Discoidin domain receptor 1 (DDR1) ablation promotes tissue fibrosis and hypoxia to induce aggressive basal-like breast cancers.

The discoidin domain receptor 1 (DDR1) is overexpressed in breast carcinoma cells. Low DDR1 expression is associated with worse relapse-free survival, reflecting its controversial role in cancer progression. We detected DDR1 on luminal cells but not on myoepithelial cells of DDR1+/+ mice. We found that DDR1 loss compromises cell adhesion, consistent with data that older DDR1-/- mammary glands had more basal/myoepithelial cells. Basal cells isolated from older mice exerted higher traction forces than the luminal cells, in agreement with increased mammary branches observed in older DDR1-/- mice and higher branching by their isolated organoids. When we crossed DDR1-/- mice with MMTV-PyMT mice, the PyMT/DDR1-/- mammary tumors grew faster and had increased epithelial tension and matricellular fibrosis with a more basal phenotype and increased lung metastases. DDR1 deletion induced basal differentiation of CD90+CD24+ cancer cells, and the increase in basal cells correlated with tumor cell mitoses. K14+ basal cells, including K8+K14+ cells, were increased adjacent to necrotic fields. These data suggest that the absence of DDR1 provides a growth and adhesion advantage that favors the expansion of basal cells, potentiates fibrosis, and enhances necrosis/hypoxia and basal differentiation of transformed cells to increase their aggression and metastatic potential.

The molecular basis of mammary gland development and epithelial differentiation.

Our understanding of the molecular events underpinning the development of mammalian organ systems has been increasing rapidly in recent years. With the advent of new and improved next-generation sequencing methods, we are now able to dig deeper than ever before into the genomic and epigenomic events that play critical roles in determining the fates of stem and progenitor cells during the development of an embryo into an adult. In this review, we detail and discuss the genes and pathways that are involved in mammary gland development, from embryogenesis, through maturation into an adult gland, to the role of pregnancy signals in directing the terminal maturation of the mammary gland into a milk producing organ that can nurture the offspring. We also provide an overview of the latest research in the single-cell genomics of mammary gland development, which may help us to understand the lineage commitment of mammary stem cells (MaSCs) into luminal or basal epithelial cells that constitute the mammary gland. Finally, we summarize the use of 3D organoid cultures as a model system to study the molecular events during mammary gland development. Our increased investigation of the molecular requirements for normal mammary gland development will advance the discovery of targets to predict breast cancer risk and the development of new breast cancer therapies.

Evaluation of early post-natal pig mammary gland development and human breast cancer gene expression.

Breast cancer is the second leading cause of death in women after lung cancer, and only 5% of patients with metastatic breast cancer survive beyond ten years of diagnosis. Considering the heterogeneous subclasses of breast cancer, current cancer models have shortfalls due to copy number variants, and genetic differences of humans and immunocompromised animal models. Preclinical studies indicate stem cell activity in early post-natal mammary development may be reactivated in the human adult as a trigger to initiate cell proliferation leading to breast cancer. The goal of the work reported herein was to compare genetic expression of early development, post-natal pig mammary glands to the literature reported genes implicated in different subclasses of human breast cancer. Differentially expressed genes associated with breast cancer and present in early developing pig samples include NUCB2, ANGPTL4 and ACE. Histological staining confirmed E-cadherin, Vimentin, N-cadherin, and Claudin-1, which are all implicated in malignant cancer. Due to the homology of gene expression patterns in the developing pig mammary gland and reported genes in human breast cancer profiles, this research is worthy of further study to address a potential model using mammary development cues to unravel breast cancer biology.

Induction of alternative NF-κB within TAg-induced basal mammary tumors in activation-resistant inhibitor of κ-B kinase (IKKα) mutant mice.

