Prepubertal and Postpubertal Mammary Development

Generally, there is little or no true lobulo‐alveolar development before conception. This period is associated with the creation of a framework to allow the proliferation of the secretory alveolar cells needed for lactation. The period is generally a time during which the duct system is extended and the growth of the adipose and connective tissue increases. Ductular growth is limited for dogs, cats, and rabbits but more extensive for rats, mice, the rhesus monkey, cattle, and sheep.

Because it is expected that most organs in growing animals would also grow in concert with rate of overall body growth, the regulated, cyclic development of mammary glands can be difficult to study under these conditions. One approach is to evaluate mammary development in terms of relative growth and to ask if growth of the mammary gland fits the law of simple allometry y = bx^α. A usual approach is to log transform variables associated with body and tissue growth and use linear regression analysis to calculate the equilibrium constant (α), which relates the difference in the growth rate of the organ under study to the growth of the body as a whole. Simple body weight or body weight^2/3 (to approximate surface area) is usually used as the independent variable in these analyses. The dependent variable (mammary gland area, mammary gland weight, DNA content, weight of parenchymal mass) serves as an index of the growth of the mammary gland. When α = 1, growth is said to be isometric. If α > 1 the growth rate is positively allometric (simple allometry) but an α < 1 indicates the growth rate of the organ is negatively allometric (enantiometry).

Data for rodents, cattle, and sheep demonstrate that from birth until before puberty, the mammary gland grows only somewhat faster than the rest of the body. Thereafter the rate of mammary gland growth becomes markedly allometric usually through the first few estrus cycles. Growth then reverts to a period of isometric growth that also waxes and wanes during the estrus cycle. As demonstrated for heifers, the classic study by Sinha and Tucker (1969) shows that positive allometric begins at about three months of age and continues until about nine months of age. Specifically, when mammary development was expressed a log of mammary DNA and body growth as a log of body weight, the equilibrium constant (α) averaged 1.6 in the period from birth to 2 months, 3.5 from 3 to 9 months, and 1.5 between 10 and 12 months of ages (Fig. 18.2). These data indicate that the mammary gland grows somewhat faster than the body during the early postpartum period but dramatically faster between three and nine months of age.

Although the absolute amount of mammary growth during the period before conception is only a fraction of the mature mammary gland late in gestation, there is compelling evidence that alterations in mammary growth during the peripubertal period can affect subsequent mammary function. For example, rapid prepubertal weight gain inhibits subsequent mammary parenchyma tissue growth and can reduce subsequent milk production (Sejrsen and Purup 1997). Not surprisingly, very few cattle studies have considered changes in mammary development in the period shortly after parturition. While minor in terms of absolute mass, our observations of mammary glands of Holstein calves between one and three months of age (Capuco and Akers, 2010; Akers, 2017) suggest that parenchymal tissue per gland increases approximately 60‐fold in only a few weeks. It is intriguing to consider that growth during even this very early postnatal period may also be critical to the success of lactation (Akers, 2017a,b; Sejrsen et al., 2000).

Geiger et al. (2016) showed that differences in feeding in calves could dramatically alter the growth of the parenchymal portion of the mammary gland between one and two months of age. This difference is dramatically illustrated in Figure 18.3. Such data suggest that mammary growth and development may be more malleable than generally appreciated and not nearly as quiescent in early life in ruminants as once supposed. Of course, a key aspect is whether changes in early mammary development significantly impact either future growth driven by puberty or pregnancy or future milk production.

It may be that induction and/or regulation of the appearance of mammary stem cells early in life can indeed impact future development and function. As described in Chapter 4, new tools and techniques are emerging to better understand the role of stem cells in the regulation of mammary development and function (Capuco et al., 2012; Choudhary and Capuco, 2021).

Figure 18.4 illustrates various stages of mammary development using the mouse mammary gland as an example. Small rodents are especially useful for this purpose because an entire mammary gland can be removed, spread onto a microscope slide, fixed, defatted, and stained. Observing structural differences in mammary glands from various physiological stages quickly illustrates the dramatic changes in the development of the mammary epithelium as the gland prepares for the onset of lactation. However, a word of caution is advised. Although the fundamental developmental processes are similar in rodents and other species, there are clearly substantial differences in the tissue composition of the mammary gland in rodents compared with other species, as well as differences in the pattern of ductular development.

