Apoptosis in the Mammary Gland

Epithelial apoptosis has a key role in the development and function of the mammary gland. It begins with the formation of the ducts in the embryonic phase and occurs again at puberty and with a stage of menses. Regulated apoptosis occurs at several stages of mammary development. In the embryo, epithelial buds emerge from ectoderm into mammary mesenchyme, which is the origin of the ductal tree. When the ducts later hollow out in puberty, extensive apoptosis occurs within the terminal bud.

Deregulated apoptosis contributes to the malignant progression in the genesis of breast cancer. Research in apoptosis continues because it may lead to new cancer treatments, but the knowledge itself will be valuable.

When suckling ceases during weaning, the alveolar component of the gland involutes by both apoptosis and tissue remodeling, which rebuilds the gland to the prepregnancy state.

Much is being learned about mammary development and function through the intense study of the breast as an experimental system. The use of novel “knockout” mouse models has been employed to study nursing failure. The apoptosis control mechanism from the angle of the signaling pathways has been studied. Further work at the level of the cell is underway, including extensive genetic analysis.

Hormonal Control of Lactation

In contrast to most organs, which are fully developed at birth, the mammary gland undergoes most of its morphogenesis postnatally, in adolescence, and in adulthood. Lactation is an integral part of the reproductive cycle of all mammals, including humans. The hormonal control of lactation can be described in relation to the five major stages in the development of the mammary gland: (1) embryogenesis; (2) mammogenesis, or mammary growth; (3) lactogenesis, or initiation of milk secretion; (4) lactation (stage III lactogenesis), or full milk secretion; and (5) involution ( Table 3-1 ).

Current terminology divides lactogenesis into two stages. Stage I takes place during pregnancy when the gland is sufficiently developed to actually produce milk. It begins about midpregnancy (approximately 16 weeks). It can be identified by measuring the levels of plasma lactose and α-lactalbumin. Should the mother deliver at this point, milk would be produced. Some mothers can express colostrum during this time. As the pregnancy proceeds, milk production is inhibited by high levels of circulating progesterone in most mammals and estrogen as well in humans.

Stage II of lactogenesis is the onset of copious milk production at delivery. In all mammals, it is associated with the drop in progesterone levels ( Figure 3-1 ). This drop occurs to herald delivery in some species so that milk is copious when the young are born. In humans, these levels drop during the first 4 days postpartum, which is reflected by the milk “coming in” during this time. The drop in progesterone is accompanied by the transformation of the mammary epithelium to produce volumes of milk by the fifth day. This change includes a change in permeability of the paracellular pathway and changes in secretion of protective factors (i.e., lactoferrin, immunoglobulins), as well as increases in all milk components that parallel increased glucose production.

Progesterone withdrawal initiates lactogenesis II in women. The increase in lactose concentrations associated with increased synthesis of milk components coincides with a rapid decrease in progesterone concentration when the placenta is removed at parturition.

(From Czan KC, Henderson JJ, Kent JC, et al: Hormonal control of the lactation cycle. In Hale TW, Hartmann PE, editors: Textbook of human lactation , Amarillo, Tex., 2007, Hale Publishing LP.)

During the next 10 days, the composition of the milk slowly changes to mature milk. Composition then changes slowly over the months of full exclusive breastfeeding.

Embryogenesis

Embryogenesis begins with the mammary band, which develops about the 35th embryonic day and progresses to a bud at the 49th day (see Chapter 2 ). Ducts continue to elongate to form a mammary sprout, which invades the fat pad, branches, and canalizes, forming the rudimentary mammary ductal system present at birth. After birth, growth of this set of small branching ducts parallels the child’s linear growth but remains limited, probably controlled by growth hormone (GH) before onset of ovarian activity.

Under the influence of sex steroids, especially the estrogens, the mammary glandular epithelium proliferates, becoming multilayered. Buds and papillae then form. The growth of the mammary gland is a gradual process that starts during puberty. The process depends on pituitary hormones. Lobuloalveolar development and ductal proliferation also depend on an intact pituitary gland.

The following six well-documented factors help explain the organization of mammary growth. Much of this work has resulted since the availability of “knockout” studies in mice and associated techniques.

• 1.
  

  Mammary ducts must grow into an adipose tissue pad if morphogenesis is to continue. Only adipose stroma supports ductal elongation. The mammary epithelium is closely associated with the adipocyte-containing stroma in all phases of development. In midgestation during human fetal development, a fat pad is laid down as a separate condensation of mesenchyma. Rudimentary ducts expand into the fat pad but do not progress. At puberty, the ducts elongate to fill the entire fat pad, terminating growth as they reach the margins of the fat pad.

  
  
• 2.
  

  Estrogen is essential to mammary growth. Ductal growth does not occur in the absence of ovaries but can be stimulated when estrogen is provided. In the ovariectomized (oophorectomized) mouse, an estrogen pellet placed in the mammary tissue stimulates growth in that gland but not in the opposite gland. When the estrogen receptor is “knocked out” in the mouse, no mammary development occurs. The increase in estrogen at puberty results in mammary development. Although estrogen is essential, it is not adequate alone.

  
  
• 3.
  

  The exact location of the estrogen receptors in human breasts is unclear. Estrogen receptors are not in the proliferating cells and have not been located in the stroma. Cells with estrogen receptors, however, secrete a paracrine factor that is responsible for the proliferation of ductal cells. This paracrine factor may hold the key to understanding both normal and abnormal breast development.

