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Dairy cattle’s breeding is an important industry, since it is providing milk and meat to population. Modern cows are significantly different from their ancestors in biology, behavior and in productivity. The scientific approach to feeding, condition of content and formation of focused heredity contributed to increasing the cows’ productivity. During the mammary gland formation and sexual cycles manifestation, puberty occurs, which makes it possible to obtain offspring and secretion of milk for feeding calves. The modern cows are capable of producing milk for their calves and for feeding people. The appearance of record cows, giving about 100 kg per day, and during lactation more than 32 tons of milk serves as an example for achieving such indicators of production. There are negative effects in reproduction biology, which takes place in modern cattle breeding. In order to breed highly productive cows, it is important to increase their livestock by intensive selection. Modernity is such that fertility is reduced due to cows’ reproductive functions inhibition. Modern practice shows that it is possible to introduce methods of obtaining several calves from one cow. It is generally accepted for this purpose use new reproduction methods, for example transplantation of embryos with high genetic potential. This technique is widely used in biotechnology of cows breeding. We have tested the method of preparing and conducting embryo transfer to heifers from 13 to 16 months old in the Kuban Agro-Association. The article presents indicators of heifer. It was revealed that not all animals reacted positively to superstimulation. Embryo transplantation showed that 35.1% of embryos survived after embryo transfer. Economic calculations have shown that for the rapid formation of a highly productive herd on the farm, it is advisable to use Ayrshire breed heifers capable of raising embryos and giving birth to healthy calves as recipients.

Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation., Mizuki Yamamoto et al. show that TRAF6 is needed for mammary stem cell maintenance and pregnancy-induced epithelial cell expansion in cells and mice. They propose a model whereby TRAF6 promotes cell proliferation via PI3K/AKT signalling and inhibits apoptosis by activating canonical NF-κB signalling.

Background. The pathogenesis of diabetic mastopathy is unknown. It is assumed that one of the important factors in the development of this disease may be hyperglycemia. The aim of this study was to evaluate the clinical manifestations and echographic features of the mammary gland structure in women with diabetes mellitus type 1. Materials and methods. The three groups of women were examined: group 1: women with type 1 diabetes, with the onset of the disease before menarche (n = 58); group 2: women with type 1 diabetes, with the onset of the disease after menarche (n = 56); group 3: women with fibrocystic mastopathy, without diabetes mellitus type 1 (n = 79). Mastalgia was evaluated using the visual analogue scale. The ultrasound examination of mammary glands was performed on day 5 to 7 of the menstrual cycle. Results. The patients of all groups complained of mastalgia. Mammary gland ultrasound revealed that the thickness of the glandular tissue in patients of the first group was significantly less than in the second and third group. The diameter of the milk ducts was also significantly different in patients in each study group. In the group of patients with fibrocystic mastopathy without diabetes mellitus type 1, the tissue echogenicity is average in 41.8% of subjects, with it elevated in 58.2% of individuals. Moreover, in groups of patients with diabetes, 90% of subjects have increased echogenicity of breast tissue. Conclusion. Diabetic mastopathy is characterized by a fibrotic echo version of the organ parenchyma, which can be caused by a violation of the mammary gland formation and function in patients with diabetes mellitus type 1.

MRCKα is a ubiquitously expressed serine/threonine kinase involved in cell contraction and F-actin turnover, which is highly amplified in human breast cancer and part of a gene expression signature for bad prognosis. Nothing is known about the in vivo function of MRCKα. To explore MRCKα function in development and in breast cancer, we generated mice lacking a functional MRCKα gene. Mice were born close to the Mendelian ratio and showed no obvious phenotype including a normal mammary gland formation. Assessing breast cancer development using the transgenic MMTV-PyMT mouse model, loss of MRCKα did not affect tumor onset, tumor growth and metastasis formation. Deleting MRCKα and its related family member MRCKβ in two triple-negative breast cancer cell lines resulted in reduced invasion of MDA-MB-231 cells, but did not affect migration of 4T1 cells. Further genomic analysis of human breast cancers revealed that MRCKα is frequently co-amplified with the oncogenes ARID4B and AKT3 which might contribute to the prognostic value of MRCKα expression. Collectively, these data suggest that MRCKα might be a prognostic marker for breast cancer, but probably of limited functional importance.

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.