Compared with the rodent pattern of development where the end buds allow for the filling of the entire mammary fat pad during the prepubertal period (Fig. 18.4), the developmental pattern in the ruminants is more compact. Specifically, in rodents, as the epithelial ducts develop, they are surrounded by stromal cells and the stromal tissue contains a sea of adipocytes. A higher power view (Fig. 18.5B) of a similar area from the mammary gland of a prepubertal heifer illustrates several epithelial structures that radiate from a mammary duct. Pre‐ and post‐puberty cross‐sectioned ducts often demonstrate a scalloped appearance, suggesting a complex tubular structure. Indeed, three‐dimensional, computer animations prepared from serial sections of prepubertal bovine mammary parenchymal tissue elegantly confirm this tissue architecture (Capuco et al., 2002). This suggests that in the ruminant, in contrast with rodents, the gland is not filled with elongated ducts during the prepubertal period. To use a plant analogy, in the peripubertal rodent, widely spaced mammary ducts fill the mammary fat pad like the bare branches of a tree. In the peripubertal ruminant mammary gland, closely packed ducts radiate from the gland cistern in broccoli‐like fashion, but the ducts generally fill only a fraction of the mammary fat pad.

As the gland continues to develop the relative tissue area occupied by the epithelium increases at the expense of the surrounding stromal tissue. This is especially evident with the growth of alveoli during gestation. It is also evident that much of the tissue area in histological sections of mammary tissue taken from cows in late gestation and lactation is also occupied by lumenal space. This does not mean that there is necessarily a loss of stroma cells as the gland develops but rather that there is a dramatic rearrangement of cells and tissue elements so that the stromal cells are less evident in histological sections. As gestation advances clusters of alveolar structures appear like scattered, round islands, until late in gestation, when histological fields are filled with closely packed alveoli. Under normal circumstances, true alveoli are not formed until well into gestation. Variation among species reflects the number of estrus cycles that occur before conception. Using cattle as an example, in the early stages of pregnancy the duct system continues to develop with the appearance of a rudimentary lobulo‐alveolar system by about five months of gestation.

Mammary Development During Pregnancy

It is estimated that 94% of mammary development for hamsters takes place as pregnancy advances. Estimates for other species range from 78% for the mice and sheep to 66% for rabbits and 60% for rats. By far the greatest promoter of natural mammary growth or mammogenesis is pregnancy and associated hormonal and physiological changes. With the influence of pregnancy, mammary growth is reinitiated after reversion to isometric growth following puberty. This growth can be described by an exponential equation with the following form: Y = a^bt, where Y = mammary size, t = day of gestation, and the terms a and b are constants. Such equations have been developed to model mammary growth in cows, goats, and guinea pigs. The term a is mammary gland mass or size at the beginning of gestation and the term b is the first‐order rate constant. The time necessary for the mammary gland to double is equal to ln2/b (Sheffield, 1988).

Measurement of mammary DNA or weight of dissected parenchymal tissue is useful to quantify rates of mammary growth, but these techniques do not distinguish differences among cell types. Problems of trying to distinguish between stromal and parenchymal tissue are especially difficult in rodents. For example, both wet weight and defatted dry weight of the inguinal mammary glands of rats are reported to vary from 50% to 120% during pregnancy but total mammary DNA changes 200%–300%. Because total DNA reflects cell number, changes in weight alone underestimate cellular development. Table 18.2 illustrates changes in mammary development of ewes during gestation. Changes in weight, total DNA, or % epithelium show a marked increase in parenchymal tissue between days 80 and 115 of gestation. However, between days 115 and 140 quantitative histology (% epithelial area) alone suggests there is no continued parenchymal development. This is clearly not true because both tissue weight and total DNA doubled in this time. Lack of change in the epithelial area between the days 115 and 140 is a reflection that alveoli are present at these stages of gestation, but an increase in lumen and decrease in stromal tissue area reflect the accumulation of some secretions and compression of stromal tissue between alveoli. In addition to the appearance of alveoli during gestation, the cells undergo progressive structural and biochemical maturation as parturition approaches. The increase in DNA labeling index on day 115 also reflects the rapid growth during this period.

	Day of Gestation
Measurement	Day 50	Day 80	Day 115	Day 140
Trimmed udder weight (g)	304 ± 43	253 ± 30	557 ± 29	1050 ± 188
Total DNA (mg)	94 ± 4	57 ± 14	1304 + 152	2324 ± 321
% Epithelium	14.2 ± 0.3	19.2 ± 2.1	41.2 ± 2.3	40.3 ± 1.8
% Stroma	85.8 ± 0.3	77.8 ± 3.2	35.7 ± 6.5	20.7 ± 3.0
% Lumen	NP	1.0 ± 0.2	23.1 ± 4.3	39.0 ± 2.9
Alveolar cell No.	NP	NP	36.4 ± 3.8	36.6 ± 1.4
Labeling index	0.16 ± 0.03	1.0 ± 0.2	3.6 ± 1.7	1.0 ± 0.4