  
  
• 4.
  

  In addition to estrogen, the pituitary gland is necessary for breast development. Kleinberg has identified GH as important to pubertal development and development of the terminal end buds in the breast. Prolactin could not replace GH in these experiments, but insulin-like growth factor-1 (IGF-1) could. It is produced in the stromal compartment of the mammary gland under stimulation by GH, and together with estradiol from the ovaries, IGF-1 brings about ductal development at puberty.

  
  
• 5.
  

  Transforming growth factor beta (TGF-β) maintains the spacing of the mammary ducts as they branch and elongate. These ducts exhibit unique behavior during growth, turning away to avoid other ducts and end buds. This avoidance behavior accounts for the orderly development of the duct system in the breast and the absence of ductal entanglements. This pattern provides ample space between ducts for later development of alveoli. TGF-β has been identified as the negative regulator and is found in many tissues, including breast tissue produced by an epithelial element. The pattern formation in ductal development depends on the localized expression of TGF-β.

  
  
• 6.
  

  Progesterone secretion brings about the side branching of the mammary ducts. The presence of progesterone receptors in the epithelial cells has been confirmed by studies in knockout mice in which mammary glands develop to the ductal stage but not to alveolar morphogenesis. Ormandy et al. established that prolactin is necessary for full alveolar development through prolactin receptor studies in knockout mice in which mammary glands do not develop beyond the ductal stage. This was further confirmed in murine mammary cultures in which full development of the alveoli depends on prolactin. Further, when prolactin is withdrawn, apoptosis of the alveolar cells occurs.

The coordination of epithelial and stromal activity in the mammary gland is complex. Hepatocyte growth and scatter factor has been associated with the process during puberty. Another growth factor, heregulin, a member of the epidermal growth factor (EGF) family, has been identified in the stroma of mammary ducts during pregnancy.

Neville has diagrammed the regulation of mammary development ( Figure 3-2 ). She notes that the concentrations of estrogen, progesterone, and lactogenic hormone in the form of prolactin or placental lactogen (PL) greatly increase, enhance alveolar development, and result in the differentiation of alveolar cells. Although many investigators have contributed pieces to the puzzle of mammogenesis, Neville succeeded in creating the current visualization.

Scheme for regulation of mammary development in the mouse.

(From Neville MC: Mammary gland biology and lactation: a short course. Presented at the annual meeting of the International Society for Research on Human Milk and Lactation, Plymouth, Mass., 1997.)

Mammogenesis: Mammary Growth

Growth During Pregnancy

Hormonal influences on the breast cause profound changes during pregnancy ( Figures 3-3 and 3-4 ). Early in pregnancy, a marked increase in ductular sprouting, branching, and lobular formation is evoked by luteal and placental hormones. PL, prolactin, and chorionic gonadotropin have been identified as contributors to the accelerated growth ( Figure 3-4 ). The dichorionic ductular sprouting has been attributed to estrogen and lobular formation to progesterone.

Hormonal preparation of breast during pregnancy for lactation.

(Modified from Vorherr H: The breast: morphology, physiology and lactation , New York, 1974, Academic Press.)

Plasma hormone levels during pregnancy. E1 , estrone; E2 , estradiol; E3 , estriol; hCG , human chorionic gonadotropin.

(From Neville MC, Morton J, Umemura S: The evidence for breastfeeding, Pediatr Clin North Am 48:42, 2001.)

Prolactin is essential for complete lobular-alveolar development of the gland. Almost complete growth of the mammary lobular-alveolar system can be obtained experimentally in the hypophysectomized-adrenalectomized rat if the animal receives estrogen, progesterone, and prolactin. Prolactin, as with other protein hormones, exerts its effect through receptors for the initiation of milk secretion located on the alveolar cell surfaces. The induction of milk synthesis requires insulin-induced cell division and the presence of cortisol. Prolactin is secreted by the pituitary, which is negatively controlled by prolactin-inhibiting factor (PIF) from the hypothalamus.

From the third month of gestation, secretory material that resembles colostrum appears in the acini. Prolactin from the anterior pituitary gland stimulates the glandular production of colostrum. By the second trimester, PL begins to stimulate the secretion of colostrum. A mother who delivers after 16 weeks’ gestation will secrete colostrum, even though she has had a nonviable infant. This demonstrates the effectiveness of hormonal stimulation on lactation.

An estrogen-mediated increase in prolactin secretion in pregnancy may produce as much as a tenfold to twentyfold increase in plasma prolactin. This effect may be partially controlled by lactogen from the placenta, which inhibits the production of prolactin. Hormonal regulation of the growth and proliferation of the mammary gland cells has been carefully studied in many species.

Studies of mice in which receptors for each of the hormones have been ablated demonstrate that progesterone and prolactin (or possibly placenta lactogen) are key to alveolar development in pregnancy. The major inhibitor of milk production during pregnancy has been shown to be progesterone.

A complex sequence of events, governed by hormonal action, prepares the breast for lactation (see Figure 3-3 ). During pregnancy, 17β-Estradiol stimulates the ductal system of epithelial cells to elongate. In contrast to puberty, however, when estrogens appear to directly and indirectly stimulate breast development, estrogens have no indispensable role in mammary development during pregnancy except as a prolactin potentiator: according to Neville, when estrogen levels are low in pregnancy, the breast still develops. Estrogen levels are normally high in pregnancy, but not for mammogenesis. Induced lactation in the cow is dependably reproduced with 7 days of estrogen and progesterone treatment. Progesterone, in turn, induces the specific epithelial cells of the tubular invaginations to produce distinct ducts, which branch from the main tubules.