BACKGROUND: The alternative NF-κB pathway is activated by the NF-κB-inducing kinase (NIK) mediated phosphorylation of the inhibitor of κ-B kinase α (IKKα). IKKα then phosphorylates p100/NFKB2 to result in its processing to the active p52 subunit. Evidence suggests that basal breast cancers originate within a subpopulation of luminal progenitor cells which is expanded by signaling to IKKα. OBJECTIVE: To determine the role of IKKα in the development of basal tumors. METHODS: Kinase dead IkkαAA/AA mice were crossed with the C3(1)-TAg mouse model of basal mammary cancer. Tumor growth and tumor numbers in WT and IkkαAA/AA mice were assessed and immunopathology, p52 expression and stem/progenitor 3D colony forming assays were performed. Nik-/- mammary glands were isolated and mammary colonies were characterized. RESULTS: While tumor growth was slower than in WT mice, IkkαAA/AA tumor numbers and pathology were indistinguishable from WT tumors. Both WT and IkkαAA/AA tumors expressed p52 except those IkkαAA/AA tumors where NIK, IKKαAA/AA and ErbB2 were undetectable. Colonies formed by WT and IkkαAA/AA mammary cells were nearly all luminal/acinar however, colony numbers and sizes derived from IkkαAA/AA cells were reduced. In contrast to IkkαAA/AA mice, virgin Nik-/- mammary glands were poorly developed and colonies were primarily derived from undifferentiated bipotent progenitor cells. CONCLUSIONS: C3(1)-TAg induced mammary tumors express p100/p52 even without functional IKKα. Therefore the development of basal-like mammary cancer does not strictly rely on IKKα activation. Signal-induced stabilization of NIK may be sufficient to mediate processing of p100NFKB2 which can then support basal-like mammary tumor formation. Lastly, in contrast to the pregnancy specific role of IKKα in lobuloalveogenesis, NIK is obligatory for normal mammary gland development.

Mammary Development and Breast Cancer: a Notch Perspective.

Mammary gland development primarily occurs postnatally, and this unique process is complex and regulated by systemic hormones and local growth factors. The mammary gland is also a highly dynamic organ that undergoes profound changes at puberty and during the reproductive cycle. These changes are driven by mammary stem cells (MaSCs). Breast cancer is one of the most common causes of cancer-related death in women. Cancer stem cells (CSCs) play prominent roles in tumor initiation, drug resistance, tumor recurrence, and metastasis. The highly conserved Notch signaling pathway functions as a key regulator of the niche mediating mammary organogenesis and breast neoplasia. In this review, we discuss mechanisms by which Notch contributes to breast carcinoma pathology and suggest potentials for therapeutic targeting of Notch in breast cancer. In summary, we provide a comprehensive overview of Notch functions in regulating MaSCs, mammary development, and breast cancer.

The miR-200 family in normal mammary gland development.

The miR-200 family of microRNAs plays a significant role in inhibiting mammary tumor growth and progression, and its members are being investigated as therapeutic targets. Additionally, if future studies can prove that miR-200s prevent mammary tumor initiation, the microRNA family could also offer a preventative strategy. Before utilizing miR-200s in a therapeutic setting, understanding how they regulate normal mammary development is necessary. No studies investigating the role of miR-200s in embryonic ductal development could be found, and only two studies examined the impact of miR-200s on pubertal ductal morphogenesis. These studies showed that miR-200s are expressed at low levels in virgin mammary glands, and elevated expression of miR-200s have the potential to impair ductal morphogenesis. In contrast to virgin mammary glands, miR-200s are expressed at high levels in mammary glands during late pregnancy and lactation. miR-200s are also found in the milk of several mammalian species, including humans. However, the relevance of miR-200s in milk remains unclear. The increase in miR-200 expression in late pregnancy and lactation suggests a role for miR-200s in the development of alveoli and/or regulating milk production. Therefore, studies investigating the consequence of miR-200 overexpression or knockdown are needed to identify the function of miR-200s in alveolar development and lactation.

Effect of photoperiod before and during first gestation on milk production and prolactin concentration in dairy heifers.