Mammary growth as measured by total DNA continues during the early days of lactation in rats, rabbits, guinea pigs, and mice. In the rat, for example, as much as 26% of the total mammary growth occurs during lactation. In the mouse, there is a transient surge in mammary cell proliferation two to three days postpartum. In fact, it is reported that the cell population doubles between the last day of gestation and day five of lactation. The guinea pig is especially interesting in that there is little change in total mammary DNA during gestation but a dramatic increase within two days of the birth of the pups. However, if suckling is not permitted in these species the increased growth just after parturition is prevented. This suggests that signals associated with suckling or milking are important for growth, especially in early lactation. Regardless it seems clear that once lactation is established the rate of mammary cell proliferation is markedly lower than during other stages of mammary development. However, recent reports show that several weeks of “extra” milking stimulation within the first two months of lactation in dairy cows increase subsequent milk production when the milking frequency returns to normal. Mechanisms for the effect are unknown but the fact that it continues after treatment supports the idea that it may involve recruitment of additional well‐differentiated cells. This might reflect overt proliferation of additional secretory cells or perhaps enhanced functional differentiation in a population of preexisting nonsecretory cells (Bar‐Peled et al., 1995). This effect contrasts with the galactopoietic treatment of cows with bovine somatotropin (bST) in that milk production is dramatically increased during the period of treatment but returns to control levels when treatment is discontinued. This pattern of response has been interpreted to indicate that bST alters metabolism rather than cell proliferation. This is clearly an area of continuing research interest.

In dairy species, it is usually concluded that there is normally little mammary growth during established lactation. However, comprehensive data for early lactation is lacking. The concentration of DNA in mammary parenchyma is relatively unchanged in early lactation in sheep, goats, or cows but it is risky to interpret this as a lack of mammary growth because it does not evaluate the entire mammary gland. Total parenchymal DNA doubles between two weeks before and after parturition (27.9 vs. 46.0 g) but these data do not determine if the growth occurs before or after parturition. Rates of thymidine incorporation are very low for mammary tissue taken from lactating ruminants, but mitotic cells are sometimes observed.

In the goat unilateral inhibition of secretion in one mammary gland results in a compensatory increase in milk production of the other gland. There are also reports of increased milk production in cows by uninfected quarters among cows with mastitis. The relative contributions of hypertrophy and hyperplasia to this effect are unknown. However, in lactating beef cows, covering one half of the udder to prevent sucking increased thymidine incorporation in lactating mammary glands compared with control glands of cows that continued suckling in all glands. Morphologically, lactating tissue from control and compensatory treatment cows was indistinguishable, and the tissue did not appear consistently different between zones within lactating quarters. About 40% of the parenchymal tissue consisted of closely packed alveoli. In some areas, the secretory cells appeared highly differentiated like cells from dairy cows. Remaining parenchymal tissue contained alveoli more widely scattered in the stromal matrix and the cells were less well differentiated. Labeled cells were observed in both regions but appeared more frequently in the less well‐differentiated tissue (Akers et al., 1990).

Figure 18.6 shows changes in the growth of mammary parenchymal beef and dairy heifers during pregnancy and into lactation. Comparisons between panels A and B illustrate those measures of udder parenchymal mass alone but can be misleading. Specifically, based on udder parenchymal mass there is marked growth of parenchymal tissue between day 260 and day 49 of lactation in both Holsteins and Herefords (Fig. 18.6A). In contrast, when udder growth is evaluated based on parenchymal DNA (Fig. 18.6B), the marked differences between breeds remain but little change occurs between day 260 of gestation and day 49 of lactation in Holsteins. This is likely explained by the increased accumulation of secretions in the Holstein heifers compared with the Hereford heifers. When lactation performance is considered, differences in milk production (3.5 vs. 20.3 kg/d) are explained by both changes in udder parenchymal tissue mass and function. For example, the total RNA, as well as RNA/DNA ratio, is greater in Holstein than in Hereford heifers. Interestingly, the ability of mammary explants from lactating animals to secrete α‐lactalbumin (a specific milk protein) closely mirrored (57 vs. 289 ng per mg of tissue per 24 h) the corresponding 5.8‐fold difference in daily milk production. The relative failure of cytological differentiation (Akers et al., 2006) in mammary tissue of beef compared with dairy heifers is puzzling because milking stimulation and management were identical between breeds in this study. Is it possible that selection for increased milk production (in dairy cattle) has allowed for the maximization of differentiation signals during the critical periparturient period?