The end result of the combined actions of estrogen and progesterone is a richly branched arborization of the gland. Highly differentiated secretory alveolar cells develop at the ends of these ducts under the influence of prolactin ( Figure 3-5 ).

Hormonal preparation of breast for lactation postpartum. PIF , prolactin-inhibitory factor.

(Modified from Vorherr H: The breast: morphology, physiology and lactation , New York, 1974, Academic Press.)

Serum growth factor, which is present in normal human serum, and insulin can stimulate the stem cells of the gland to proliferate. These dividing cells are further directed to the formation of alveoli by corticosteroid hormones. At least two types of cells are identified in the epithelial layer of the gland: stem cells and secretory alveolar cells. At this point in the pregnancy, prolactin influences the production of the constituents of milk.

TGF-β influences pattern formation in the developing mammary gland and may negatively regulate ductal growth as well. The pattern of mammary ductal development varies widely among species and is a function of both genotype and hormonal status. Normal human breast cells secrete TGF-β and are themselves inhibited by it, suggesting an autoregulatory feedback circuit that may be modulated by estradiol. Growth and patterning of the ductal tree are regulated in part by TGF-β operating through an autocrine feedback mechanism and by paracrine circuits associated with epithelial-stromal interactions.

The high circulating levels of prolactin in pregnancy are not associated with milk production partly because of the progesterone antagonism of the stimulatory action of prolactin on casein messenger ribonucleic acid (mRNA) synthesis. During late pregnancy, the lactogenic receptors, which have similar affinities for both prolactin and human placental lactogen (hPL), are predominantly occupied by hPL. High doses of estradiol impair the incorporation of prolactin into milk secretory cells.

Prolactin is prevented from exerting its effect on milk excretion by the elevated levels of progesterone. Following the drop in progesterone and estrogen at delivery, copious milk secretion begins. The key hormone requirements for lactation to begin are prolactin, insulin, and hydrocortisone. A high level of plasma prolactin is essential to lactogenesis in humans as well. There is a question as to whether it is a surge in prolactin that is necessary for lactogenesis at parturition. Prolactin levels are now described as biphasic in humans for the initiation of lactogenesis at birth. Prolactin stabilizes and promotes transcription of casein mRNA and stimulates synthesis of a lactalbumin that is the regulatory protein of the lactose-synthetase enzyme system. Prolactin further increases the lipoprotein lipase activity in the mammary gland. Prolactin exists in three heterogenic forms of varying biologic activity. The monomer is in greatest quantity and is the most active form.

Lactogenesis: Initiation of Milk Secretion

Stage I lactogenesis starts approximately 12 weeks before parturition and is heralded by significant increases in lactose, total proteins, and immunoglobulin and by decreases in sodium and chloride, and the gathering of substrate for milk production. The composition of prepartum secretion is fairly constant until delivery, as monitored by the milk protein α-lactalbumin.

Lactogenesis is initiated in the postpartum period by a fall in plasma progesterone, but prolactin levels remain high ( Figure 3-6 ). The initiation of the process does not depend on suckling by the infant until the third or fourth day, when the secretion declines if milk is not removed from the breast.

Prolactin levels in the postpartum period in women who are lactating (open circles) and nonlactating (dots). Levels in lactating women vary with intensity of suckling.

(From Neville MC, Morton J, Umemora S: The evidence for breastfeeding, Pediatr Clin North Am 48:44, 2001.)

Stage II lactogenesis includes the increase in blood flow and oxygen and glucose uptake as well as the sharp increase in citrate concentration, considered a reliable marker for lactogenesis stage II. Stage II at 2 to 3 days postpartum begins clinically when the secretion of milk is copious and biochemically when plasma α-lactalbumin levels peak (paralleling the period when “the milk comes in”). The major changes in milk composition continue for 10 days, when “mature milk” is established. The establishment of the mature milk supply, once called galactopoiesis, is now referred to as stage III of lactogenesis ( Figures 19-2 to 19-4 and 3-6 ).

The profound changes in milk composition have been established for the period of transition to mature milk in relationship to increase in milk volume. Detailed studies of successfully lactating women were performed by Neville et al., who report that a significant fall in sodium, chloride, and protein and a rise in lactose precede the major increase in milk volume during early lactogenesis. At 46 to 96 hours postpartum, copious milk production is accompanied by an increase in citrate, glucose, free phosphate, and calcium concentrations and a decrease in pH.

The breast, one of the most complex endocrine target organs, has been prepared during pregnancy and responds to the release of prolactin by producing the constituents of milk (see Figure 3-5 ). The lactogenic effects of prolactin are modulated by the complex interplay of pituitary, ovarian, thyroid, adrenal, and pancreatic hormones ( Figure 3-7 ).

Hormonal action necessary for phases of the lactation cycle.

(From Czank C, Henderson JJ, Kent JC, et al: Hormonal control of lactation cycle. In Hale TW, Hartmann PE, editors: Textbook of human lactation , Amarillo, Tex., 2007, Hale Publishing LP, p 91.)