Holstein heifers (n = 45) were subjected to treatments according to a 2 × 2 factorial design where the main effects were the photoperiod treatments during the second isometric (ISO, 52-61 wk of age) and the second allometric (ALLO, 62 wk of age to 8 wk before calving) periods of mammary gland development. During the ISO period, heifers were subjected to either a short-day photoperiod (SDP; 8 h light, 16 h dark; n = 22) or a long-day photoperiod (LDP; 16 h light, 8 h dark; n = 23). During the ALLO period, the photoperiodic treatments were either maintained (SDP:SDP, n = 11; LDP:LDP, n = 11) or switched (SDP:LDP, n = 11; LDP:SDP, n = 12). The treatments ended 8 wk before calving. All animals were then subjected to about 16 h of light per day. Serum prolactin (PRL) concentration during the ISO period was greater in heifers exposed to LDP than in those exposed to SDP. For the first 20 wk of the ALLO period, heifers exposed to LDP had greater serum concentration of PRL than those exposed to SDP. On the other hand, previous exposure to LDP during the ISO period reduced the concentration of PRL compared with those exposed to SDP during that period. During the second 20 wk of the ALLO period, PRL concentration remained greater in the serum of heifers then exposed to LDP than SDP, but serum PRL was greater in heifers exposed to LDP during the ISO period. During the last weeks before calving, when all animals were exposed to LDP, previous exposure to LDP during the ALLO period reduced serum PRL. Early-lactation milk (wk 1-5) and energy-corrected milk (wk 2-6) production were higher in the heifers exposed to SDP than in those exposed to LDP during ALLO. Photoperiod had no effect on milk production after that period. In conclusion, the results do not support to the hypothesis that photoperiod affects mammary gland development during the second allometric phase. However, they confirm that a short-day photoperiod in late gestation enhances milk production in the following lactation in primiparous heifers. Using serum PRL as an indicator of the photoperiodic response, we can conclude that responsiveness to the photoperiodic signal is still conditioned by a previous photoperiod several months after it ends.

Chronic prepartum light-dark phase shifts in cattle disrupt circadian clocks, decrease insulin sensitivity and mammary development, and are associated with lower milk yield through 60 days postpartum.

Circadian and metabolic systems are interlocked and reciprocally regulated. To determine if the circadian system regulates glucose homeostasis and mammary development, the function of the circadian system was disrupted by exposing cattle to chronic light-dark cycle phase shifts from 5 wk before expected calving (BEC) to parturition. Multiparous Holstein cows were exposed to 16 h of light and 8 h of dark (CON, n = 8) or phase shifting (PS, n = 8) the light cycle 6 h every 3 d beginning 35 d BEC. After calving, both treatments were exposed to CON lighting. Mammary biopsies were taken at 21 d BEC and 21 d in milk (DIM), and histological analysis indicated PS treatment decreased the ratio of lumen to alveolar area and percentage of proliferating epithelial cells in the prepartum period. Intravenous glucose tolerance test was performed at 14 d BEC and 7 DIM by administering 50% dextrose. Blood glucose, ß-hydroxybutyrate, insulin, and nonesterified fatty acids were consequently measured over 3 h. At 14 d BEC no treatment differences were observed in baseline glucose or insulin. Treatment had no effect on blood glucose or glucose area under the curve at 14 d BEC and 7 DIM. Insulin area under the curve was higher in PS versus CON at 14 d BEC and 7 DIM. The PS cows produced less milk than CON cows through 60 DIM (40.3 vs. 42.6 kg/d). Exposure to chronic light-dark PS in late gestation decreased mammary development and increased insulin resistance in periparturient cows, which may have caused subsequent lower milk yield.

Effect of whole-milk allowance on liveweight gain and growth of parenchyma and fat pads in the mammary glands of dairy heifers at weaning.

The rates of development of 2 tissues in mammary glands, parenchyma (PAR) and the mammary fat pad (MFP), in response to nutrition in early life might have a major bearing on lifetime milk production. Historical studies reported that feeding greater amounts of dietary nutrients from postweaning to puberty increased growth rates of heifers and stimulated the growth of MFP at the expense of PAR, which might suggest compromised mammary development and future milk production. The current study sought to determine if a higher volume of whole milk (8 vs. 4 L/d) offered to calves would increase rates of growth and development of PAR in mammary glands at weaning (1 to 12 wk). To measure these tissues, we developed 2 simple methods to assess the size of PAR and MFP at the time of screening using ultrasound. We report that calves offered 8 L/d of whole milk had greater rates of growth until weaning (0.86 ± 0.06 vs. 0.81 ± 0.09 kg/d), compared with calves offered 4 L/d. Ultrasonography showed that despite the faster rates of growth in calves offered 8 L/d of milk/d, the ratio of PAR:MFP depth was 40% less at weaning in the front glands (34%) compared with calves offered 4 L of milk/d. Rear glands were less impaired. The ultrasound methods developed here might be useful to monitor the development of mammary glands in response to different nutritional regimens during the preweaning period.