Realization in the early 1960s that the DNA content of cells is essentially constant (except for the generally small proportion of cells that are undergoing DNA synthesis in preparation for cell division at a given moment) ushered in a host of studies to estimate mammary cell number based on total DNA content. This method is especially valuable when combined with careful dissection of the mammary gland to distinguish the parenchymal portion from the stromal tissue of the mammary gland. Even with care dissection of the mammary gland to remove apparent connective tissue, there are clearly nonglandular cellular elements, that is, blood vessels, lymphatic vessels, nerves, fibroblasts, adipocytes, and white blood cells, which contribute to the DNA content of the parenchymal tissue compartment. Regardless, classic studies in a variety of lactating species give direct evidence that the number of mammary epithelial cells is proportional to milk production. The correlation between total parenchymal DNA and milk production averages about 0.85. Consequently, any activity which reduces the number or function of the mammary alveoli will also reduce milk production (Tucker, 1981, 1987).

In addition to the non‐secreting epithelial cells and various stromal cells, extracellular secretions, that is, proteins and proteoglycans, that surround the mammary ducts and alveoli are also critical for development. Collagen is the major extracellular protein component of the mammary stromal tissue. Moreover, the amino acid hydroxyproline is a specific and major component of collagen. Thus, the assay of tissue content of hydroxyproline provides a quantitative measure of stromal tissue. When coupled with the measurement of fat, the relative amounts of connective tissue associated with the parenchymal tissue can be estimated.

Data in Tables 18.1 and 18.3 illustrate the dramatic changes in mammary growth from birth to lactation in Holstein heifers and crossbred ewes. Measured as trimmed udder weight or parenchymal DNA, mammary growth is greatest during gestation. However, the relative lack of change in DNA from late gestation into lactation compared with trimmed udder weight suggests that DNA is a better measure of cell growth because increased weight may accumulate as secretions.

	Stage of Development
Measure	Prepuberty	Postpuberty	Mid Gest	Late Gest	Lactation
Heifers
DNA (g)	1.1	2.6	16.3	39.3	38.8
Wt. (g)	495	957	5110	8560	16,350
Ewes
DNA (g)	0.02	0.09	1.3	2.3	2.6
Wt. (g)	15	78	557	1057	1340

Measurement of parenchymal tissue RNA and/or protein indicates synthetic capacity and is useful to evaluate the fully developed mammary gland. The reason for this is simple. As the secretory cells differentiate in concert with parturition, there is a marked increase in the presence of rough endoplasmic reticulum (RER) and the corresponding production of mRNA for specific milk proteins. Consequently, the onset of milk synthesis and secretion corresponds with a marked increase in the mammary tissue RNA. On a per‐cell basis, increased synthetic capacity can be evaluated by comparing the ratio of RNA/DNA in mammary parenchymal tissue. Late in gestation after alveoli have formed but before the onset of copious milk synthesis and secretion this ratio is generally about one. During established lactation, the RNA content of the secretory cells increases dramatically so that this ratio more than doubles. Changes in parenchymal tissue protein follow a similar pattern; however, care must be taken to account for the milk protein, which may be trapped in the tissue. Finally, any of these measures are more useful if they can be applied on a whole mammary gland basis. For rodents or other animals, in which an entire mammary gland (or parenchymal tissue) can be isolated and sampled, these measures can be related to milk production.

Alveolar Cell Differentiation and Lactogenesis

A consistent theme that we have emphasized is that structure follows function. The structural differentiation of the alveolar cells around the time of parturition illustrates this principle. As parturition approaches, these cells become polarized, with the appearance of abundant arrays of RER, numerous mitochondria, and competent tight junctions, just as full‐scale milk synthesis and secretion are initiated. This process is called lactogenesis. Two events are critical. The structural differentiation of the alveolar epithelial cells allows for packaging of milk components for secretion while biochemical differentiation allows for synthesis of milk constituents. We will begin by reviewing some of the structural changes.

The mammary gland begins to reach its mature structural state late in gestation. That is alveoli and mammary ducts are in place. Indeed, soon after the alveoli are formed some accumulation of secretions begins. For example, in cows during first gestation, small but variable volumes of secretions can be expressed from the mammary gland several months before parturition. Prepartum milking of dairy heifers is sometimes initiated during the month before parturition as a management technique to relieve intramammary pressure. Irrespective of prepartum milking, secretions obtained before calving are generally high in protein and low in lactose compared with normal milk but with relatively small concentrations of specific milk proteins. Because extensive removal of mammary secretions before calving can alter the course of mammary development, that is, premature mammary cell differentiation, it is recommended that prepartum milking once initiated should continue until the onset of normal milking at calving.

High concentrations of protein in secretions obtained prepartum in cows reflect the accumulation of immunoglobulins transferred into secretions from the bloodstream. As the alveolar cells differentiate, the accumulation of immunoglobulins is reduced. However, the accumulated immunoglobulins in the secretions obtained with the first milking or suckling postpartum, that is, colostrum, provide passive immunity to the offspring. This is critical for those species that lack immunoglobulin transfer to the fetus in utero. With the onset of regular milking or suckling, the composition of mammary secretions progressively changes to reflect normal milk composition.