Prolactin

Stricker and Grueter discovered the pituitary hormone prolactin in 1928. They observed that extracts of the pituitary gland induced lactation in rabbits.

Human prolactin is a significant hormone in pregnancy and lactation. Prolactin also has a range of actions in various species that is greater than any other known hormone. Prolactin has been identified in many animal species whether they nurse their young or not. Because of the original association with lactation, the term describes its action, “support or stimulation of lactation.” Prolactin, however, has been shown to control nonlactating responses in other species and has been identified with more than 300 different physiologic processes, unrelated to lactation. Study of prolactin was hampered until 1970, when it became possible to separate prolactin from human growth hormone (hGH) and to isolate and characterize prolactin from human pituitary glands.

Before 1971, hGH and prolactin in humans were considered the same hormone. Until 1971, in fact, it was thought that prolactin did not exist in humans. However, hGH is present in the human pituitary gland in an amount 100 times that of prolactin.

Although prolactin is secreted by the anterior pituitary gland, the brain is exposed to it. Prolactin is found in the cerebrospinal fluid and may even be produced by neurons in the portal vessels of the hypothalamus. Prolactin increases the activity of tuberoinfundibular neurons, which control dopamine.

Prolactin, the lactogenic hormone, is essential for glucocorticoid stimulation of the milk-protein genes. Little is known about the biochemical pathway of action of this important polypeptide hormone, which is required for both morphogenesis and expression of functional differentiation of the parenchyma of the breast (see Figures 3-8 and 3-9 ).

Breast growth and placental growth are closely associated.

(Modified from Cox DB, Kent JC, Casey TM, et al: Breast growth, Exp Physiol 84:421-434, 1999.)

Relationship between lactose excretion into urine and prolactin concentration in the blood during pregnancy.

(Modified from Cox DB, Kent JC, Casey TM, et al: Breast growth, Exp Physiol 84:421-434, 1999.)

Synthesis and secretion is not restricted to the anterior pituitary gland, but includes multiple sites in the brain (cerebral cortex, hippocampus, amygdala, cerebellum, brainstem, and spinal cord). It is also produced in the placenta, amnion, decidua, and uterus. Evidence suggests that lymphocytes from the immune system, thymus, and spleen release bioactive prolactin. Prolactin is found in epithelial cells of the lactating mammary gland and the milk itself. Prolactin reaches the milk by crossing the mammary epithelial cell basement membrane, attaches to a specific prolactin binding protein, and ultimately moves by exostosis through the apical membranes into the alveolar lumen. Prolactin mRNA in milk contains more prolactin variants than serum. Milk prolactin participates in the maturation of the neuroendocrine and immune systems.

The information generated by the use of knockout mice with prolactin knockouts or prolactin receptor knockouts has refined the understanding of mammary morphogenesis and subsequent lactogenesis. It has been confirmed that prolactin does not operate alone but depends on estrogen, progesterone, and glucocorticoids, as well as insulin, thyroid hormone, parathyroid hormone, and even oxytocin. Prolactin also stimulates uptake of some amino acids, uptake of glucose, and synthesis of milk sugar and milk fats (see Figure 3-7 ).

Plasma prolactin varies in relation to psychosocial stress. Utilizing four different real-life stress studies in a longitudinal design, Theorell found that changing situations associated with passive coping are accompanied by increased plasma prolactin levels. Changing situations associated with active coping are associated with unchanged or even lowered prolactin levels. The regulation of plasma prolactin is part of a dopaminergic system (see the list of pharmacologic suppressors in the next section).

In vitro, prolactin stimulates the synthesis of the mRNA of specific milk proteins by binding to membrane receptors of the mammary epithelial cells. Prolactin has been demonstrated to penetrate the cytoplasm of these cells and even their nuclei. These specific actions in the gland require the presence of extracellular calcium ions. Some prolactin actually appears in the milk substrate itself, the functional significance of which is uncertain, although it is thought to influence fluid and ion absorption from the neonatal jejunum.

The effect of the stimulation of protein synthesis by allowing the expression of milk protein genes is not a direct effect of the hormone, but rather the consequence of the activation of sodium/potassium adenosinetriphosphatase (Na/K ATPase) in the plasma membrane. The intracellular concentration of potassium is kept high and that of sodium low compared with the concentrations in extracellular fluid. As a result, the Na/K ratio is high both in the milk and in the intracellular fluid. Further action of prolactin has been identified in the development of the immune system in the mammary gland and, possibly more directly, in the lymphoid tissue. In conjunction with estrogen and progesterone, prolactin attracts and retains immunoglobulin A (IgA) immunoblasts from the gut-associated lymphoid tissue for the development of the immune system for the mammary gland. A very sensitive bioassay has been developed using the in vitro biologic effect of prolactin to stimulate the growth of cell cultures for malignant niobium rat lymphomas.

The baseline levels of prolactin are essentially the same in normal male and female humans ( Table 3-2 ). Moreover, both men and women experience a rise in prolactin levels during sleep. There is also a normal diurnal variation in levels in both men and women. At puberty, the increase in estrogens causes a slight but measurable increase in prolactin. Prolactin increases during the proliferative phase of the menstrual cycle but not during the secretory phase. A number of factors, including some that are significant for the nursing mother, such as psychogenic influence and stress, increase prolactin levels. Anesthesia, surgery, exercise, nipple stimulation, and sexual intercourse also produce increased amounts in both lactating and nonlactating women. Prolactin levels increase as serum osmolality increases.