In Utero Exposure to Bisphenol a Promotes Mammary Tumor Risk in MMTV-Erbb2 Transgenic Mice Through the Induction of ER-erbB2 Crosstalk.

Bisphenol A (BPA) is the most common environmental endocrine disrupting chemical. Studies suggest a link between perinatal BPA exposure and increased breast cancer risk, but the underlying mechanisms remain unclear. This study aims to investigate the effects of in utero BPA exposure on mammary tumorigenesis in MMTV-erbB2 transgenic mice. Pregnant mice were subcutaneously injected with BPA (0, 50, 500 ng/kg and 250 µg/kg BW) daily between gestational days 11-19. Female offspring were examined for mammary tumorigenesis, puberty onset, mammary morphogenesis, and signaling in ER and erbB2 pathways. In utero exposure to low dose BPA (500 ng/kg) induced mammary tumorigenesis, earlier puberty onset, increased terminal end buds, and prolonged estrus phase, which was accompanied by proliferative mammary morphogenesis. CD24/49f-based FACS analysis showed that in utero exposure to 500 ng/kg BPA induced expansion of luminal and basal/myoepithelial cell subpopulations at PND 35. Molecular analysis of mammary tissues at PND 70 showed that in utero exposure to low doses of BPA induced upregulation of ERα, p-ERα, cyclin D1, and c-myc, concurrent activation of erbB2, EGFR, erbB-3, Erk1/2, and Akt, and upregulation of growth factors/ligands. Our results demonstrate that in utero exposure to low dose BPA promotes mammary tumorigenesis in MMTV-erbB2 mice through induction of ER-erbB2 crosstalk and mammary epithelial reprogramming, which advance our understanding of the mechanism associated with in utero exposure to BPA-induced breast cancer risk. The studies also support using MMTV-erbB2 mouse model for relevant studies.

Symposium review: Integration of postweaning nutrient requirements and supply with composition of growth and mammary development in modern dairy heifers.

To optimize first lactation and lifetime milk yield, growth benchmarks were established to help meet the appropriate growth objectives of breeding weight and age at an economically viable time and to achieve the optimum body size and composition at first calving. These guidelines provide a framework that helps to minimize overfeeding and, thus, potential overconditioning of heifers, which can lead to postpartum metabolic issues and reduced milk yield. Concerns still exist that mammary development is impaired when body weight gain exceeds a certain threshold, which would negatively affects milk yield. The objective of this review was to integrate concepts of nutrient requirements, body growth and composition, mammary development, and milk yield to provide a systems-based perspective on first-lactation milk differences that have been associated with mammary development. Work in the early 1980s described the effect of high energy intake on mammary development and the relationship with circulating growth hormone linked the relationship between prepubertal growth, mammary development, and future milk yield. The primary outcome of that research was to provide an intuitive mechanism to explain why rapid growth during the prepubertal phase resulted in reduced milk yield. The observation of reduced mammary development could be repeated in almost every experiment, leading to the conclusion that high energy intake and increased average daily gain reduced mammary development through altered hormone status or some signaling processes. However, further work that looked at mammary development over the entire prepubertal growth phase recognized that mammary development was not reduced by high energy intake, and instead accumulated at a constant rate; thus, overall mammary parenchymal growth was a function of the time to reach puberty and the associated signals to change from allometric mammary growth. The mammary gland, similar to most reproductive organs, grows in proportion to the size of the body and not in proportion to nutrient intake during the postweaning, prepubertal phase. First-lactation milk yield, mammary development, and body composition will be further discussed in the context of mechanisms and opportunities.

Epigenomics of mammary gland development.

Differentiation of stem cells into highly specialised cells requires gene expression changes brought about by remodelling of the chromatin architecture. During this lineage-commitment process, the majority of DNA needs to be packaged into inactive heterochromatin, allowing only a subset of regulatory elements to remain open and functionally required genes to be expressed. Epigenetic mechanisms such as DNA methylation, post-translational modifications to histone tails, and nucleosome positioning all potentially contribute to the changes in higher order chromatin structure during differentiation. The mammary gland is a particularly useful model to study these complex epigenetic processes since the majority of its development is postnatal, the gland is easily accessible, and development occurs in a highly reproducible manner. Inappropriate epigenetic remodelling can also drive tumourigenesis; thus, insights into epigenetic remodelling during mammary gland development advance our understanding of breast cancer aetiology. We review the current literature surrounding DNA methylation and histone modifications in the developing mammary gland and its implications for breast cancer.

