the defective ductal morphogenesis is due to defects in stromal cells. Specifically, when injected into the cleared fat pad of wild-type recipients, mixed populations of wild-type epithelial cells and Alx4-deficient stromal cells give rise to retarded ductal morphogenesis. Wild-type stromal cells mixed with Alx4-deficient epithelial cells result in normal progression of ductal development. Defective branching morphogenesis in Alx4-deficient females is not due to a loss in expression of HGF, since the level of HGF message in mammary stromal cells is similar in mutant and wild-type littermates. MMP3 is similarly expressed while a 40% increase in MMP2 and a 50% decrease in MMP9 message levels in Alx4-deficient mice relative to their wild-type littermates is observed. Thus, the activity of the stromally restricted homeodomain factor, Alx4, is required for normal branching morphogenesis of the ductal epithelia during pubescent mammary gland development. [Copyright &y& Elsevier]

Epidemiological data have raised concerns about the relationship between exposure to power frequency magnetic fields (MFs) and breast cancer. We have shown previously that 50-Hz MFs at microtesla flux densities enhance mammary gland tumor development and growth in the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in female Sprague-Dawley (SD) rats. We also demonstrated that MF exposure results in an enhanced proliferative activity of the mammary epithelium of SD rats, which is a likely explanation for the cocarcinogenic or tumor-promoting effects of MF exposure in the DMBA model. Comparison of different SD substrains indicated that the genetic background plays a pivotal role in these effects of MF exposure. This prompted us to compare the effects of MF exposure (100 μT, 2 weeks) on cell proliferation in the mammary gland in eight different strains and substrains of outbred and inbred rats. Proliferation of epithelial cells in the mammary tissue and adjacent skin was examined by labeling proliferating cells with bromodeoxyuridine (BrdU). In addition to the MF-sensitive SD substrain (SD1) previously used in our experiments, Fischer 344 rats were the only strain in which MF exposure significantly enhanced BrdU labeling in the mammary epithelium, indicating a marked increase in cell proliferation. The MF-induced increase in BrdU labeling in Fischer 344 rats was similar to that seen after DMBA application. Furthermore, whole mount analysis of mammary tissue from Fischer 344 rats demonstrated that MF exposure increased the number of terminal end buds, i.e. the site of origin of mammary carcinomas. By comparison with MF-insensitive inbred rat strains, Fischer 344 rats may serve to evaluate the genetic factors underlying sensitivity to cocarcinogenic or tumor-promoting effects of MF exposure. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The ERBB2/HER2/NEU receptor tyrosine kinase gene is amplified in up to 30% of human breast cancers. The frequent and specific selection of this receptor kinase gene for amplification in breast cancer implies that it has important normal functions in the mammary gland. To investigate the functions of ErbB2 during normal mouse mammary gland development we transplanted mammary buds from genetically rescued ErbB2-/- embryos that express ErbB2 in the cardiac muscle. ErbB2-/- mammary buds transplanted to a wild-type mammary fat pad support outgrowth of an epithelial tree that advances only slowly through the mammary fat pad at puberty. This penetration defect is associated with structural defects in terminal end buds, characterized by a decrease in body cell number, an increased presence of cap-like cells in the prelumenal compartment, and the presence of large luminal spaces. Lobuloalveolar development was not affected in glands that developed from ErbB2-/- transplanted tissue. The results may have implications for the aggressive phenotypes associated with ERBB2-overexpressing mammary carcinomas. [ABSTRACT FROM AUTHOR]

There are suspected links between an animal’s diet, differentiation status of a target tissue, and sensitivity to chemically induced cancer. We have demonstrated that rats fed AIN93G diets made with soy protein isolate (SPI) or whey protein hydrolysate (WPH) had a lower incidence of 7,12-dimethylbenz(a)anthracene (DMBA)-induced adenocarcinoma than rats fed the same diet made with casein (CAS). The current study was conducted to determine the differentiation status of the mammary glands during development. Offspring of rats (n = 5–10/group) were fed diets made with SPI, WPH, or CAS throughout life (beginning on gestation day 4) and were sacrificed on postnatal day (PND) 21, PND 33, PND 50 or on metaestrous between PND 48 and PND 51. There were no significant differences between the numbers of mammary terminal end buds (TEBs) or lobuloalveoli (LOB) between any of the diets groups at PND 21 or PND 33, but at PND 50 there was an 75% decrease in the mean numbers of TEBs/mm2 in the SPI- or WPH-fed rats, compared with the CAS-fed rats (p = 0.09 and p = 0.06, respectively). In rats sacrificed in metaestrous, there were no significant differences in the proliferation index (PI) in the TEBs or LOB between any of the diet groups. In metaestrous rats, there were twice as many cells expressing estrogen receptor β (ERβ; ∼60%) compared with estrogen receptor α (ERα; ∼30%) in the LOB and 1.5 times more ERβ (∼60%) compared with estrogen receptor α (ERα, ∼40%) in the TEBs. There were no diet-dependent differences in expression of ERα and ERβ. Similarly, there were no differences between the diet groups in progesterone receptor (PR) expressing LOB cells. However, in the TEBs there was a diet-dependent difference in PR positive cells with a 34% increase (p < 0.05) in the SPI-fed rats and a 38% increase (p < 0.05) in the WPH-fed rats compared with the CAS-fed rats. These results show that the type of dietary protein alters the phenotype of mammary epithelia in the TEBs. The SPI- and WPH-dependent changes in mammary differentiation may contribute to the reduced sensitivity to DMBA-induced mammary cancer in rats fed these proteins. [ABSTRACT FROM PUBLISHER]