Although prolactin levels in maternal serum are well established, less is known about prolactin levels in the milk and their role in the newborn. Prolactin in milk is known to be biologically potent and is absorbed by the newborn. In the intestine, prolactin influences fluid, sodium, potassium, and calcium transport. Prolactin content is highest in the early transitional milk just after the colostrum in the first postpartum week (levels of 43.1 ± 4 ng/mL). Levels drop to 11.0 ± 1.4 ng/mL in mature milk over time until approximately 40 weeks postpartum.

Prolactin-Inhibiting Factor

PIF controls the secretion of prolactin from the hypothalamus. Prolactin is unusual among the pituitary hormones because it is inhibited by a hypothalamic substance. Catecholamine levels in the hypothalamus control the inhibiting factor, which is poured into the circulation as a result of dopaminergic impulses. Drugs and events that decrease catecholamines also decrease the inhibiting factor, causing a rise in prolactin. Dopamine itself can act directly on the pituitary gland to decrease prolactin secretion. Agents that increase prolactin by decreasing catecholamines, and thus the PIF level include the phenothiazines and reserpine.

Thyrotropin-releasing hormone (TRH) is a strong stimulator of prolactin secretion, but its physiologic role is not clear, because thyrotropin levels do not rise during normal nursing. In the postpartum period, a dose of TRH will cause a marked increase in prolactin. Even a nonnursing postpartum mother will experience engorgement and milk release when stimulated with TRH. Ergot, which is frequently prescribed for postpartum patients, inhibits prolactin secretion either by direct inhibition or by its effect on the hypothalamus.

Prolactin response to breast stimulation in lactating women is not mediated by endogenous opioids. Neither baseline nor stimulated prolactin values were affected by naloxone.

The following factors affect prolactin release in normal humans:

• •
  

  Physiologic stimuli

  
  
• •
  

  Nursing in postpartum women: breast stimulation

  
  
• •
  

  Sleep

  
  
• •
  

  Stress

  
  
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  Sexual intercourse

  
  
• •
  

  Pregnancy

  
  
• •
  

  Pharmacologic stimuli

  
  
• •
  

  Neuroleptic drugs

  
  
• •
  

  TRH

  
  
• •
  

  Metoclopramide (procainamide derivative)

  
  
• •
  

  Estrogens

  
  
• •
  

  Hypoglycemia

  
  
• •
  

  Phenothiazines, butyrophenones

  
  
• •
  

  Norepinephrine

  
  
• •
  

  Histamine

  
  
• •
  

  Acetylcholine

  
  
• •
  

  Pharmacologic suppressors

  
  
• •
  

  Apomorphine, bromocriptine, cabergoline

  
  
• •
  

  l -Dopa

  
  
• •
  

  Ergot preparations (2-Br-α-ergocryptine)

  
  
• •
  

  Clomiphene citrate

  
  
• •
  

  Large amounts of pyridoxine

  
  
• •
  

  Monoamine oxidase inhibitors

  
  
• •
  

  Pramipexole

  
  
• •
  

  Prostaglandins E and F2α

  
  
• •
  

  Ropinirole, rotigotine, selegiline

In pregnancy, prolactin levels begin to rise in the first trimester and continue to rise throughout gestation. In a nonnursing mother, prolactin levels drop to normal in 2 weeks, independent of therapy to suppress lactation.

At delivery, with the expulsion of the placenta, levels of PL, estrogens, and progesterone abruptly decline ( Figure 3-8 and 3-9 ).

PL disappears within hours. Progesterone drops over several days, and estrogens fall to baseline levels in 5 to 6 days (see Figures 3-6 and 19-2 to 19-4 ). Prolactin in nonlactating women requires 14 days to reach abaseline. Progesterone is considered the key inhibiting hormone, and decline in plasma progesterone levels is considered the lactogenic trigger for stage II lactogenesis. However, progesterone does not inhibit established lactation because breast tissue does not contain progesterone-binding sites. Estrogens enhance the effect of prolactin on mammogenesis but antagonize prolactin by inhibiting secretion of milk. After delivery, there are low estrogen and high prolactin levels. Suckling provides a continued stimulus for prolactin release. If prolactin, essential for lactation, is diminished by hypophysectomy or medication, lactation ceases. Baseline prolactin levels do eventually diminish to more normal levels months after parturition, although lactation may continue.

The surge in prolactin over baseline levels, however, is critical to milk production, not the baseline levels ( Figures 3-6, 3-10 , and 3-11 ). Although prolactin is necessary for milk secretion, the volume of milk secreted is not directly related to the concentration of prolactin in the plasma. Local mechanisms within the mammary gland that depend on the amount of milk removed by the infant are responsible for the day-to-day regulation of milk volume. Suckling stimulates the release of adenohypophyseal prolactin and neurohypophyseal oxytocin. These hormones stimulate milk synthesis and production of milk-ejection metabolic hormones, which are also necessary in the process of milk synthesis. Thus suckling, emptying the breast, and receiving adequate precursor nutrients are essential to effective lactation ( Figures 3-12 and 3-13 ).

Prolactin levels after suckling.

(From Battin DA, Marrs RP, Fleiss PM, et al: Effect of suckling on serum prolactin, luteinizing hormone, follicle-stimulating hormone, and estradiol during prolonged lactation, Obstet Gynecol 65:785, 1985.)