For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several in vitro assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for in vivo mammary gland formation assay, including preparation of "cleared" recipients and mammary cells for implantation, the surgery process and how to assess the experimental results. Combined with manipulation of mammary cells via gene editing and /or drug treatment, this protocol could be very useful in the researches of mammary stem cells and mammary development.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a higher risk in younger women and a poorer prognosis and without targeted therapies available currently. Cancer stem cells (CSCs) are increasingly recognized as the main cause of treatment failure and tumor recurrence. The present paper reports the encapsulation of lovastatin (LV) into cerasomes. Compared with free LV, cerasome-encapsulated LV (C-LV) nanohybrids showed cytotoxicity to MDA-MB-231 CSCs in a dose- and time-dependent manner. Furthermore, intravenous injection of C-LV nanohybrids resulted in a significant tumor size reduction in a dose-dependent manner in xenograft tumors derived from subcutaneous inoculation of MDA-MB-231 cells. Furthermore, histopathological and/or immunohistochemical analysis revealed that C-LV nanohybrids significantly induced mammary gland formation and apoptosis and inhibited angiogenesis, the CSC phenotype, and the epithelial-to-mesenchymal transition in xenograft tumors. Most importantly, C-LV nanohybrids were found to be more effective than free LV in inhibiting the growth of breast cancer xenografts and the stemness properties in vivo. To the best of our knowledge, ours is the first demonstration of nanohybrids for efficient inhibition of CSCs derived from TNBC, offering a new option for the TNBC treatment.

The size, support, and health of udders limit the productive life of beef cows, especially those with background, because, in general, such cows have a reputation for problems with udders. Genomic association studies of bovine udder traits have been conducted in dairy cattle and recently in Continental European beef breeds but not in cows with background. The objective of this study was to determine associations of SNP and udder support scores, teat length, and teat diameter in half (Nellore), half (Angus) cows. Udders of cows ( = 295) born from 2003 to 2007 were evaluated for udder support and teat length and diameter ( = 1,746 records) from 2005 through 2014. These included a subjective score representing udder support (values of 1 indicated poorly supported, pendulous udders and values of 9 indicated very well-supported udders) and lengths and diameters of individual teats in the 4 udder quarters as well as the average. Cows were in full-sibling or half-sibling families. Residuals for each trait were produced from repeated records models with cow age category nested within birth year of cows. Those residuals were averaged to become the dependent variables for genomewide association analyses. Regression analyses of those dependent variables included genotypic values as explanatory variables for 34,980 SNP from a commercially available array and included the genomic relationship matrix. Fifteen SNP loci on BTA 5 were associated (false discovery rate controlled at 0.05) with udder support score. One of those was also detected as associated with average teat diameter. Three of those 15 SNP were located within genes, including one each in (), (), and (). These are notable for their functional role in some aspect of mammary gland formation or health. Other candidate genes for these traits in the vicinity of the SNP loci include () and (). Because these were detected in Nellore-Angus crossbred cows, which typically have very well-formed udders with excellent support across their productive lives, similar efforts in other breeds should be completed, because that may facilitate further refinement of genomic regions responsible for variation in udder traits important in multiple breeds.

MicroRNAs are differentially expressed in breast cancer cells and have been implicated in cancer formation, tumour invasion and metastasis. We investigated the miRNA expression profiles in the developing mammary gland. MiR-137 was expressed prominently in the developing mammary gland. When the miR-137 was over-expressed in the embryo, the mammary epithelium became thickened. Moreover, genes associated with mammary gland formation such as Tbx3 and Lef1 were not expressed. This suggests that miR-137 induces gland formation and invasion. When miR-137 was over-expressed in MDA-MB-231 cells, their ability to form tumours in adult mice was significantly reduced. These data support miR-137 decides epithelial cell behavior in the human breast cancer. It also suggests that miR-137 is a potential therapeutic target for amelioration of breast cancer progression.

The heritable component of breast cancer accounts for only a small proportion of total incidences. Environmental and lifestyle factors are therefore considered to among the major influencing components increasing breast cancer risk. Endocrine-disrupting chemicals (EDCs) are ubiquitous in the environment. The estrogenic property of EDCs has thus shown many associations between ongoing exposures and the development of endocrine-related diseases, including breast cancer. The environment consists of a heterogenous population of EDCs and despite many identified modes of action, including that of altering the epigenome, drawing definitive correlations regarding breast cancer has been a point of much discussion. In this review, we describe in detail well-characterized EDCs and their actions in the environment, their ability to disrupt mammary gland formation in animal and human experimental models and their associations with exposure and breast cancer risk. We also highlight the susceptibility of early-life exposure to each EDC to mediate epigenetic alterations, and where possible describe how these epigenome changes influence breast cancer risk.