ABSTRACT Physical activity has been associated with decreased risk for developing breast cancer yet to date, the mechanism remains unknown. The purpose of this investigation was to evaluate the effects of moderate exercise training on the normal mammary gland in an attempt to identify alterations or differences that might be associated with tumour inhibition. A total of 170 female Sprague–Dawley rats were randomized to baseline (n =10), exercise (EX; n =80), or sham-exercise groups (SHAM; n =80). Treadmill training (20–25 m min–1 , 15% grade, 30 min day–1 , 5 days week–1 ) was started at 28 days of age (DOA). Animals were killed at 28, 42, 56, 70 and 84 DOA. Mammary glands were evaluated by histology and immunohistochemistry. Terminal end buds (TEB), structures susceptible to carcinogenesis, were counted. Sexual maturation, estradiol and progesterone, and organ and muscle weights were also evaluated. No differences in growth, sexual maturation, or steroid hormones were observed in response to training. No difference in the number of TEBs was observed at any timepoint between EX and SHAM. Proliferation was significantly increased at 56 DOA and tended to be increased at 42 and 70 DOA in the EX animals whereas cell death was significantly increased at 70 DOA and tended to be increased at 84 DOA in the EX animals. These data suggest no difference in the number of carcinogen-susceptible structures as a result of moderate exercise. The changes in cell proliferation and apoptosis with exercise training suggest altered cell turnover that will necessitate future study particularly with relevance to carcinogenesis. [ABSTRACT FROM AUTHOR]

Breast cancers display dynamic reprogrammed metabolic activities as cancers develop from premalignant lesions to primary tumors, and then metastasize. Numerous advances focus on how tumors develop pro-proliferative metabolic signaling that differs them from adjacent, non-transformed epithelial tissues. This leads to targetable oncogene-driven liabilities among breast cancer subtypes. Other advances demonstrate how microenvironments trigger stress-response at single-cell resolution. Microenvironmental heterogeneities give rise to cell regulatory states in cancer cell spheroids in three-dimensional cultures and at stratified terminal end buds during mammary gland morphogenesis, where stress and survival signaling juxtapose. The cell-state specificity in stress signaling networks recapture metabolic evolution during cancer progression. Understanding lineage-specific metabolic phenotypes in experimental models is useful for gaining a deeper understanding of subtype-selective breast cancer metabolism. [ABSTRACT FROM AUTHOR]

The first step in pubertal mammary development is the appearance of terminal end buds arising from pleuropotent stem cells present in the immature ductal tree of the prepubertal animal. Work from this laboratory indicates that growth hormone is the pituitary hormone responsible for terminal end bud development. Growth hormone likely acts through the production of IGF-1.3 This minireview focuses on the hormonal control of early mammary development with special emphasis on the roles of growth hormone and IGF-1. [ABSTRACT FROM AUTHOR]

The Cub Sushi Multiple Domains-1 (CSMD1) protein is a tumour suppressor which has been shown to play a role in regulating human mammary duct development in vitro. CSMD1 knockdown in vitro demonstrated increased cell proliferation, invasion and motility. However, the role of Csmd1 in vivo is poorly characterised when it comes to ductal development and is therefore an area which warrants further exploration. In this study a Csmd1 knockout (KO) mouse model was used to identify the role of Csmd1 in regulating mammary gland development during puberty. Changes in duct development and protein expression patterns were analysed by immunohistochemistry. This study identified increased ductal development during the early stages of puberty in the KO mice, characterised by increased ductal area and terminal end bud number at 6 weeks. Furthermore, increased expression of various proteins (Stat1, Fak, Akt, Slug/Snail and Progesterone receptor) was shown at 4 weeks in the KO mice, followed by lower expression levels from 6 weeks in the KO mice compared to the wild type mice. This study identifies a novel role for Csmd1 in mammary gland development, with Csmd1 KO causing significantly more rapid mammary gland development, suggesting an earlier adult mammary gland formation. [ABSTRACT FROM AUTHOR]

