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INTRODUCTION

Milk production is necessary for the post-partum survival of mammalian offspring. By providing for the transfer of nutrients to newborns, the breast preserves the metabolic investment a mother has made in her young in utero, and allows for successful reproduction. Therefore, it is no surprise that the normal development of the breast is inextricably tied to the reproductive cycle; a relationship coordinated by circulating hormones. Breast development occurs in distinct steps that are initiated by changes in the reproductive status of the female. The first is the formation of the embryonic mammary bud and its outgrowth into the mammary fat pad to establish a rudimentary ductal system. After birth, breast growth is minimal until the second step, the formation of the mature ductal tree, during puberty. In concert with the other changes occurring during puberty, this stage represents the acquisition of reproductive competence by the breast, and it is driven by pubertal changes in circulating hormones. The next stage occurs only if the female becomes pregnant, and involves a hormonally-driven expansion of the mammary epithelium followed by its full functional differentiation. Hormonal changes at parturition subsequently lead to the acquisition of a secretory phenotype by the mammary epithelial cells and lactation. Finally, upon weaning, the mammary gland remodels itself back to a mature virgin-like state, in order to allow for future cycles of pregnancy and lactation and the support of additional offspring. In this review, we will discuss the hormonal regulation of these developmental stages in order to provide a framework by which to understand developmental and functional abnormalities of the human breast.

One of the earliest findings to suggest that endocrine hormones regulate mammary development came late in the 19th century when it was discovered that breast cancers regressed in some patients following ovariectomy 1. However, it was the seminal work of a few investigators in the mid-20th century that identified the major hormones involved in postnatal mammary gland development 2, 3. In these studies, rodents were ovariectomized, hypophysectomized, and adrenalectomized to eliminate confounding variables associated with cross talk between these organs. Various hormones were then systematically replaced to study their effects on mammary morphogenesis. It was determined that estrogen (E) and growth hormone (GH) stimulate ductal elongation, while progesterone (P) is necessary for alveolar development. Furthermore, these studies established that prolactin (PRL), GH, and adrenal steroids are involved in lobuloalveolar differentiation, milk synthesis, and lactation. Although additional insights have recently been gleaned from the study of genetic models in which individual hormones or their receptors have been "knocked out" or overexpressed in mice, these original studies remain the framework for our understanding of the endocrine regulation of breast development. Far less is known about the hormonal regulation of human breast development. Therefore, in the discussions that follow, we will outline what is known about the endocrine regulation of each stage of mammary development in rodents, then compare and contrast these findings to breast development in humans.

MAMMARY GLAND DEVELOPMENT IN THE EMBRYO

The first sign of embryonic mammary development is the mammary streak, also known as the milk line 4. This is an epidermal ridge that develops on the ventral surface of the embryo between the anterior and posterior limb buds. Ectodermal cells within this ridge migrate to specific locations along the line and then invaginate into the surrounding mesenchyme to form mammary buds. The mammary buds elongate into the mammary fat pad precursor and begin to form branches and, by birth, a rudimentary ductal tree is created. Concurrent with the outgrowth of the mammary bud, the overlying epidermis is transformed into the nipple and nipple sheath or areola. In humans, the mammary bud becomes septated and generates several primary ducts that each form a ductal tree within a fatty stroma surrounded by a fibrous sheath 5. This arrangement gives rise to the individual lobules of the human breast. In mice, the bud generates one primary duct that then branches upon entering the mammary fat pad. Thus, in mice all the ducts in each mammary gland are contained within one contiguous stromal compartment.