Figure 18.7 illustrates the major structure of a mammary alveolus, while Figure 18.8 and Figure 18.9 illustrate the dramatic change in alveolar cell structure that accompanies lactogenesis. Much of this change is coordinated by alterations in circulating hormones and changes in hormone signaling on mammary target cells.

Ultrastructure of the mammary epithelium during lactation has now been documented in many species. The percentages of various cellular organelles within the cytoplasm of alveolar secretory cells from lactating rats and cows are illustrated in Table 18.4. This generally uniform structure includes basal and paranuclear cytoplasm occupied by parallel arrays of RER. The supranuclear Golgi apparatus typically consists of stacks of smooth membranes whose terminal cisternae release casein and lactose‐containing secretory vesicles. Lipid droplets and secretory vesicles seemingly fill the apical ends of the cells and microtubules are most frequently observed oriented perpendicular with respect to the apical plasma membrane. Mitochondria and free ribosomes are abundant throughout the basal‐lateral cytoplasm. The basal plasma membrane is often thrown into complex folds, which are believed to indicate active pinocytosis. Myoepithelial cells frequently occur interspersed between the basal plasma membrane and the basal lamina (Safayi et al., 2012), which forms a loose barrier between the simple cuboidal epithelial cells and the underlying stromal tissues. It is now apparent that parenchymal tissue from glands of high‐yielding animals has an abundance of structurally highly differentiated polarized alveolar cells.

Hormonal Control of Lactogenesis and Lactation

The endocrine system, perhaps more than any other physiological system, plays a central role in all aspects of mammary development (mammogenesis), the onset of lactation (lactogenesis), and the maintenance of milk secretion (galactopoiesis). Lactogenesis is frequently described as a two‐stage process. Stage I consists of limited structural and functional differentiation of the secretory epithelium during the last third of pregnancy. Stage II involves the completion of cellular differentiation during the immediate periparturient period coinciding with the onset of copious milk synthesis and secretion. During lactation, the secretory cells synthesize and secrete copious amounts of carbohydrates, proteins, and lipids. Production of this complex mixture of nutrients depends on coordination between biochemical pathways to supply metabolic intermediates and secretory pathways for secretion. For example, the disaccharide lactose is the predominant milk sugar. The enzyme complex necessary for lactose synthesis, membrane‐bound galactosyltransferase, and the whey protein α‐lactalbumin combine in the Golgi apparatus to form lactose synthetase, which links glucose and galactose producing lactose. Activation of the α‐lactalbumin gene and synthesis of α‐lactalbumin is most closely associated with stage II of lactogenesis.

Experiments beginning in the 1920s (Stricker and Grueter, 1928) showed that milk secretion could be induced in virgin rabbits by injecting a pituitary extract. In 1933, Riddle, Bates, and Dykshorn purified the protein responsible for the milk secretion response observed by Stricker and Grueter, they named it prolactin (Prl). Even now, the widely touted and utilized galactopoietic effect of somatotropin to increase milk production in lactating dairy cows had its foundation in studies by Asimov and Krouze in the 1930s. They showed that injections of pituitary extracts consistently increased milk production in lactating cows (Asimov and Krouze, 1937). Scientists describing and quantifying the potent effects of pregnancy on mammary growth and changes in the mammary gland at puberty spurred others to isolate and identify the steroid hormones estrogen and progesterone. Advances in purification techniques and understanding of steroid hormone chemistry allowed further studies, leading to the production of these steroids for widespread animal testing.

Although the existence of mammogenic and lactogenic substances from the pituitary had long been known, the efforts of Li et al. in the 1940s to purify larger quantities of Prl and growth hormone (GH) were essential (Riddle et al., 1933). Soon thereafter, specific roles for these hormones in the regulation of mammogenesis in rodents were delineated in classic ablation replacement experiments (Lyons, et al., 1958; Nandi, 1958). In an extensive series of studies, triply operated (adrenalectomized, ovariectomized, and hypophysectomized) rats and mice were treated with various combinations of purified hormones to see if normal mammary development could be restored. Injections of estrogen and GH together caused proliferation of mammary ducts. However, treatment with estrogen, progesterone, Prl, and GH was needed for lobulo‐alveolar development. The maximum ductular and lobulo‐alveolar development, although still less than in pregnancy, was obtained in animals also given glucocorticoids. For some strains of mice, GH and Prl were both capable of stimulating lobulo‐alveolar development.

