Establishing a framework for the functional mammary gland: from endocrinology to morphology

From its embryonic origins, the mammary gland in females undergoes a course of ductal development that supports the establishment of alveolar structures during pregnancy prior to the onset of lactogenesis. This development includes multiple stages of proliferation and morphogenesis that are largely directed by concurrent alterations in key hormones and growth factors across various reproductive states. Ductal elongation is directed by estrogen, growth hormone, insulin-like growth factor-I, and epidermal growth factor, whereas ductal branching and alveolar budding is influenced by additional factors such as progesterone, prolactin, and thyroid hormone. The response by the ductal epithelium to various hormones and growth factors is influenced by epithelial–stromal interactions that differ between species, possibly directing species-specific morphogenesis. Evolving technologies continue to provide the opportunity to further delineate the regulation of ductal development. Defining the hormonal control of ductal development should facilitate a better understanding of the mechanisms underlying mammary gland tumorigenesis.     

Historical Perspectives of Prolactin and Growth Hormone as Mammogens,Lactogens and Galactagogues—Agog for the Future!

Around 80 years ago researchers first established that the pituitary gland regulates mammary gland function as demonstrated by the ability of its extracts to promote both mammogenesis and lactogenesis in animal models. Little did they realize that in fact two hormones, prolactin (PRL) and growth hormone (GH), were contributing to these effects. By the mid 1930s PRL had been purified as a distinct lactogen, while the galactopoietic effect of GH was confirmed after its purification in the 1940s. Interest in these hormones initially centered about their potential for increasing milk production, while in the latter half of the twentieth century it became obvious that these hormones also had the potential to influence mammary cancer development. During the past 50 years large strides have been made into understanding how these hormones signal to, and within, cells of the mammary gland, paralleling rapid developments in the fields of cellular and molecular biology. In compiling this review we have summarized the progress that has been made to date regarding roles for these hormones in the mammary gland, with a goal of ensuring that some of the seminal literature is not diluted or forgotten. In doing so it is clear that there are lessons to be learned from past experiences, where new methods and technologies will continue to present exciting new opportunities to revisit lingering questions regarding these fascinating hormones and this fascinating organ.     

Physiology of the pituitary, thyroid and adrenal glands

The pituitary gland is made of clusters of cells producing specific hormones that control growth (growth hormones, GH), thyroid function (TH), adrenal function (ACTH), gonadal function (FSH and LH). In addition, the neurons that join the posterior pituitary (neurohypophysis) secrete vasopressin - the antidiuretic hormone involved in maintaining water balance.The negative feedback loop is the basic mechanism to control the regulation of all endocrine glands. Hypothalamic peptides - releasing hormones (e.g. TRH, CRH) reach the hypophysis via the portal venous system and induce the secretion of specific stimulating hormones (e.g. TSH, ACTH) that drive the end-target endocrine cells to secrete hormones (e.g. thyroid hormones - T3 and T4 or adrenal hormones - cortisol, DHEAS). The plasma levels of these circulating hormones inhibit the pituitary (short feedback) or the hypothalamus (long feedback) and limit the further release of releasing- and stimulating- hormones.The effects of circulating hormones on different tissues are mediated via specific receptors on the cell membrane (e.g. vasopressin receptors), in the cytoplasm (steroid receptor for cortisol) or in the nucleus (e.g. thyroid hormone receptors). Understanding the physiological effects of peripheral hormones helps understanding the mechanisms by which clinical signs and symptoms developed in diseases characterised by excessive hormone secretion (e.g. thyrotoxicosis, Cushing syndrome, phaeochromocytomas) or lack of hormone secretion (e.g. diabetes insipidus).     

Influence of prenatal waterpipe tobacco smoke exposure on reproductive hormones and oxidative stress of adult male offspring rats

Male infertility is adversely affected by tobacco cigarette smoking. Herein, the effects of prenatal waterpipe tobacco smoke (WTS) exposure on reproductive hormones and oxidative stress of adult offspring rats were evaluated. Pregnant rats received either fresh air or mainstream WTS (2 hr daily). Pregnancy outcomes, circulatory levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin, testicular levels of oestrogen, testosterone and oxidative stress biomarkers [catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and thiobarbituric acid reactive substances (TBARS)] were assessed in their adult male offspring rats. Prenatal WTS exposure reduced the number of born offspring, female to pups ratio and birthweight (p < 0.05). Prenatal WTS exposure increased the circulatory levels of FSH and the testicular levels of oestrogen, testosterone and TBARS and catalase activity compared with control group (p < 0.05). However, GPx activity was reduced by WTS exposure (p < 0.05). There appeared to be a trend of increased LH and prolactin levels with prenatal WTS exposure; however, it was not statistically significant compared with control group (p > 0.05). The activity of SOD was not affected by prenatal WTS exposure (p > 0.05). In conclusion, prenatal WTS exposure altered reproductive hormones as well as oxidative stress biomarkers in adult male offspring rats.     