Very little is known about the regulation of early embryonic mammary development. It appears that this phase of breast development is the least responsive to hormonal regulation. Instead, embryonic development is primarily regulated by the action of a series of local growth factors and developmental regulatory genes. Genetic models have established that parathyroid hormone-related protein (PTHrP) and the PTH/PTHrP Type I receptor (PTH1R) 6, Fgf10 and its receptor Fgfr2b 7, the transcription factor Lef1 8, and the homeobox genes Hoxc6 9, Msx1 10 and Msx2 11 are all critical for proper embryologic development of the mammary glands in mice. Of these factors, only PTHrP signaling has been shown to be necessary for embryonic breast development in humans. Blomstrand's chondrodysplasia is a disease caused by inactivating mutations in the PTH1R. Like their mouse counterparts, human fetuses with Blomstrand mutations lack mammary glands and nipples 12.
Although, embryonic morphogenesis appears to be more dependent upon local rather than systemic endocrine signals, hormones do have effects at this stage. Male mice have no nipples and only remnants of mammary ducts. This is because circulating androgens act to destroy the mammary bud in utero 4. Human fetuses do not display this pattern of sexual dimorphism and men are capable of breast development and milk production (see discussion of galactorrhea). The embryonic mammary epithelium in both humans and mice responds to mammotropic hormones. For instance, when isolated embryonic mouse mammary epithelium is transplanted under the kidney capsule of a female mouse, the embryonic epithelium proliferates and produces milk when the recipient mouse becomes pregnant 13. To the same end, enlarged breasts and transient secretion of a milk-like substance known as "witch's milk" is frequently seen in neonates. In this case, in response to maternal and placental hormones, embryonic epithelial cells proliferate and form alveolar structures that secrete milk 14. Therefore, even at an early developmental stage, the mammary epithelium can respond appropriately to hormones. Therefore, some investigators have suggested that the hormonal environment during embryogenesis may influence the risk for breast cancer later in life. In support of this idea, neonatal or fetal exposure to diethylstilbesterol (DES) has been shown to alter the growth of the mammary gland and to alter sensitivity to carcinogens in mature female rodents 15-17. There is currently great interest in whether early exposure to synthetic estrogenic compounds in the environment may also alter the future risk for breast cancer.

Figure 1. Overview of the regulation of mammary gland development. During embryonic development, signaling molecules important in epithelial-mesenchymal interactions include PTHrP, FGF-10, LEF-1, and Msx2. Under the influence of maternal PRL and PL, the neonatal mammary gland undergoes transient functional differentiation and produces witch's milk. Mammary gland development proceeds slowly after birth until puberty, when E and GH stimulate rapid ductal elongation. During pregnancy, progesterone stimulates alveologenesis and lactogenesis 1. At parturition, the withdrawal of progesterone is required for initiation of lactogenesis 2. Prolactin promotes lactogenesis 2 and, along with oxytocin, maintains lactation. The withdrawal of prolactin and oxytocin causes involution of the mammary gland to a mature virgin-like state. MFP, mammary fat pad; TEB, terminal end bud.

POSTNATAL MAMMARY GLAND DEVELOPMENT

Birth to Puberty 
From the time of birth until puberty, the mammary gland remains largely dormant. Growth of the rudimentary ductal system is proportional to the growth of the whole body (isometric growth) 14. Very little is known about the regulation of mammary growth at this stage. Epidermal growth factor (EGF) may play a role in ductal elongation, since EGF receptor knockout (EGFRKO) mice exhibit an elongation defect at an early age 18.

Puberty
At puberty, the mammary gland begins to grow rapidly so that its growth rate exceeds that of the body surface area (allometric growth). In the mouse, club-shaped terminal end buds (TEB) form at the distal ends of the rudimentary ducts and serve as the loci for cellular proliferation, differentiation and apoptosis 19. These structures are composed of 4-6 layers of cuboidal epithelial cells. The outermost or "cap cells" give rise to the myoepithelial cell layer beginning at the neck of the TEB. The body cells make up the central portion of the structure. As the TEBs proliferate and progress through the fat pad, some body cells give rise to luminal epithelial cells while the innermost cells undergo apoptosis to generate the ductal lumen. The ducts branch in a dichotomous pattern and continue to elongate until they read the edge of the mammary fat pad. At that point, the TEB regress and growth stops. The end result of this phase of growth is a network of branched tubes filling out the mammary fat pad.