At the beginning of the 21st century, developmental biologists together with medical researchers in a wide range of fields are witnessing rapid progress in molecular developmental biology. For example, conditional gene knockout systems are being designed to tackle questions about organogenesis and body plan formation in experimental mouse models and experimental designs include several compound mutant analyses and genome modification strategies. On the other hand, several fields remain relatively unexplored. Molecular mechanisms of sex differentiation are one of the unexplored huge area. Unanswered questions include the molecular genetic cascade of gonad formation, reproductive organ formation, uterus, external genitalia and mammary gland formation, and also the molecular mechanisms of signal transduction, and gene regulation by nuclear hormone receptors. This special thematic review series entitled, "Reproductive/urogenital organ development and molecular genetic cascades: glamorous developmental processes of bodies," covers such a wide range of topics. For this special issue, I have asked active researchers to contribute reviews of these topics which I believe will be useful not only for molecular developmental biologists, but also for researchers in biochemistry and cell biology. It will be my great pleasure if this special thematic issue encourages scientists to study this exciting research field.

Hepatocyte growth factor/scatter factor (HGF/SF), acting through the receptor tyrosine kinase Met, stimulates cells derived from a variety of different organs to form elongated hollow tubules when grown in three-dimensional gels. In vivo data also indicate a role for HGF/SF and Met in tubule formation during liver and kidney regeneration and mammary gland formation. Activation of Met results in the recruitment of a myriad of signal transducers that regulate dissociation of adherens junctions and the stimulation of cellular motility, survival, proliferation and morphogenesis during tubule formation. Among these many signal transducers, the Gab1 adaptor protein and its effector, the SHP2 tyrosine phosphatase, have been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAP kinase pathway. Here, we discuss the contribution of these and other signaling pathways and the role of HGF/SF and Met in the formation of epithelial cell tubules both in vitro in branching-morphogenesis assays and in vivo during organogenesis.

TNF and TNFR family proteins play important roles in the control of cell death, proliferation, autoimmunity, the function of immune cells, or the organogenesis of lymphoid organs. Recently, novel members of this large family have been identified that have critical functions in immunity and that couple lymphoid cells with other organ systems such as bone morphogenesis and mammary gland formation in pregnancy. The TNF-family molecule RANK-L (RANK-L, TRANCE, ODF) and its receptor RANK are key regulators of bone remodeling, and they are essential for the development and activation of osteoclasts. Intriguingly, RANK-L/RANK interactions also regulate T cell/dendritic cell communications, dendritic cell survival, and lymph node formation; T cell–derived RANK-L can mediate bone loss in arthritis and periodontal disease. Moreover, RANK-L and RANK are expressed in mammary gland epithelial cells, and they control the development of a lactating mammary gland during pregnancy and the propagation of mammalian species. Modulation of these systems provides us with a unique opportunity to design novel therapeutics to inhibit bone loss in arthritis, periodontal disease, and osteoporosis.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translational repression or RNA degradation. The aberrant expression of miRNAs might be involved in human diseases, including cancer. The expression of miR-206 in estrogen receptor alpha (ER-α)-positive human breast cancer tissues is well known. However, the expression and regulation of miR-206 in the developing mammary gland has not yet been studied. To understand the effects of miR-206 on mammary gland development, we have profiled gene expression in scramble-transfected and miR-206-overexpressing developing mammary buds. The genes that are potentially regulated by miR-206 in the mammary epithelium and/or mesenchyme, such as Tachykinin1 and Gata3, are known to be breast cancer markers. The expression of Wnt, which is involved in gland positioning, and of the transcription factors Tbx3 and Lef1, which are essential for mammary gland development, changes after miR-206 overexpression. Using a mammary bud in vitro culture system, we have demonstrated that miR-206 acts downstream of ER-α during mammary gland growth. Thus, miR-206 might be a novel candidate for morphogenesis during the initiation of mammary gland formation and the regulation of genes related to mammary gland development and breast cancer.