Percent increase in prolactin over baseline after suckling.

(From Battin DA, Marrs RP, Fleiss PM, et al: Effect of suckling on serum prolactin, luteinizing hormone, follicle-stimulating hormone, and estradiol during prolonged lactation, Obstet Gynecol 65:785, 1985.)

Immunoreactive prolactin determined in plasma samples collected from 11 mothers (at 1, 2, and 4 months) and from nine mothers (at 6 months), immediately before suckling (□) and 45 minutes after the commencement of suckling (•). Number of observations is shown in parenthesis. Twenty-four hour milk production (mL/24 h) of the same mothers determined by test weighing. Results are mean values + SEM.

(From Cox DB, Owens RH, Hartman PE: Prolactin and milk synthesis in women, Exp Physiol 81:1007–1020, 1996.)

Plasma prolactin measured by radioimmunoassay before, during, and after a period of nursing in three mothers, 22 to 26 days postpartum. Prolactin levels rose with suckling and not with infant contact.

(Modified from Josimovich JB, Reynolds M, Cobo E: Lactogenic hormones, fetal nutrition, and lactation. In Josimovich JB, Reynolds M, Cobo E, editors: Problems of human reproduction , vol 2, New York, 1974, John Wiley & Sons.)

When milk is not removed, secretion ceases in a few days, and the composition of the mammary secretion returns to a colostrum-like fluid. When the composition of the breast secretion of breastfeeding and nonbreastfeeding women was followed by Kulski and Hartmann, it was the same for 3 to 4 days. Thereafter, the sodium and chloride concentrations in the nonbreastfeeding women increased rapidly.

The regulation of milk production in full lactation is based primarily on infant demand. Maternal nutrition, age, body composition, and parity have only secondary impact. Suckling is a powerful stimulus to prolactin synthesis and secretion, and prolactin is necessary for milk secretion. The pulsatile nature of prolactin secretion makes it difficult to measure over time. Milk yield is not directly correlated to prolactin levels.

Two local mechanisms have been associated with milk volume control. An inhibitor of milk secretion builds up as milk accumulates. The actual volume of milk secreted may be reduced if the breast is not drained adequately. Distention or stretching of the alveoli also affects production and secretion of milk. Evidence indicates that a proteinaceous factor in milk itself actually inhibits milk production and is associated with residual milk in the breast. This has been identified as a feedback inhibitor of lactation (FIL).

It has been assumed that prolactin levels control the rate of milk synthesis. When 24-hour milk production was measured by Cox et al., however, the results were different. The short-term rates of milk synthesis (i.e., between feeds) and the concentration of prolactin in the blood and in the milk were measured from 1 to 6 months in 11 women. The 24-hour milk production remained constant (708 ± 54.7 g per 24 hours at 1 month and 742 ± 79.4 g per 24 hours at 6 months). Marked variation in short-term milk synthesis between breasts was observed. The baseline and suckling-stimulated prolactin levels declined over time but the peak over base remained. The concentration of prolactin in milk was related to the fullness of the breasts, being highest when the breasts were full. Cox et al. found no relationship between the concentration of prolactin in the plasma and the rate of milk synthesis in either the short or long term.

Evidence indicates that a proteinaceous factor in milk itself actually inhibits milk production and is associated with residual milk in the breast. This has been identified as a FIL. Prolactin circadian rhythm persists throughout lactation. Prolactin levels are notably higher at night than during the day, despite greater nursing times during the day. The highest levels in the study by Stern and Reichlin were when the least nursing occurred.

The most effective and specific stimulus to prolactin release is nursing. The stimulation is a result of nipple or breast manipulation, especially suckling, not a psychologic effect of the presence of the infant (see Figures 3-13 and 3-14 ). The prolactin-release reflex during nipple stimulation is suppressed in some adult women, being evidenced only during pregnancy and lactation.

The sense of milk coming in. Secretory activation precedes the sense of milk “coming in.” The distribution of the times when women first sensed the “coming in” of milk after normal delivery is compared with the changes in lactose and sodium concentrations of breast milk over the first postpartum days. The number of women for each time point was expressed as a cumulative percentage of the total number ( n = 107) of women. Lactose and sodium concentrations obtained from left and right breasts for each woman were averaged and presented as percentages of the maximum lactose and sodium concentrations over the 5 days.

(From Pang WW, Hartman PL: Initiation of human lactation: secretory differentiation and secretory activation, J Mammary Gland Biol 12:211–221, 2007.)

During human pregnancy, when serum prolactin rises steadily to 150 to 200 ng/mL at term, there is a brief drop in levels hours before delivery and then a rise again as soon as the neonate is suckled. The response to nipple stimulation can be abolished by applying local anesthetic. On the other hand, trauma or surgery to the chest wall can initiate a prolactin rise and, in some reported cases, milk production.

Although it was initially reported that the high levels of prolactin measured in the first days and weeks of lactation dwindled to normal baseline by 6 months and showed no response to suckling stimulus, later studies clearly showed a different picture with more sensitive assays. Prolactin does not drop to normal, but further stimulus causes a doubling of levels over baseline at all stages of lactation through the second year (see Table 3-2 ).