Early studies demonstrated that ovariectomy caused TEB regression and halted ductal growth, suggesting that E was necessary for the pubertal growth of the mammary ducts 20. These results have now been confirmed in knockout mouse models. At puberty, estrogen receptor a knockout (ERKOa) mice do not form TEB and the ducts do not elongate 21. Interestingly, it is specifically the stromal ERa that is required for ductal morphogenesis during puberty 22. However, experiments in the triply operated rodent demonstrated that although E was necessary for pubertal mammary growth, it was not sufficient. Growth hormone (GH) is also necessary 2, 3. Like ERKOa mice, growth hormone receptor knockout (GHRKO) mice have a defect in ductal elongation 23. GH also acts on the mammary stroma, where it leads to the production of insulin-like growth factor 1 (IGF-1) 24. IGF-1 then stimulates proliferation of the mammary epithelium, acting through its receptor (IGF-1R) 25. As would be expected, ductal elongation is also impaired in IGF-1R knockout mice 26.

After reaching the borders of the fat pad, the mammary ducts undergo cyclical side branching that appears to be regulated by the estrous cycle. The development of these short branches is driven by the actions of progesterone. This is illustrated by the mammary ducts of the P receptor knockout (PRKO) mouse, which have few side branches, but grow through the fat pad in a normal fashion 27. It has been suggested that prolactin may also contribute to the development of these side branches, for the mammary glands in prolactin receptor KO mice have defects in side branching 28. Transplantation experiments suggest that prolactin's effects on side branching may be mediated through its ability to alter systemic hormone levels rather than through direct actions on the mammary ducts themselves 29.

In humans, pubertal breast development is slightly different from rodents 14. At puberty, primary and secondary ducts grow by both dichotomous branching and sympodial branching. Dichotomous branching is the bifurcation of a duct into two branches, while sympodial branching results from lateral budding off of a duct. Like mice, branching ducts and alveolar buds arise from TEB-like structures. The principal difference in human development is the development of some lobuloalveolar structures concurrent with the formation of the duct system. About 11 alveolar buds form in clusters around each terminal duct to make up what is known as a lobule, or terminal ductal lobular unit (TDLU) 14. The TDLU is analogous to the lobuloalveolus of the mouse 30. However, there is a spectrum of TDLU that differ in their mitotic activity, their retention of TEB like structures and their degree of differentiation. The lobule described above is a virginal lobule, or TDLU type 1, according to the nomenclature of Russo and Russo 14. This type is the most mitotically active and retains numerous TEB like structures. Unlike the mouse, the human breast continues to develop slowly after the pubertal growth spurt, and the character of the TDLU changes with alterations in hormonal status. With recurrent menstrual cycles, type 1 TDLU gradually develop into type 2 and 3 TDLU 31. In type 2 and 3, there are fewer end bud-like structures and less mitosis but more ductules and mature looking lobuloalveolar structures. Type 2 and type 3 TDLU are also more frequently found in parous women. These differences in the character of the TDLU may be important for the genesis of breast carcinomas, most of which have been suggested to arise from the most immature and mitotically active, or type 1, TDLU 31.

As in the rodent, E is important for ductal elongation. The level of breast development in pubertal girls correlates with serum E levels 32, 33, and exogenous E stimulates breast development in girls with E deficiency 34. However, unlike the mouse, the human stroma does not express ER 35. ER and PR are expressed mainly in the epithelial cells. As in the mouse, GH also appears to contribute to ductal morphogenesis in the human. Systemic GH is elevated during puberty 36 and stromal cells in human breast tissue express IGF-1, particularly those immediately adjacent to the mammary epithelium 37.

Figure 2. Upper panel, The percentage of human mammary epithelial cells that are estrogen or progesterone receptor positive (ER/PR +), the percentage that are proliferating, and the percentage that are both ER/PR + and proliferating 39, 40. This graph illustrates that steroid receptor expression and proliferation infrequently occur in a single mammary epithelial cell at a given time. Rather, receptor positive cells are found in close juxtaposition to proliferating cells, suggesting a paracrine mechanism for the mitogenic action of estrogen and progesterone on mammary epithelial cells. Stimulation of ER/PR + cells (lower panel) could release a paracrine factor that either stimulates adjacent luminal epithelial cells to proliferate or causes proliferation of the responder cells by eliciting a secondary response through stromal cells.