A 100-Year Review: Mammary development and lactation.

What is old is new again-and with respect to the study of the mammary development and function in dairy animals, the expression resonates. Many of the mammary and milk production questions raised in the early years of the Journal of Dairy Science apply today. To be sure, scientists have filled in many details regarding, for example, identification of hormones and growth factors important in the control of mammary growth, the onset of copious milk production at calving, and maintenance of lactation. Early years focused on identification and subsequent availability of classic mammogenic, lactogenic, and galactopoietic hormones (e.g., steroids, prolactin, and growth hormone). The advent of sensitive assays to measure concentrations of these hormones and, subsequently, myriad growth factors in blood, milk, and tissues, allowed creation of multiple hypotheses to explain mammary cell proliferation and regulation of function. It is also apparent that we understand many of the fundamentals of milk removal, milking frequency, milking management, and milk ejection for successful lactation. However, some questions remain. Are the principles that were identified when cows produced markedly less milk still valid for the high-producing cows of today and the future? What mechanism(s) explain the positive effects of early increased milking frequency on subsequent milk production? Can the persistency of lactation be improved (secretory cell number vs. secretory cell function) or does early management "program" future mammary development or productivity (epigenetics, immune responsiveness, other)? The explosion of tools and techniques (Southern and Northern blots, PCR, and the "-omics" revolution) has driven an almost overwhelming evaluation of cellular and molecular functions in the mammary gland and other tissues. One key may be the discovery of a "Rosetta stone" that will allow understanding of this mass of detailed information on gene expression, cell signaling, and so on. Many scientists can now better appreciate the difficulty of the dairy farmer seeking to process DHIA or Dairy Comp 305 data, milking data, weights, feeding reports, pedometer readings, or genomic evaluations to manage their operations.

Stromal regulation of embryonic and postnatal mammary epithelial development and differentiation.

The stroma, which is composed of supporting cells and connective tissue, comprises a large component of the local microenvironment of many epithelial cell types, and influences several fundamental aspects of cell behaviour through both tissue interactions and niche regulation. The significance of the stroma in development and disease has been increasingly recognised. Whereas normal stroma is essential for various developmental processes during vertebrate organogenesis, it can be deregulated and become abnormal, which in turn can initiate or promote a disease process, including cancer. The mouse mammary gland has emerged in recent years as an excellent model system for understanding stromal function in both developmental and cancer biology. Here, we take a systematic approach and focus on the dynamic interactions that the stroma engages with the epithelium during mammary specification, cell differentiation, and branching morphogenesis of both the embryonic and postnatal development of the mammary gland. Similar stromal-epithelial interactions underlie the aetiology of breast cancer, making targeting the cancer stroma an increasingly important and promising therapeutic strategy to pursue for breast cancer treatment.

CLOCK regulates mammary epithelial cell growth and differentiation.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.

Consumption of endophyte-infected fescue seed during the dry period does not decrease milk production in the following lactation.

Ergot alkaloids in endophyte-infected grasses inhibit prolactin (PRL) secretion and may reduce milk production of cows consuming these grasses. We investigated the effects of consuming endophyte-infected fescue seed during late lactation and the dry period on mammary growth, differentiation, and milk production. Twenty-four multiparous Holstein cows were randomly assigned to 3 treatment groups. Starting at 90±4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (0.1mg/kg of body weight, positive control; BROMO), or endophyte-infected fescue seed (INF) as 10% of the diet on an as fed basis. Although milk yield of groups did not differ before treatment, at dry off (-60 d prepartum) INF and BROMO cows produced less milk than CON. Throughout the treatment period, basal concentrations of PRL and the prepartum increase in plasma PRL were reduced in INF and BROMO cows compared with CON cows. Three weeks after the end of treatment, circulating concentrations of PRL were equivalent across groups. In the subsequent lactation milk yield was not decreased; in fact, BROMO cows exhibited a 9% increase in milk yield relative to CON. Evaluation of mammary tissue during the dry period and the subsequent lactation, by quantitative histology and immunohistochemical analysis of proliferation markers and putative mammary stem or progenitor cell markers, indicated that feeding endophyte-infected fescue seed did not significantly affect mammary growth and development. Feeding endophyte-infected grasses during the dry period may permit effective utilization of feed resources without compromising milk production in the next lactation.