Animal models for the study of milk secretion

Milk secretion is regulated by a complex interaction of galactopoietic hormones which is not yet fully understood. Recent studies have demonstrated that this systemic control is modulated within the mammary gland by local mechanisms responsive to the frequency and completeness of milk removal. New insights into the endocrine and local (paracrine and autocrine) regulation of milk secretion have come from the adaptation of traditional endocrinological techniques to take advantage of new molecular tools, and from technical advances in other fields. This paper reviews recently developed animal models for the study of milk secretion and describes their application to provide new information into the roles of two key galactopoietic hormones, growth hormone and prolactin, and the modulation of their actions by local, intramammary mechanisms.     

Prolactin Regulation of Mammary Gland Development

Mammary morphogenesis is orchestrated with other reproductive events by pituitary-driven changes to the systemic hormone environment, initiating the formation of a mammary ductal network during puberty and the addition of secretory alveoli during pregnancy. Prolactin is the major driver of development during pregnancy via regulation of ovarian progesterone production (in many species) and direct effects on mammary epithelial cells (in all species). Together these hormones regulate two aspects of development that are the subject of intense interest: (1) a genomic regulatory network that integrates many additional spatial and temporal cues to control gene expression and (2), the activity of a stem and progenitor cell hierarchy. Amalgamation of these two aspects will increase our understanding of cell proliferation and differentiation within the mammary gland, with clear application to our attempts to control breast cancer. Here we focus on providing an over-view of prolactin action during development of the model murine mammary gland. Samantha R. Oakes and Renee L. Rogers contributed equally to this work. Financial Support: Australian Research Council, National Health and Medical Research Council, The Cancer Institute NSW and Cancer Council NSW.     

Local IGF-I Axis in Peripubertal Ruminant Mammary Development

The regulation of mammary growth and development in heifers is accomplished by complexinteractions of hormones, growth factors, and extracellular matrix molecules. Many of thesegrowth stimulators are believed to be locally produced in the mammary gland and to beaffected by developmental and nutritional status. Although estrogen and growth hormone areconsidered critical to pubertal mammogenesis, results summarized in this review suggest thatIGF-I⁶ and IGF binding proteins are especially important locally-produced growth regulatorsin peripubertal ruminants. This assertion is supported by studies of ovariectomized heifers, inwhich increased stromal IGFBP-3 and reduced IGF-I correspond with a failure of udderdevelopment. Similarly, reduced mammary development with overfeeding coincides withreduced mitogenic activity of mammary tissue extracts and altered concentrations of IGF-Iand IGFBPs. In vitro studies convincingly demonstrate that much of the mitogenic activity ofmammary extracts or serum can be attributed to IGF-I and that alterations in IGFBP-3 modulateits effectiveness. Thus by analogy to second messenger mechanisms of action for proteinhormones, local mammary-derived growth factors likely explain many of the effects attributedto the classic mammogenic hormones.     

EGF-Related Peptides in the Pathophysiology of the Mammary Gland

Normal mammary gland development is the result of complex interactions between a number of hormones and growth factors. Normal and malignant human mammary epithelial cells are able to synthesize and to respond to various different, locally acting growth factors and growth inhibitors. Among these, the EGF-related peptides play an important role in regulating the proliferation and differentiation of human mammary epithelial cells. EGF⁴ and TGF are able to stimulate the lobulo-alveolar development of the mammary gland in vivo as well they are involved in the pathogenesis of human breast cancer. Experimental evidence suggests that estrogen-induced proliferation of breast carcinoma cells is mediated in part by EGF-related growth factors. It has also been demonstrated that activation of certain cellular protooncogenes such as c-Ha-ras in human mammary epithelial cells results in cellular transformation and in an increased production of several EGF-related growth factors such as TGF and amphiregulin. Coexpression of both EGF-related peptides and their own receptors frequently occurs in human breast carcinomas and in human breast cancer cell lines, suggesting that an autocrine pathway of uncontrolled cell growth sustains neoplastic transformation.     