PREGNANCY AND LACTATION

Alveolar Development
When pregnancy occurs, mammary development rapidly accelerates. During the first stages of pregnancy, buds repeatedly form and elongate perpendicular to existing ducts to form small terminal ducts. When the mammary fat pad is filled so that there is little space between nascent buds, sac-like alveoli are formed at the end of the terminal ducts. In the classic hormone ablation/replacement experiments, the most effective hormonal regimen to induce full mammary development was a period of E, GH, and adrenal steroid treatment followed by P and PRL 3. These results imply that P and PRL are important for alveologenesis in the later stages of mammary gland development during pregnancy. Recent genetic ablation studies have confirmed and expanded upon these data. For example, epithelial cells from PR knockout (PRKO) mice are incapable of forming terminal ducts and initiating the formation of alveoli 38. Likewise, studies using prolactin receptor deficient mice have demonstrated that prolactin signaling is necessary for lobuloalveolar morphogenesis 28. Interestingly, unlike pubertal development, alveologenesis is driven by P action directly on the epithelial cells and not on stromal cells 29. However, receptor/proliferation co-localization studies in rodents and humans, have found that cells expressing PR seldom proliferate 39, 40. Rather, the proliferating cells are adjacent to the receptor positive cells, suggesting that the hormone-responsive epithelial cells send a paracrine signal to a distinct "responder" population of epithelial cells that are responsible for proliferating and forming alveoli 41.

Lactogenesis
Mammary gland differentiation is also called lactogenesis, and leads to full lactation 42. Lactogenesis is traditionally divided into two stages 43. Lactogenesis 1 starts around mid-pregnancy, when some of the genes encoding milk proteins are first expressed. In the mouse, cytoplasmic lipid droplets are seen at this stage, while in humans a-lactoglobulin can be detected in the maternal serum 44. Lactogenesis 2 occurs at about the time of parturition, and is characterized by increased expression of milk proteins, the formation of tight junctions between mammary epithelial cells, and the expulsion of lipid droplet and casein micelles into the lumen 42. As discussed in the previous section, PRL promotes lactogenesis 1. Furthermore, PRL is required for lactogenesis 2 and for maintenance of lactation. Lowering prolactin levels using dopamine agonists such as bromocriptine will prevent lactogenesis 2 and suppresses milk production in both rodents and women 45, 46. In rodents, PRL and PRLR levels are decreased during pregnancy, and placental lactogen, acting through the GHR and/or PRLR, substitutes for the lactogenesis-promoting role of PRL 47. However, the importance of placental lactogen to human lactation is questionable because women with placental lactogen deficiency can lactate normally 48, 49, whereas women with low PRL levels cannot lactate (see below). In dairy cows, GH (bovine somatotropin) increases milk yield 50, but this effect is not seen in other species. In fact, GH is dispensable for lactogenesis in mice and humans, as GHR knockout mice 51 and human dwarfs with mutations in either GH or GHR can lactate 52, 53.

P, predominantly from the placenta during pregnancy, is an absolute requirement for lactogenesis 1. However, P prevents the onset of lactogenesis 2, and it is the withdrawal of P at parturition that is responsible for the initiation of milk production 54. In rodents, P levels begin to fall and trigger lactogenesis 2 before birth . However, P falls only after delivery of the placenta in humans, causing a delay in full lactation of about 2-4 days 44. PR expression is down regulated late in pregnancy and is absent during lactation, further reinforcing the drop in P levels 55.