Localization and quantitation of macrophages, mast cells, and eosinophils in the developing bovine mammary gland.

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is highly regulated and in mice is known to be affected by the presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. For the first time, we determined the presence, location (relative to mammary ductal structures), and changes in numbers of eosinophils, mast cells, and macrophages in prepubertal bovine mammary tissue, and evaluated the effects of age, ovariectomy, and exogenous estrogen on numbers of each cell type. Chemical stains and immunofluorescence were used to identify the 3 cell types in formalin-fixed, paraffin-embedded mammary tissue from prepubertal female calves from 3 archived tissue sets. The ontogeny tissue set included samples of mammary tissue from female calves (n=4/wk) from birth to 6 wk of age. The ovary tissue set contained samples from ovary intact and ovariectomized heifers allowing us to investigate the influence of the ovaries on immune cells in the developing mammary gland in prepubertal heifers. Nineteen animals were intact or ovariectomized 30 d before sampling; they were 90, 120, or 150 d old at the time of sampling. A third tissue set, the estrogen set, allowed us to determine the effect of exogenous estrogen on innate immune cells in the gland. Eosinophils were identified via Luna staining, mast cells by May-Grunwald Giemsa staining, and macrophages with immunofluorescence. Key findings were that more eosinophils and mast cells were observed in near versus far stroma in the ontogeny and ovary tissue sets but not estrogen. More macrophages were observed in near versus far stroma in ontogeny animals. Eosinophils were more abundant in the younger animals, and fewer macrophages tended to be observed in ovariectomized heifers as compared with intact heifers and estrogen treatment resulted in a reduction in cell numbers. In summary, we show for the first time that innate immune cells are present in prepubertal bovine mammary tissue, localization varies by immune cell type, and abundance is related to proximity of epithelial structures and physiological state. We suggest a likely role for these cells in control of bovine mammary growth and ductal development.

Effect of prepartum photoperiod and melatonin feeding on milk production and prolactin concentration in dairy heifers and cows.

Holstein multiparous cows (n = 29) and primiparous heifers (n = 32) calving over a 1-yr period were subjected to photoperiod-melatonin treatments according to a 2 × 3 factorial design. Starting 8 wk before expected calving, all animals were subjected to 1 of the following treatments: 8h of light and 16 h of dark (8L:16D), 16 h of light and 8h of dark (16L:8D), or 16L:8D plus melatonin feeding (16L:8D-melatonin). Each day at 1355 h, the animals in the melatonin treatment received orally a gelatin capsule containing 25mg of melatonin. The treatments ended at calving, when the animals were moved to the lactation barn; all animals were then subjected to about 16 h of light per day. At the beginning and end of the treatment period before calving, blood samples were taken from 6 heifers and 6 cows through a jugular cannula for 24h at 30-min intervals to monitor serum melatonin and prolactin concentrations. Milk production in the heifers was not affected by the photoperiod treatments. Early-lactation milk production was higher in the cows exposed to the short-day photoperiod than in those exposed to a long-day photoperiod (16L:8D and 16L:8D-melatonin), with averages of 36.7 ± 0.9, 33.1 ± 0.8, and 34.1 ± 0.9 kg/d for 8L:16D, 16L:8D, and 16L:8D-melatonin, respectively. Photoperiod had no effect on late-lactation milk production in the cows. During lactation, the dry matter intake of heifers was not affected by the treatments, but dry matter intake of the cows exposed to a short-day photoperiod was greater than that of the cows exposed to a long-day photoperiod. Feed efficiency of heifers was improved by short-day photoperiod. During the treatment period, prolactin concentration was lower in the animals exposed to a short-day photoperiod than in those exposed to a long-day photoperiod, was lower with the 16L:8D-melatonin treatment than with the 16L:8D treatment, and tended to be lower with the 8L:16D treatment than with the 16L:8D-melatonin treatment, with averages of 3.5 ± 0.8, 9.9 ± 0.8, and 6.0 ± 0.8 ng/mL for 8L:16D, 16L:8D, and 16L:8D-melatonin, respectively. In early lactation, prolactin concentration was lower in the heifers exposed to the 16L:8D photoperiod during the dry period than in those exposed to the 8L:16D photoperiod or fed melatonin. In conclusion, a short-day photoperiod during the dry period transiently increases milk production of cows and the feed efficiency of heifers in the following lactation. However, melatonin cannot be used to mimic a short-day photoperiod during the dry period.