British researchers focused on efforts to improve and maintain milk supplies during World War II, initiated many endocrine studies on mammary development and function in dairy animals (Cowie et al., 1980). For example, the effects of estrogen and progesterone on mammogenesis were extensively evaluated in attempts to induce lactation in nonpregnant animals. Although difficulties with needed surgeries and expense continue to limit the use of ablation replacement experiments to study mammary development and function in cattle, Cowie et al. (1966) studied hypophysectomized–ovariectomized goats and showed that mammary development comparable to mid‐gestation could be obtained in animals treated with a combination of estrogen, progesterone, Prl, GH, and adrenocorticotrophic hormone (ACTH). Such experiments served to confirm that at least general effects attributed to these hormones on mammary development in rodents, applied to mammary development in dairy animals.

Although it should be clear that mammogenesis involves more than increased secretion of estrogen and progesterone, which occurs during pregnancy, this is nonetheless critical. During the estrus cycle, estrogen concentrations increase because of follicle growth but with the appearance of the dominant follicle and ovulation concentrations of estrogen decline and progesterone concentrations increase. Because estrogen and progesterone are both important for final duct growth and lobulo‐alveolar development, the lack of a sustained simultaneous increase of both steroids during the estrus cycle likely explains the lack of marked parenchymal tissue development at this time. Furthermore, few of the studied species show evidence of lobulo‐alveolar development before conception. With conception, the corpus luteum is maintained, and along with increasing production by the placenta in many species, blood concentrations of progesterone are elevated throughout gestation. Concentrations of estradiol are higher (relative to the estrus cycle) with a gradual increase during gestation until a more dramatic increase during the final few weeks before birth. Consequently, concentrations of estrogen and progesterone are both simultaneously elevated during much of gestation, especially during the later portion of gestation. This is believed to be responsible for much of the mammary growth during gestation. Prl and possible prolactin‐like activity associated with the secretion of placental lactogen (PL) are also important in mammogenesis in some ruminants (sheep and goats). In cattle, Prl is most likely a permissive agent for the mammogenic effects of steroids and other growth factors. There are no specific changes in the secretion of Prl in cattle associated with mammogenesis and lobulo‐alveolar formation during pregnancy.

An interesting accidental discovery reported by Smith and Schanbacher (1973) created a flurry of activity on hormonal induction of lactation in cattle. These researchers were studying the effects of steroids on the secretion of immunoglobulins into mammary secretions of non‐lactating cows, that is, animals that had failed to conceive. They observed that injections of estradiol‐17‐β and progesterone for only seven days, at doses that mimicked blood concentrations in animals near calving, caused the udders of some of the animals to “bag up”. When these animals were milked the initial colostrum‐like secretions rapidly gave way to secretion of milk. As reviewed (Akers, 1985; Akers 2017), subsequent studies showed that lactation could be induced in about 70% of these nonpregnant cows and that milk yields for the successful animals averaged about 70% of normal. Others found positive correlations between the success of induced lactations and concentrations of Prl and well as improved yields when cows were also given drugs to induce Prl secretion. Moreover, greater milk yields measured for cows induced into lactation during the spring and summer were attributed to higher concentrations of Prl in serum compared with cows treated during winter months.

Although changes in blood concentrations of mammogenic hormones are important in explaining changes in mammary growth, changes in tissue sensitivity and availability of biologically active hormones are also important (Purup et al., 1993). In circulation, the steroid hormones are bound to transport proteins. Even in tissues, the steroids can become sequestered with cellular lipids and therefore effectively unavailable (Capuco et al., 1982). This effect coupled with a decrease in progesterone receptor concentration and a change in isoforms of the receptor explains the disappearance of the negative effect of progesterone on lactogenesis at the time of parturition in cattle. This illustrates the concept that the biological effectiveness of circulating hormones may change independent of the total blood concentration of the hormone. In addition, changes in expression, synthesis, or availability of hormone receptors in target cells also clearly impact biological response.

With the advent of techniques to radiolabel hormones (see Chapter 12), it became possible to measure the apparent number of receptors in tissues and cells. This typically requires the isolation and homogenization of tissues to isolate cell membranes (the usual site for protein hormone receptors) or cell nuclei for evaluation of steroid receptors. These studies were powerful and served to emphasize that changes in the numbers of receptors in target cells did indeed change in correspondence with the physiological effects of many important mammogenic or lactogenic hormones. For example, data from an assay of ovine mammary tissue shows that expression of the progesterone receptor occurs in close correspondence with the appearance of lobulo‐alveolar development (Table 18.2). Serum concentrations of progesterone are consistently elevated during gestation with higher concentrations in later stages of gestation and in ewes with more than one fetus. Table 18.5 illustrates changes in serum concentrations of some important mammogenic hormones during gestation, as well as mammary tissue receptor concentration (Akers and Keys, 1984).