Copyright @ 2012 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and 85 reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel University Open Access Publishing Fund. Introduction: Estrogens regulate the proliferation of normal and neoplastic breast epithelium. Although the intracellular mechanisms of estrogens in the breast are largely understood, little is known about how they induce changes in the structure of the mammary epithelium, which are characteristic of breast cancer. In vitro three dimensional (3D) cultures of immortalised breast epithelial cells recapitulate features of the breast epithelium in vivo, including formation of growth arrested acini with hollow lumen and basement membrane. This model can also reproduce features of malignant transformation and breast cancer, such as increased cellular proliferation and filling of the lumen. However, a system where a connection between estrogen receptor (ER) activation and disruption of acini formation can be studied to elucidate the role of estrogens is still missing. Methods/Principal Findings: We describe an in vitro 3D model for breast glandular structure development, using breast epithelial MCF-12A cells cultured in a reconstituted basement membrane matrix. These cells are estrogen receptor (ER)α, ERβ and G-protein coupled estrogen receptor 1 (GPER) competent, allowing the investigation of the effects of estrogens on mammary gland formation and disruption. Under normal conditions, MCF-12A cells formed organised acini, with deposition of basement membrane and hollow lumen. However, treatment with 17β-estradiol, and the exogenous estrogens bisphenol A and propylparaben resulted in deformed acini and filling of the acinar lumen. When these chemicals were combined with ER and GPER inhibitors (ICI 182,780 and G-15, respectively), the deformed acini recovered normal features, such as a spheroid shape, proliferative arrest and luminal clearing, suggesting a role for the ER and GPER in the estrogenic disruption of acinar formation. Conclusion: This new model offers the opportunity to better understand the role of the ER and GPER in the morphogenesis of breast glandular structure as well as the events implicated in breast cancer initiation and progression. This study was partly funded by a School of Pharmacy Studentship.

A reduced complement of cardiomyocytes in early life can adversely affect life-long cardiac functional reserve. In the present study, using a cross-fostering approach in rats, we examined the contributions of the prenatal and postnatal environments in the programming of cardiomyocyte growth. Rat dams underwent either bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of gestation. One day after birth, Control and Restricted pups were cross-fostered onto Control (normal lactation) or Restricted (impaired lactation due to impaired mammary gland formation) mothers. In male offspring, genes involved in cardiomyocyte differentiation, proliferation, hypertrophy and apoptosis were examined at gestational day 20 and postnatal days 1 and 7 to assess effects on cardiomyocyte growth. At postnatal day 7 cardiomyocyte number was determined stereologically. Offspring were examined at age 6 mo for evidence of hypertension and pathological cardiac gene expression. There was an increase in Igf1 and Igf2 mRNA expression in hearts of Restricted pups at gestational day 20. At postnatal day 7, Agtr1a and Agtr1b mRNA expression as well as Bcl2 and Cmyc were elevated in all hearts from offspring that were prenatally or postnatally growth restricted. There was a significant reduction (−29%) in cardiomyocyte number in the Restricted-on-Restricted group. Importantly, this deficit was prevented by optimization of postnatal nutrition (in the Restricted-on-Control group). At 6 mo, blood pressure was significantly elevated in the Restricted-on-Restricted group, but there was no difference in expression of the cardiac hypertrophy, remodeling or angiogenic genes across groups. In conclusion, the findings reveal a critical developmental window, when cardiomyocytes are still proliferating, whereby improved neonatal nutrition has the capacity to restore cardiomyocyte number to normal levels. These findings are of particular relevance to the preterm infant who is born at a time when cardiomyocytes are immature and still dividing.

Neogenin, a close relative of the axon guidance receptor Deleted in Colorectal Cancer (DCC), has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule (RGM) families. While Netrin-1-Neogenin interactions result in a chemoattractive axon guidance response, the interaction between Neogenin and RGMa induces a chemorepulsive response. Evidence is now accumulating that Neogenin is a multi-functional receptor regulating many diverse developmental processes, including neural tube and mammary gland formation, myogenesis and angiogenesis. Little is known of the function of Neogenin in the adult, however, a novel role in the regulation of iron homeostasis is now emerging. While the signal transduction pathways activated by Neogenin are poorly understood, it is clear that the functional outcome of Neogenin activation, at least in the embryo, depends on both the developmental context as well as the nature of the ligand.