Parity affects mammary development in late-pregnant swine.

The goal of this project was to determine whether various measures of mammary development differed between gilts and multiparous sows at the end of gestation. During gestation, Yorkshire × Landrace gilts (n = 19) and sows (second and third gestations, n = 17) were fed one daily meal of a conventional corn-based diet, where the amount fed was based on body weight (BW) and backfat thickness (BF) at mating. On day 110 ±â€…1 of gestation, a jugular blood sample was obtained from all gilts and sows to measure insulin-like growth factor-1 (IGF-1), glucose, free fatty acids, and urea. On that same day, BW and BF were measured and animals were euthanized. Mammary glands from one side of the udder were dissected for compositional analyses. The fifth gland of the contralateral row of mammary glands was sampled for histology and immunohistochemical localization of Ki67. There was less total parenchyma (1,437.4 vs. 2,004.7 ±â€…127.1 g; P < 0.001) and total extraparenchymal tissue (1,691.0 vs. 2,407.0 ±â€…125.3 g; P < 0.001) in mammary glands of gilts compared to those from sows. When these values were expressed per kg BW (226.0 and 284.0 ±â€…2.7 kg for gilts and sows, respectively), parenchymal mass did not differ (P > 0.10), while extraparenchymal tissue weight tended to be less in gilts than sows (P = 0.07). All components within the parenchyma differed by parity (P < 0.001). Specifically, parenchymal tissue from gilts contained a greater proportion of fat and dry matter (DM), a lower proportion of protein, and lower concentrations of DNA (6.59 vs. 9.35 ±â€…0.53 mg/g DM) and RNA (7.76 vs. 12.33 ±â€…0.70 mg/g DM) than that from sows. On the other hand, the circumference of alveolar lumens was greater in gilts than sows (P < 0.001), while the percentage of epithelial cells that were positive for Ki67, a marker of cell proliferation, was greater in sows than gilts (P < 0.05). Circulating concentrations of IGF-1 were greater in gilts than in multiparous sows (45.0 vs. 27.3 ±â€…2.8 ng/mL, P < 0.001). None of the other blood variables were changed by parity. Results show a marked effect of parity on mammary gland development in swine. At the end of gestation, the mammary glands of gilts had less parenchyma with lower epithelial proliferation than glands from multiparous sows. These differences could alter the response of mammary tissue to various nutritional or endocrine signals. This information is crucial for the development of management strategies designed to maximize sow milk yield.

Sympathetic nerve signals: orchestrators of mammary development and stem cell vitality.

The mammary gland is a dynamic organ that undergoes significant changes at multiple stages of postnatal development. Although the roles of systemic hormones and microenvironmental cues in mammary homeostasis have been extensively studied, the influence of neural signals, particularly those from the sympathetic nervous system, remains poorly understood. Here, using a mouse mammary gland model, we delved into the regulatory role of sympathetic nervous signaling in the context of mammary stem cells and mammary development. Our findings revealed that depletion of sympathetic nerve signals results in defective mammary development during puberty, adulthood, and pregnancy, accompanied by a reduction in mammary stem cell number. Through in vitro three-dimensional culture and in vivo transplantation analyses, we demonstrated that the absence of sympathetic nerve signals hinders mammary stem cell self-renewal and regeneration, while activation of sympathetic nervous signaling promotes these capacities. Mechanistically, sympathetic nerve signals orchestrate mammary stem cell activity and mammary development through the ERK signaling pathway. Collectively, our study unveils the crucial roles of sympathetic nerve signals in sustaining mammary development and regulating mammary stem cell activity, offering a novel perspective on the involvement of the nervous system in modulating adult stem cell function and organ development.