As is often true, technical advances have allowed more detailed evaluation of the expression of many hormone receptors in specific cells in various tissues, including the mammary gland. The creation of very specific antibodies against hormone receptors (and other cell markers), fluorescent tags, fluorescence microscopy, advanced computing power, and multispectral analysis, coupled with rapid improvements in quantitation of digital images, is allowing evaluation not just identification of populations of cells expressing hormone receptors but simultaneous identification of multiple receptors and levels of expression. As envisioned by Ellis et al. (2012) these technical advances offer tremendous opportunities to advance understanding of the intricacies of cell signaling and tissue development.

Specific to mammary development in prepubertal calves, Tucker et al. (2016) reported the results of an experiment in which prepubertal calves were administered the antiestrogen tamoxifen. As anticipated, based on the measurement of tissue mass or DNA, mammary development was markedly impaired (50% lower) in calves given tamoxifen. There was no effect on serum estradiol. The proportion of epithelial cells expressing either estrogen or progesterone receptor was not affected by treatment but the level of expression of estrogen receptor was 6.2‐fold lower in tamoxifen‐treated heifers (see Figure 12.8 in Chapter 12). Likewise, messenger RNA expression of the estrogen receptor was significantly reduced by tamoxifen treatment. The intensity of progesterone receptor expression was greater in tamoxifen‐treated heifers. Such data supports the utility of advanced imaging tools and specialized reagents to better understand the intricacies of hormone receptor action.

As its name might suggest, Prl has undoubtedly been the most intensely studied hormone related to lactation and mammary growth. Despite understanding that the presence of the pituitary is essential for normal mammary development, whether Prl or GH predominate in mammogenesis is not clear. The answer to this question is likely species dependent.

In addition to the ovary and corpus luteum, the placenta produces estrogen, progesterone, and a prolactin‐like hormone called PL. PL was first recognized by its Prl‐like biological effects. However, the PLs of many species have both GH and Prl‐like activities. For example, Prl and non‐primate GH molecules interact only with their specific receptors, called lactogenic and somatogenic, respectively. Human GH in contrast recognizes both receptor classes. This coincidently probably explains the inappropriate breast development sometimes observed in human males with pituitary tumors that overproduce GH. It also explains its utility as a ligand to measure Prl receptors in ruminant tissues. Both ovine and bovine PL behave like human GH, they compete for both somatogenic and lactogenic receptors. Although PL is implicated in the preparation of the mammary gland for lactation, stimulation of steroidogenesis, fetal growth, and alteration of maternal metabolism, direct evidence for its effects in cattle is lacking. The best evidence for the role of Pl in mammogenesis is in rodents and in sheep and goats.

While the significance of Prl in mammogenesis in all species is unclear, there is no doubt about the importance of Prl in lactogenesis. Some of the best evidence for the importance of increased periparturient secretion of Prl in stage II lactogenesis has come from experiments in which the administration of a dopamine agonist was used to inhibit Prl secretion and correspondingly impair lactation. In ruminants where postpartum milking continues, administration of the dopamine agonist α‐bromoergocryptine (CB154) reduced basal prolactin concentration by about 80% and prevented the usual periparturient rise, as well as the milking‐induced Prl rise during the first week postpartum. Milk production was reduced by 45% during the first 10 days postpartum. Lost milk production was associated with reduced synthesis of α‐lactalbumin, lactose, and fatty acids, as well as impaired structural differentiation of the mammary secretory cells. Selected effects are summarized in Table 18.6. Cows treated with exogenous Prl, in addition to the agonist (to replace the periparturient surge in prolactin), showed no loss of milk production or negative effects on milk component biosynthesis or alveolar cell differentiation. The effect of Prl suppression and replacement during the periparturient period on milk production in multiparous cows is shown in Figure 18.9. Clearly, Prl is important in mammary cell differentiation and lactogenesis (Akers et al. 1981a,b) (Fig. 18.10).

Aside from circumstantial evidence related to changes in circulating hormone concentrations, there is a marked increase in the numbers of mammary cell receptors for Prl, IGF‐I, and cortisol during late gestation. Progesterone receptor concentration is also correspondingly reduced with the onset of lactation. Thus, simultaneous, coordinated changes in circulating hormones and receptors serve to regulate the timing of lactogenesis. It is believed that high concentrations of progesterone during gestation act to inhibit the onset of lactation. Near the time of parturition, progesterone concentrations begin to wane and estrogen concentrations increase. Removal of the negative effects of progesterone, along with the positive effects of Prl and glucocorticoids, set the stage for the onset of copious milk production (stage II of lactogenesis).