Acute prolactin and oxytocin responses were measured by Zinaman et al., who compared various mechanical pumping devices with manual expression and infant suckling. Prolactin response to mechanical expression in quantity and duration depended on the device used, with a full-size pulsatile electric pump eliciting the greatest response. This compared equally with infant suckling. There was no difference seen in oxytocin response with various devices. These data confirm that results in studies of milk production and release in humans also depend on the equipment used to stimulate the breast. Eight fully lactating women were followed through the first 6 months postpartum at 10, 40, 80, 120, and 180 days, recording serum prolactin, luteinizing hormone, follicle-stimulating hormone, and estradiol (zero time only) obtained just before the initiation of suckling and during the next 120 minutes. Samples were obtained at 0, + 15, + 30, + 60, and + 120 minutes. Prolactin levels were high the first 10 days (90.1 ng/mL) but slowly declined over 180 days (44.3 ng/mL). The stimulus of suckling doubled the baseline values. Mean estradiol levels were low at 10 days (7.2 pg/mL), then gradually rose to a mean of 47.3 pg/mL at 180 days postpartum in the subjects whose menses had resumed. In the amenorrheic subjects the estradiol levels remained low (4.25 pg/mL), whereas baseline prolactin remained high (63.6 ng/mL). The subjects were breastfeeding on demand, averaging 11 feedings (range 8 to 16) per day at 10 days and 8 feedings (range 5 to 12) at 120 and 180 days. All infants had stopped one night feeding, and two infants had started some solids between the third and fourth months.

When specific binding sites for prolactin were looked for in the tammar wallaby, many sites were demonstrated in the lactating mammary gland but not the inactive gland. Mammary prolactin receptors were also identified in the rabbit. Thus the increased binding capacity would enhance tissue responsiveness, which may explain the maintenance of full lactation in the face of falling concentrations of prolactin. Prolactin also plays a critical role in increasing maternal bile secretory function postpartum.

Human Placental Lactogen and Human Growth Hormone

Three main hormones are recognized in the lactogenic process: hPL, hGH, and prolactin. The progressive rise in prolactin during pregnancy parallels the rise in hPL, becoming measurable at 6 weeks’ gestation and increasing to 6000 ng/mL at term (see Figure 3-4 ). This parallel action contributed to the belief that prolactin and hPL were the same. Although the principal function of hPL and prolactin in humans is a lactogenic one, no lactation ordinarily appears before delivery, although some women report being able to express a few drops of colostrum.

First described in 1962, hPL has been studied more than lactogens from any other species. Extensive immunologic and structural homology exists between hGH and hPL, which probably explains their similar biologic activities. Concentrations of hPL increase steadily during gestation and decrease abruptly with the delivery of the placenta. A large-molecular-weight substance, hPL is derived from the chorion. Receptor sites that bind lactogen also bind protein and hGH. hPL has been associated with mobilization of free fatty acid and inhibition of peripheral glucose utilization and lactogenic action.

hGH is secreted from the anterior pituitary eosinophilic cells. These cells have been identified by staining techniques that distinguish them from those that produce prolactin. Toward the end of pregnancy, the cells that produce prolactin are noticeably more numerous, whereas those that produce hGH are “crowded out.” The role of hGH in the maintenance of lactation is poorly defined and may be synergistic with prolactin and glucocorticoids.

Prolactin, hGH, PL, and chorionic somatotropin form a family of polypeptide hormones from the same ancestral gene, even though prolactin and hGH are produced by the pituitary and PL and chorionic somatotropin by the placenta. The suckling stimulus in postpartum lactation causes a rapid increase in serum hGH and prolactin. hGH and prolactin evolve from the same precursor, and, although the hormones are distinct, the acute interruption of hGH secretion does not interfere with the milk secretion.

The possible role of TSH as a physiologic prolactin-releasing factor has been disproved by Gehlbach et al., who state that TSH is not responsible for the brisk release of prolactin with suckling. Normal lactation is possible in women with ateliotic dwarfism in the absence of detectable quantities of hGH. For any hormone to exert its biologic effects, however, specific receptors for the hormone must be present in the target tissue. Changes in serum concentration have no effect if receptors are not present in the mammary gland to bind the hormone.

Oxytocin was the first hormone studied in relation to breastfeeding and to the let-down reflex. Studies first explored its role in the initiation and progression of labor. Because it was measurable, isolated in the laboratory, and finally manufactured synthetically, our knowledge of oxytocin was more extensive than it was for prolactin until the last two decades.

Oxytocin is not just a female hormone; it is produced by both male and female humans, and it is increased not just during reproduction in women. It is now credited with producing increased responsiveness to receptivity, closeness, openness to relationships, and nurturing. The oxytocin circulating during breastfeeding has been credited with producing calm, lack of stress, and an enhanced ability to interact with infants. The calm and connectedness system is part of a system of nerves and hormones that together trigger these effects.

Oxytocin is a polypeptide found in all mammalian species and works though a mechanism through which it activates receptors on the outer surface of the cell membrane. Oxytocin is produced in the supraoptic and paraventricular nuclei of the hypothalamus. Receptors have been identified for oxytocin in the uterus and the breast as well as the brain. It acts via the bloodstream and as a signaling substance in the nervous system. Substances that act to stimulate the release of oxytocin include serotonin, dopamine, noradrenaline, and glutamate. Other substances, such as opiates, enkephalin, and β-endorphin, inhibit its release. Spinal anesthesia has been associated with the inhibition of oxytocin release after childbirth. Estrogen can increase the number of receptors and stimulate the production of oxytocin. The release of oxytocin by repetitive soothing touches or when given via injection produces a calming reaction and lowers blood pressure and pulse rate. Uvnäs Moberg has studied oxytocin extensively and calls it the hormone of calm, love, and healing.