The mammary gland: A unique organ for the study of development and tumorigenesis

The microanatomy and development of the mammary gland are unique and a reflection of its function to synthesize and deliver milk to the newborn offspring. The uniqueness of the mammary gland resides in several factors. First, the mammary parenchyma undergoes the vast majority of its growth postpubertally, thus enabling experiments on development to be performed in the juvenile or adult and presenting opportunities for experimental manipulation of the gland not available with other organs. On the basis of this characteristic, the fat pad transplantation method was developed, which resulted in the elaboration of important concepts in senescence, immortalization, and preneoplasia. Second, the accessibility of the gland and the ductal organization allows delivery and localization of specific molecules to mammary parenchyma cells, the cells which are the site of origin of neoplastic development. Third, the organ is the target of viral, chemical, and physical carcinogens, allowing development of unique and complex models for neoplastic development. Finally, the complexity of hormone and growth factor regulation of mammary gland function allows a sophisticated approach to the study of hormone action. The purpose of this review is to illustrate some unique properties of the gland which provide the basis for specialized approaches to developmental, neoplastic, and functional problems.     

Interactions of Prolactin and Growth Hormone (GH) in the Regulation of Mammary Gland Function and Epithelial Cell Survival

The relative importance of GH³ and prolactin in mammary gland function varies between species with prolactin playing a major role in rodents and GH taking lead role in ruminants. In rodents, however, GH appears to play a vital role in maintaining a high-fat/low volume milk in the absence of prolactin and a similar finding has been demonstrated in goats where prolactin deficiency causes a more modest (15%) decrease in milk yield. Surprisingly GH-deficiency in goats induced no further decline in milk yield whereas exogenous GH or prolactin both stimulated milk output considerably. Although direct effects of prolactin on mammary epithelial cells are well-documented effects of GH are believed to be mediated indirectly via IGF-1 production from the liver. We have been unable to confirm this hypothesis in rats and believe this to be because it is too simplistic. By considering prolactin and GH to be survival factors for the mammary gland we now propose a mechanism by which they interact through the IGF system. Involution of the mammary gland involves apoptosis and, in rats, it is induced by prolactin-deficiency or milk accumulation. Coincidentally with this process mammary epithelial cells synthesize and secrete an IGF binding protein, IGFBP-5. We hypothesize that GH stimulates IGF-1 production, possibly from the mammary parenchyma. IGF-1 then acts as a survival factor for the mammary gland. Prolactin plays an essential role since it suppresses the secretion of IGFBP-5 which would otherwise inhibit IGF-1 action and lead to the induction of cell death.     

Mammogenesis and lactogenesis in hypophysectomized, ovariectomized, adrenalectomized rats

Summary  In the hypophysectomized-ovariectomized-adrenalectomized hooded Norway rat, growth of the mammary ducts can be induced with oestrone + somatotrophin + corticoids; lobule-alveolar development ensues if in addition to the above combination progesterone + prolactin is also administered. On withdrawing these hormones the local injection of prolactin then causes secretion in the underlying mammary gland, but only when corticoids are administered systemically.     

Hormonal Regulation of the Immune Microenvironment in the Mammary Gland

It is well established that the development and homeostasis of the mammary gland are highly dependent upon the actions of ovarian hormones progesterone and estrogen, as well as the availability of prolactin for the pregnant and lactating gland. More recently it has become apparent that immune system cells and cytokines play essential roles in both mammary gland development as well as breast cancer. Here, we review hormonal effects on mammary gland biology during puberty, menstrual cycling, pregnancy, lactation and involution, and dissect how hormonal control of the immune system may contribute to mammary development at each stage via cytokine secretion and recruitment of macrophages, eosinophils, mast cells and lymphocytes. Collectively, these alterations may create an immunotolerant or inflammatory immune environment at specific developmental stages or phases of the menstrual cycle. Of particular interest for further research is investigation of the combinatorial actions of progesterone and estrogen during the luteal phase of the menstrual cycle and key developmental points where the immune system may play an active role both in mammary development as well as in the creation of an immunotolerant environment, thereby affecting breast cancer risk.     