Regulatory CD4(+)CD25(+) T cells are important in suppressing immune responses. The requirements for the maintenance of peripheral CD4(+)CD25(+) T cells remain incompletely understood. Receptor activator of NF-kappaB (RANK) and its ligand (RANKL; also known as CD254, OPGL and TRANCE) are key regulators of bone remodeling, mammary gland formation, lymph node development and T-cell/dendritic cell communication. Here we report that RANKL is expressed in keratinocytes of the inflamed skin. RANKL overexpression in keratinocytes resulted in functional alterations of epidermal dendritic cells and systemic increases of regulatory CD4(+)CD25(+) T cells. Thus, epidermal RANKL expression can change dendritic cell functions to maintain the number of peripheral CD4(+)CD25(+) regulatory T cells. Epidermal RANKL mediated ultraviolet-induced immunosuppression and overexpression of epidermal RANKL suppressed allergic contact hypersensitivity responses and the development of systemic autoimmunity. Therefore, environmental stimuli at the skin can rewire the local and systemic immune system by means of RANKL.

The steroid hormones oestrogen and progesterone have a role in sickness and in health. In breast tissue, both roles probably work through a single mechanism: controlling the number and activity of mammary stem cells. The ovarian hormones oestrogen and progesterone are involved in a complicated series of interactions in the mammary glands from the beginning of puberty to the menopause. Many of these changes are associated with cell proliferation, and breast cancer can result when errors occur. Two studies in this issue examine the effects of oestrogen and progesterone on mouse mammary stem cell (MaSC) function. They find that MaSC numbers decrease in virgin mice in the absence of both hormones due to ovariectomy or drug blockade, but increase with oestrogen and progesterone treatment. In addition, both groups implicate RANKL, a progesterone target known to be involved in bone remodelling and mammary gland formation, as an intermediary in the MaSC response to progesterone.

Mammary glands are skin appendages whose development is initiated through continuous cell-cell interactions between ectoderm and adjacent mesenchyme. Considerable progress has been made in uncovering the molecular basis of these interactions in mammary gland formation in mouse embryos, including the network of initiating signals comprising Fgfs and Wnts, Bmps involved in gland positioning and transcription factors, Tbx3 and Lef1, essential for mammary gland development. Here we provide evidence that retinoic acid signaling may also be involved. Raldh2 is expressed in E10-E10.5 mouse embryos in ventral hypaxial extensions of somites adjacent to the flank region where mammary buds 2, 3 and 4 develop. This pattern of expression overlaps with that of Fgf10, known to be required for mammary gland formation. Manipulation of retinoic acid levels in organ cultures of E10.5 mouse embryo flank by adding either retinoic acid or citral, a retinoic acid synthesis inhibitor, resulted in changes in expression of genes involved in retinoic acid homeostasis and also showed that retinoic acid signaling is required for Tbx3 expression while high levels of retinoic acid signaling inhibit Bmp4 expression and repress Wnt signaling. The results of experiments using RNAi against Tbx3 and Wnt10b suggest that manipulating RA signaling in the mammary gland forming regions. Furthermore, RA affects expression of genes involved in mammary gland initiation. We produce a molecular model for mammary gland initiation incorporating retinoic acid signaling. Citation Format: Kyoung-Won Cho, Woo-Kyung Moon. Retinoic acid signaling and initiation of mammary gland development. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A121.

The extracellular matrix (ECM), once thought to solely provide physical support to a tissue, is a key component of a cell's microenvironment responsible for directing cell fate and maintaining tissue specificity. It stands to reason, then, that changes in the ECM itself or in how signals from the ECM are presented to or interpreted by cells can disrupt tissue organization; the latter is a necessary step for malignant progression. In this review, we elaborate on this concept using the mammary gland as an example. We describe how the ECM directs mammary gland formation and function, and discuss how a cell's inability to interpret these signals -- whether as a result of genetic insults or physicochemical alterations in the ECM -- disorganizes the gland and promotes malignancy. By restoring context and forcing cells to properly interpret these native signals, aberrant behavior can be quelled and organization re-established. Traditional imaging approaches have been a key complement to the standard biochemical, molecular, and cell biology approaches used in these studies. Utilizing imaging modalities with enhanced spatial resolution in live tissues may uncover additional means by which the ECM regulates tissue structure, on different length scales, through its pericellular organization (short-scale) and by biasing morphogenic and morphostatic gradients (long-scale).