Lactation requires a significant transfer of nutrients from mother to offspring via milk, a process facilitated by changes in maternal hormones. Like P, E levels rise during pregnancy then fall off. Lactation is an E-deficient state, and in this respect is similar to the post-menopausal period. Osteoporosis is one of the major problems associated with E-deficiency in menopause, and significant bone resorption also occurs during lactation, presumably to free calcium for milk production 56. This raises the possibility that the connection between E and bone resorption may be an adaptation to ensure adequate calcium delivery to offspring through milk. Thyroid hormones are required for efficient milk production in rodents 57. However, in humans, plasma thyroxine levels are lower in lactating than in non-lactating women 58. Likewise, glucocorticoids may influence lactation indirectly by regulating nutrient flux, but levels of cortisol during lactation are lower than baseline levels 59. Insulin does not seem to play a direct role in lactogenesis or lactation 60 but low insulin levels during lactation may divert nutrients from storage pathways to make them available for milk production 59.

Oxytocin (OT) and Milk Letdown
OT is the principal regulator of milk ejection, or the "let down" reflex . In the mammary gland during lactation, the myoepithelial cells form a mesh-like network around the secretory alveoli, and form a longitudinal network around the mammary ducts. Upon suckling at the nipple, afferent nerve impulses travel to the brain via the spinal cord and cause OT release from the posterior pituitary 61. OT travels through the bloodstream and binds OT receptors (OTR) on myoepithelial cells, leading to their contraction and milk expulsion 62. The release of OT and the subsequent myoepithelial contractions are pulsatile in nature 63. Milk letdown is a "neuroendocrine reflex" that can be triggered by the sight, sound, or even the mere thought of an infant 63. In addition, mental stress 64, alcohol 65, and opioids 66 all appear to interfere with milk letdown by inhibiting OT release. The importance of OT and the "let down" reflex is underscored by the failure of lactation in OT knockout mice 67.

Figure 3. A, time course of plasma prolactin or human growth hormone (HGH) levels in eight nursing mothers from 8-41 days postpartum and six women between 63 and 194 days postpartum (mean +/- standard error). A sharp suckling-induced increase in prolactin was seen in the 8-41 days postpartum group, while this response was diminished in the 63-194 days postpartum group. HGH levels did not increase with suckling. B, profile of prolactin concentrations in three women between 22 and 26 days postpartum who played with their infants before nursing. In all three women, milk let down began shortly after they started interacting with the infants. However, prolactin levels did not rise until suckling began. (G.L. Noel, H.K. Suh, and A.G. Frantz, Prolactin release during nursing and breast stimulation in postpartum and nonpostpartum subjects. J Clin Endocrinol Metab, 1974, 38:413-423, The Endocrine Society)

Prolactin and the Maintenance of Lactation
Prolactin is necessary for the maintenance of lactation, and suckling causes prolactin secretion. Unlike oxytocin, prolactin is not released by psychological stimuli in anticipation of suckling, but levels begin to rise within 10 minutes after suckling begins and peak by 30 - 60 minutes after the nursing stimulus 68. Initially, prolactin levels are elevated after parturition and suckling causes further elevations. However, within 1 - 2 months after parturition, mean prolactin levels decline, but suckling continues to result in transient elevations 69. It appears that the continuous elevations of prolactin early after parturition are important to the initiation of lactation, but that once begun, nursing can be maintained with the lower, transient elevations of prolactin. However, further lowering of prolactin levels with dopamine agonists will terminate milk production 70.

INVOLUTION

The last stage of the mammary life cycle involves the removal of the differentiated mammary epithelial cells and the remodeling of the gland to a duct system similar to that in the mature virgin. When no longer needed, the milk-producing machinery is destroyed, to be recapitulated in a subsequent pregnancy in preparation for another round of lactation. Involution of the mammary gland is triggered by the combination of milk stasis and a fall in prolactin levels 71. Lack of suckling and milk stasis results in a rapid, but reversible induction of apoptosis within the differentiated population of mammary epithelial cells. If the lack of suckling is prolonged, prolactin levels decline below a threshold level and apoptosis is accompanied by a tissue-remodeling phase involving the induction of matrix-degrading enzymes and inflammatory cell infiltration. Once the transition to the alveolar remodeling phase begins, the process of involution cannot be reversed 72. The end result of this process is the elimination of all lobuloalveolar structures leaving behind a simple ductal tree.