Stage III Lactogenesis (Galactopoiesis): Maintenance of Established Lactation

Early studies in the past 100 years established that milk was synthesized in the mammary gland from substances removed from the maternal arterial blood supply. Then it was confirmed that milk ejection was the removal of stored milk and not from the rapid synthesis of milk. The enzymes and hormones involved have been identified. Understanding the molecular biochemistry and physiology of the gland has revealed the details of the production of milk. A number of genes encode for components that are part of the intricate signaling pathways. Complex interactions of signaling molecules with epigenetic factors interact at the level of gene expression. Intracellular signaling is basic to understanding normal human mammary development.

The basic features of milk production are the identification of the cell surface and intracellular receptors for extra cellular signals (12 hormones autocrine and paracrine factors according to Martin and Czank). Chain reactions convey the signal to a site of action. A class of compounds that regulate gene expression depend on modification to their structures and the nature of their binding to the genetic material.

The maintenance of established milk secretion, originally called galactopoiesis, is now labeled stage III lactogenesis, or simply lactation. An intact hypothalamic-pituitary axis regulating prolactin and oxytocin levels is essential to the initiation and maintenance of lactation. The process of lactation requires milk synthesis and milk release into the alveoli and the lactiferous sinuses. When the milk is not removed, the increased pressure lessens capillary blood flow and inhibits the lactation process. Lack of sucking stimulation means lack of prolactin release from the pituitary gland. Basal prolactin levels that are enhanced by the spurts that result from sucking are necessary to maintain lactation in the first postpartum weeks. Without oxytocin, however, a pregnancy can be carried to term, but the woman will fail to lactate because she will fail to let-down.

Sensory nerve endings, located mainly in the areola and nipple, are stimulated by suckling. The afferent neural reflex pathway, via the spinal cord to the mesencephalon and then to the hypothalamus, produces secretion and release of prolactin and oxytocin. Hypothalamic suppression of earlier PIF secretion causes adrenohypophyseal prolactin release. When prolactin is released into the circulation, it stimulates milk synthesis and secretion. A conditioned milk ejection can occur in lactating women without a concomitant release of prolactin, so that indeed the releases are independent, which may be significant in treating apparent lactation failure ( Figure 3-15 ).

Neuroendocrine control of milk ejection.

(Modified from Vorherr H: The breast: morphology, physiology and lactation , New York, 1974, Academic Press.)

Hormonal Regulation of Prolactin and Oxytocin

The release of prolactin is inhibited by PIF. The PIF has not been described but is closely associated with dopamine. There is also evidence of either serotonin release of prolactin or catecholamine-serotonin control of prolactin release. TSH has also been shown to stimulate the release of prolactin. The amount of prolactin is proportional to the amount of nipple stimulation during early stages of lactation after the first 4 days. Milk synthesis proceeds for the first 4 days whether or not the breast is stimulated. At this time, prolactin levels are the same for lactators and nonlactators ( Figure 3-16 ).

An overview of the regulation of prolactin secretion. Prolactin secretion is paced by a light-entrained circadian rhythm, which is modified by environmental input, with the internal milieu and reproductive stimuli affecting the inhibitory or stimulatory elements of the hypothalamic regulatory circuit. The final common pathways of the central stimulatory and inhibitory control of prolactin secretion are the neuroendocrine neurons producing prolactin-inhibiting factors (PIF), such as dopamine (DA), somatostatin (SST), and γ-aminobutyric acid (GABA), or prolactin-releasing factors (PRF), such as thyrotropin-releasing hormone (TRH), oxytocin (OT), and neurotensin (NT). PIF and PRF from the neuroendocrine neurons can be released either at the median eminence into the long portal veins or at the neurointermediate lobe, which is connected to the anterior lobe of the pituitary gland by the short portal vessels. Thus lactotrophs are regulated by bloodborne agents of central nervous system or pituitary origin (α-melanocyte stimulating hormone) delivered to the anterior lobe by the long or short portal veins. Lactotrophs are also influenced by PRF and PIF released from neighboring cells (paracrine regulation) or from the lactotrophs themselves (autocrine regulation).

(From Freeman ME, Kanyicska B, Lerant A, Nagy G: Prolactin: structure, function and regulation of secretion, Physiol Rev 80:1523–1630, 2000.)

Although both oxytocin and prolactin release are stimulated by nipple stimulation, some oxytocin is released by other sensory pathways, such as visual, tactile, olfactory, and auditory. Thus a woman may release milk on seeing, touching, hearing, smelling, or thinking about her infant. Prolactin, however, is released only on nipple stimulation so that milk production is not initiated by other sensory pathways. Oxytocin is also released under physical stress, such as pain, exercise, cold, heat, changes in plasma osmolality, or hypovolemia, but these responses are blunted or reversed during lactation.

When suckling occurs, oxytocin is released. It enters the circulation and rapidly causes ejection of milk from alveoli and smaller milk ducts into larger lactiferous ducts and sinuses. This is the pathway of the let-down, or ejection, reflex. Oxytocin also causes contraction of the myometrium and involution of the uterus ( Figure 3-17 ).

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