Data from culture experiments also supports the roles of these hormones in lactogenesis. For example, additions of estradiol or cortisol markedly enhance Prl‐induced secretion of α‐lactalbumin (a specific milk protein) by mammary explants taken from pregnant cows. Mammary explants from estrogenprimed or pregnant mice also require insulin, cortisol, and Prl for the accumulation of casein and α‐lactalbumin. However, some caution is advised with wholesale extrapolation of results from culture experiments to the intact animal, as well as uncritical extrapolation between species. For example, induction of the various milk proteins in culture does not necessarily reflect the timing of events in vivo. Neither do existing culture methods allow consistent synthesis and secretion of milk lipids. Finally, hormone concentrations employed may not accurately reflect the situation at the level of the mammary cell in the animal. Culture systems by their very nature represent relatively uncomplicated regulation compared with the intact animal. Nonetheless, results shown in Figure 18.11 illustrate the positive effects of Prl, cortisol, and estradiol on synthesis and secretion of α‐lactalbumin by explants of bovine mammary tissue.

Application of molecular techniques to mammary gland biology has solidified knowledge that Prl and glucocorticoids are the primary stimulators of mammary cell differentiation. Both Prl and glucocorticoid response elements are found within the promoter regions of the genes for several mammary‐specific milk proteins. Induction of both mRNA and specific milk proteins in the presence of Prl or glucocorticoids in isolated mammary epithelial cells confirms the importance of these hormones in lactogenesis and provides details for mechanisms of action of these hormones in the control of milk protein gene expression (Rosen et al., 1999; Akers, 2006).

The term galactopoiesis was originally coined to describe the enhancement of an established lactation. With this strict definition, only exogenous GH (bST) and thyroid hormones are undisputed galactopoietic hormones in dairy animals. Responses also suggest that these hormones are endogenously rate limiting. However, more generally, galactopoiesis can also be described as the maintenance of lactation. In this sense, a larger number of hormones and growth factors are candidates as galactopoietic agents. Continued secretion of galactopoietic hormones, growth factors, and regular milk removal are essential for regulation and maintenance of lactation following lactogenesis.

The pituitary gland and its hormones are essential integrators of the endocrine regulation of milk secretion. This is dramatically shown by the loss of milk production in hypophysectomized lactating goats. However, milk yield can be fully restored to prehypophysectomy levels by the combined administration of Prl, GH, glucocorticoids, and triiodothyronine (T₃) see Figure 18.12. Although species differences exist, endocrine organ ablation/replacement studies show that Prl, GH, glucocorticoids, and thyroid hormones are typically required for the full maintenance of lactation (Topper and Freeman, 1980). This does not mean that additional hormones and growth factors (humoral and local, identified and unidentified) might not also be important or mediate the effects of these hormones.

Secretion of oxytocin at milking or suckling is necessary in most species for efficient removal of accumulated milk and in some species it is essential to obtain any milk at all. The importance of regular milk removal to prevent mammary involution has been known for many years but it has also been hypothesized that the secretion of galactopoietic hormones with milking or sucking was also important. Secretion of Prl and oxytocin, as well as related secretions of norepinephrine or epinephrine, which can impact the secretion (or action) of oxytocin, are most often associated with hormone secretion at milking and its effects on milk synthesis. However, glucocorticoids are also secreted in response to milking or suckling, as is GH in some species.

Colostrum

The secretions that accumulate in the mammary gland during gestation, especially just before parturition, are critically important to the survival of the offspring. This is especially true for animals in which there is no transfer of antibodies to the developing fetus. Because it takes time for the immune systems of newborns to develop, the protection provided by ingestion of colostrum is essential. In most bovine dairies, calves are removed from their dams soon after birth. This is being done for management reasons. It would be difficult to arrange for milking in modern milking parlors if calves were maintained with dams. In addition, it is apparent that there is a premium on harvesting milk, the primary income source for most dairies. Regardless, calves required colostrum. This means that the first milkings are recovered and the colostrum is preserved to supply the colostrum for calves. Westhoff et al. (2024) have reviewed the variety of factors that influence the quality of colostrum available for calf feeding. Some of the sources that impact colostrum include individual animal variability, seasonal changes, prepartum nutrition of the cow, storage conditions, the validity of measures to assess the quality of the colostrum, and others. Silva et al. (2024) provide a comprehensive review of the myriads of physiologically important components present in bovine colostrum.

In addition, to the antibodies and antimicrobial agents in colostrum (lactoferrin, lysozyme, and lactoperoxidase), colostrum also contains a large variety of hormones and growth factors in much higher concentrations than in milk. This at least in part explains the interest in colostrum (Liu et al., 2024) as a source of bioactive agents (see Box 18.2).