• 1,25D3-MARRS responds to changes in vitamin D3 doses in the diet. • Mammary growth reduction due to a loss of MARRS is reversed with a high D3 dose. • No changes to number of TEBs observed when animals are on a D3 deficient diet. 1,25 dihydroxyvitamin D3 (1,25D3) is the most potent biologically active form of vitamin D3. Its actions on the mammary gland include cell growth inhibition and anti-cancer effects. This study's purpose was to explore the role of the 1,25D3-membrane associated rapid response steroid (MARRS) receptor in the mammary gland using a tissue-specific knockout mouse model and a vitamin D3 dietary intervention. Three genotype groups were created using the Cre/loxp system to knock-down (+/−) and knockout (−/−) the MARRS receptor in epithelial cells of mammary glands (MG). Abdominal MGs were collected from 6-week old female mice (n = 94) on diets of 10,000 IU/kg (excess), 1,000 IU/kg (sufficient) or 0 IU/kg (deficient) of D3. There was a significant interaction between genotype and diet regarding number of terminal end buds (TEBs) (p = 0.001) and ductal coverage of the fat pad (p = 0.03). MARRS -/- mice on the sufficient diet had significantly fewer TEBs (p = 0.001) compared to MARRS +/+ on the same diet, but the opposite effect was seen in mice on the excess diet. There were no effects of genotype on TEBs when animals were vitamin D3 deficient. These results suggest that there is an effect of MARRS on mammary gland development that is dependent on 25(OH)D status, specifically, altering the number of highly proliferative TEBs. Increased numbers of TEBs have been correlated with increased breast cancer risk later in life. Therefore the results of this study warrant further examination of 25(OH)D status and recommendations in adolescent humans to reduce dietary effects on future breast cancer risk. [ABSTRACT FROM AUTHOR]

Development of the mammary gland requires coordination of hormone signaling pathways including those mediated by estrogen, progesterone, androgen and prolactin receptors. These hormones play important roles at several distinct stages of life including embryonic/fetal development, puberty, pregnancy, lactation, and old age. This also makes the gland sensitive to perturbations from environmental agents including endocrine disrupting chemicals (EDCs). Although there is evidence from human populations of associations between EDCs and disruptions to breast development and lactation, these studies are often complicated by the timing of exposure assessments and the latency to develop breast diseases (e.g., years to decades). Rodents have been instrumental in providing insights-not only to the basic biology and endocrinology of the mammary gland, but to the effects of EDCs on this tissue at different stages of development. Studies, mostly but not exclusively, of estrogenic EDCs have shown that the mammary gland is a sensitive tissue, that exposures during perinatal development can produce abnormal mammary structures (e.g., alveolar buds, typically seen in pregnant females) in adulthood; that exposures during pregnancy can alter milk production; and that EDC exposures can enhance the response of the mammary tissue to hormones and chemical carcinogens. Other studies of persistent organic pollutants have shown that EDC exposures during critical windows of development can delay development of the gland, with lifelong consequences for the individual. Collectively, this work continues to support the conclusion that EDCs can harm the mammary gland, with effects that depend on the period of exposure and the period of evaluation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dietary phytoestrogens may prevent certain human diseases, but endocrine activity has been reported in animal studies. Sprague-Dawley rats were exposed perinatally to a 1-, 10- or 100-fold "high human dietary intake" mixture of 12 phytoestrogens consisting of mainly the lignan secoisolarici resinol and the isoflavones genistein and daidzein. This mixture induced persistent adverse effects, as adult male mammary glands showed hypertrophic growth. A reduced anogenital distance in newborn males indicated an anti-androgenic mode of action. Testosterone levels, testis and prostate weights, and expression of selected genes in testis and prostate were unaffected. Decreased serum estradiol was seen in genistein-exposed dams. This study indicated adverse effects at high intake levels in rats, but does not provide evidence for risk of phytoestrogen-mediated endocrine disruption at normal human dietary consumption levels. Further studies are warranted to increase the knowledge upon which risk assessment on dietary phytoestrogen exposure during pregnancy and infancy is based., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Biological determinants of breast density, a strong predictor of human breast cancer risk, are postulated to be influenced by prenatal exposures to mitogens. We investigated the extent to which prenatal exposures to insulin-like growth factor-1 (IGF-1) would affect body weight, breast density, and levels of breast stem/progenitor cells in the prepubescent offspring of wild type C57BL/6J and IGF-1 deficient mice. We found that administration of IGF-1 to pregnant mice resulted in significantly heavier birth and postnatal body weights of the offspring when compared to PBS controls. Morphometric analysis of whole mount carmine alum staining of the left fourth inguinal mammary gland revealed that a prenatal dose of 5 μg IGF-1 resulted in significantly longer ductal elongation in wild type mice and significantly higher breast density in both mouse strains. Furthermore, 5 μg IGF-1 also resulted in the highest number of putative CD49f(+)CD24(+) and CD49f(+)CD24(+)CD29(+) breast stem/progenitor cells in the wild type offspring when compared to PBS controls, as assessed by flow cytometric analysis of dissociated cells from the right fourth inguinal mammary gland, while significantly higher numbers of these cell populations as well as CD24(+)CD29(+) and CD49f(+)EpCAM(+) cells were observed in IGF-1 deficient mice. These findings provide direct evidence for a prenatal modulation of breast density in the offspring by IGF-1, possibly involving populations of breast stem/progenitor cells.