Role of Prolactin and Vasoinhibins in the Regulation of Vascular Function in Mammary Gland

The formation of new blood vessels has become a major focus of mammary gland research stimulated by the therapeutic opportunities of controlling angiogenesis in breast cancer. Normal growth and involution of the mammary gland are profoundly affected by the expansion and regression of blood vessels, whereas dysregulation of angiogenesis is characteristic of breast cancer growth and metastasis. Prolactin stimulates the growth and differentiation of the mammary gland under normal conditions, but its role in breast cancer is controversial. Its action is complicated by the fact that prolactin itself is angiogenic, but proteases cleave prolactin to generate vasoinhibins, a family of peptides that act on endothelial cells to suppress angiogenesis and vasodilation and to promote apoptosis-mediated vascular regression. This review summarizes our current knowledge about the vascular effects of prolactin and the generation and action of vasoinhibins, and discusses their possible contribution to the regulation of blood vessels in the normal and malignant mammary gland.     

Mechanistic Aspects of Crosstalk Between GH and PRL and ErbB Receptor Family Signaling

Growth hormone (GH) and prolactin (PRL) are anterior pituitary hormones that have multiple roles in growth and metabolism. Both hormones are important in mammary development and breast cancer. The epidermal growth factor (EGF) family of peptides and the receptors that they activate (the ErbB family) are also major players in mammary biology and pathophysiology. Recent studies in signal transduction have highlighted the interplay between signaling pathways referred to as crosstalk. In this review, cell biological and signaling studies related to crosstalk between GH and PRL and the ErbB family are discussed. In particular, the role of GH- and PRL-induced phosphorylation of ErbB receptors in regulating EGF responsiveness is highlighted with attention to potential pathophysiological relevance.     

Effects of Anabolic Implants of Oestradiol Alone or in Combination with Trenbolone Acetate on the Ultrastructure of Mammary Glands in Female Lambs Regarding their Interference in Prolactin Secretion

The side‐effects of anabolic steroid implants on mammary gland ultrastructure were evaluated in female lambs treated with oestradiol (n=10) and with oestradiol plus trenbolone acetate (n=10). Ten non‐implanted lambs were used as controls. Apart from the ultrastructural study of the mammary gland, an assessment of the prolactin pituitary cell population was carried out by immunological methods. Our results showed that oestrogenic implants exert stimulating effects on mammary gland development, both by activating the synthesis process at mammary gland cell levels and by increasing prolactin pituitary production. Nevertheless, there was no evidence of secretory products in the lumen of the gland. Implants containing trenbolone acetate counteracted the mammary stimulus of oestrogens showing ultrastructural images of cell autolysis and necrosis.     

Hypothalamic and pituitary function

The endocrine system consists of groups of cells (glands) that secrete messengers (hormones), which affect distant groups of cells (target organs). It controls mainly basal processes. Hormonal action may be on receptors in the target cell membrane (e.g. leading to alterations in membrane channel properties), in which case it is rapid, or it may affect gene function and thus protein synthesis, in which case the onset of action is relatively slow. Endocrine function is controlled via single and multiple feedback mechanisms from products of the various target organs. It is largely under the control of the hypothalamus via the pituitary gland. Releasing factors and hormones from the hypothalamus act on the pituitary, which produces its own hormones (antidiuretic hormone, oxytocin, growth hormone and prolactin) as well as hormones and releasing factors that affect other endocrine glands (adrenocorticotrophic hormone, thyroid stimulating hormone, luteinizing hormone and follicle stimulating hormone). Growth hormone controls skeletal growth via the release of insulin-like growth factors from the liver; it promotes anabolism, but also antagonizes the hypoglycaemic effect of insulin. Antidiuretic hormone secretion is stimulated by changes in osmolality and is a sensitive mechanism for conserving fluid via its action on the kidney. Oxytocin stimulates uterine contraction, and prolactin stimulates milk production. Luteinizing and follicle stimulating hormones affect the growth of the gonads.     

Initiation of Human Lactation: Secretory Differentiation and Secretory Activation

Theories for the origin of milk have been recorded since the time of Ancient Greeks. In those times it was believed that milk was derived from special vessels that connected the uterus to the breasts. The “chyle theory” on the origin of milk was another prominent theory which persisted well into the nineteenth century before the realisation that milk components were derived from blood and some milk constituents were actually synthesized within the breasts. The demonstration that milk ejection was the expulsion of milk that had already been secreted and that milk secretion was a separate continuous process, set the background for the development for the current understanding of milk synthesis and secretion. Today we know that there are two stages in the initiation of lactation- secretory differentiation and secretory activation. Secretory differentiation represents the stage of pregnancy when the mammary epithelial cells differentiate into lactocytes with the capacity to synthesize unique milk constituents such as lactose. This process requires the presence of a ‘lactogenic hormone complex’ of the reproductive hormones, estrogen, progesterone, prolactin and some metabolic hormones. Secretory activation on the other hand, is the initiation of copious milk secretion and is associated with major changes in the concentrations of many milk constituents. The withdrawal of progesterone triggers the onset of secretory activation but prolactin, insulin and cortisol must also be present. This review describes the works of pioneers that have led to our current understanding of the biochemical and endocrinological processes involved in the initiation of human lactation.     

Perinatal ethinyl oestradiol alters mammary gland development in male and female Wistar rats

Increased attention is being paid to human mammary gland development because of concerns for environmental influences on puberty onset and breast cancer development. Studies in rodents have showed a variety of changes in the mammary glands after perinatal exposure to endocrine disrupting chemicals, indicating progressed development of mammary glands when exposed to oestrogens early in life. However, laboratories use different parameters to evaluate the development of mammary glands, making studies difficult to compare. Moreover, studies of whole mounts in Wistar rats are lacking. In the present study, Wistar rats were exposed to 0, 5, 15 or 50 μg/kg of ethinyl oestradiol per day during gestation and lactation. A wide range of morphological parameters were evaluated in whole mounts of mammary glands from male and female offspring PD21-22. This study showed that in both male and female pre-pubertal Wistar rats, mammary gland development was accelerated after perinatal oestrogen exposure with increase in size, density and number of terminal end buds (TEBs). In female rats, the most sensitive parameters were the distance to the fifth gland, the relative growth towards the lymph node and the overall density. The sensitive endpoints in male rats were TEB numbers, both in the whole gland and in the zone C, the overall- and the highest density. The overall density was sensitive in both male and female rats and was considered a good representative of both branching and budding of the gland. The number of TEBs in zone C was representative of the number of TEBs in the whole gland. Further studies in older Wistar rats and with weak oestrogenic compounds could be performed to validate mammary gland examination as an endpoint in reproductive toxicity studies and to examine how early life environmental exposures may alter mammary gland development, disrupt lactation and alter susceptibility to breast cancer.     

Rational Design of Competitive Prolactin/Growth Hormone Receptor Antagonists

There is increasing evidence that prolactin (PRL) and growth hormone (GH) act as growth-promoters of breast tumors. Recent arguments have accumulated to suggest that when they are locally-produced within the mammary tissue, these hormones, acting by an autocrine-paracrine mechanism may have enhanced, or even specific functions compared to endocrine PRL and GH. Classical drugs blocking pituitary hormone production (dopamine and somatostatin analogs) are ineffective on extrapituitary expression of PRL/GH genes, therefore the undesirable effects of these locally-produced hormones remain a target of interest for alternative strategies. This has encouraged the development of competitive PRL and/or GH receptor antagonists, which involve engineered variants of natural receptor ligands (PRL or GH) aimed at blocking receptor activation rather than hormone production in peripheral tissues. This article overviews the rational design of this new class of molecules, their specific molecular features (receptor specificity, biological properties, etc.) and whenever available, the data that have been obtained in cell or animal models of breast cancer.     

Targeting Mucosal Immunity in the Battle to Develop a Mastitis Vaccine

The mucosal immune system encounters antigens that enhance and suppress immune function, and serves as a selective barrier against invading pathogens. The mammary gland not only encounters antigens but also produces a nutrient evolved to protect and enhance mucosal development in the neonate. Efforts to manipulate antibody concentrations in milk to prevent mastitis, an infection of the mammary gland, have been hampered both by complexity and variation in target pathogens and limited knowledge of cellular immunity in the gland. Successful vaccination strategies must overcome the natural processes that regulate types and concentrations of milk antibodies for neonatal development, and enhance cellular immunity. Furthermore, the need to overcome dampening of immunity caused by non-pathogenic encounters to successfully prevent establishment of infection is an additional obstacle in vaccine development at mucosal sites. A significant mastitis pathogen, Staphylococcus aureus, not only resides as a normal flora on a multitude of species, but also causes clinical disease with limited treatment options. Using the bovine model of S. aureus mastitis, researchers can decipher the role of antigen selection and presentation by mammary dendritic cells, enhance development of central and effector memory function, and subsequently target specific memory cells to the mammary gland for successful vaccine development. This brief review provides an overview of adaptive immunity, previous vaccine efforts, current immunological findings relevant to enhancing immune memory, and research technologies that show promise in directing future vaccine efforts to enhance mammary gland immunity and prevent mastitis.