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Mammary Gland Development
Postpubertal Period


Mammary Gland
Development During the
Postpubertal Period

Cross section of a beef heifer's udder.

Puberty to Conception

From work with rodents and other species like the rabbit, the mammary gland duct formation occurs by outgrowth from the base of the nipple and laterally, conforming to the contours of the body wall. Thus, the mammary gland of these species are thin but broad in width and length. By cutting the skin near the mammary glands of a mouse or rat, the underlying mammary glands can be peeled off of the skin. These whole glands can be placed upon a microscope slide, fixed, defatted, and stained to show the ductal structures. These preparations are called whole mounts. Using this type of whole mount preparation, the branching of ducts originating from the base of the nipple can be observed. At the actively growing end of these ducts, where the outer-most limits of ductal elongation invade the fat pad are actively growing structures called Terminal End Buds (TEB). Terminal end buds represent the structures where elongation and branching of the ducts is occurring and estrogen stimulated cell division is occurring. In general, estrogen causes cell multiplication at the tip of the TEB and enlargement of ducts (lengthening and branching of ducts), while progesterone causes duct and ductule cells to multiply, leading to ductule development and duct enlargement or widening.

Structures specifically like the TEBs are not seen in species like ruminants. However, the principle of an actively elongating and branching structure probably holds for those species, as well. In ruminants and probably many other species, the fundamental developmental unit inthe mamamry tissue probably is the terminal ductule lobular unit, or TDLU. These structures are characteristic of postpubertal mammary development in the human breast and similar structures can be observed in the ruminant udder. A TDLU makes up a developmental and functional unit in the parenchymal tissue. It resembles a cluster of grapes on the end of a stalk. Epithelial components of a TDLU are held together by loose intralobular connective tissue and are surrounded by a denser interlobular connective tissue sheath. While the lobular organization of mammary tissue is apparent in the cycling heifer, it is during pregnancy that a TDLU will develop further to form a cluster of alveoli in what is recognizable histologically as a lobuloalveolar unit.

This is a whole mount of a virgin, cycling ewe's mammary gland. The udder was removed, extraneous fat pad cut away, the tissue fixed, then defatted and stained. Note the branchings of the growing tissue around the fringes of the darker, more densly developed portion of the gland. These would be the TDLUs. [Image kindly provided by RM Akers, VPI] An example of a TDLU from a cow.
This is a low magnification image of a TDLU from a cow. The image is actually from a cow that had lactated and was undergoing involution. This TDLU had not previously developed into a lactating lobule during the lactation, but probably would have developed into a lacating lobule if the cow had gone through pregnancy and lactation again. Note the intralobular duct within the TDLU. D = an interlobular duct. S = stromal tissue between lobules. Cross-section through the udder of a beef heifer.
This is a cross-section through the udder of a beef heifer. The area where the teat would have been is outlined by the black line at the bottom of the image. Note the extensive fat pad and the branchings of the parenchymal tissue into the fat pad. FP = fat pad; P - parenchymal tissue; CP = cisternal ducts. An example of a TDLU from a cow.
This is a histological section of mammary tissue from a nonpregnant gilt. A = adipose tissue; C = connective tissue sheaths running through the adipose tissue; D - ducts growing up through the connective tissue sheaths. Histological section of mammary tissue from a gilt.
This is another histological section of mammary tissue from a nonpregnant gilt. A = adipose tissue; C = connective tissue sheaths running through the adipose tissue; D =ducts growing up through the connective tissue sheaths and branching into TDLUs; LS = lactiferous sinous (see Mammary Gland Structure Lesson if you do not remember this structure). Histological section of mammary tissue from a gilt.
Another example of a histological section of mammary tissue from an ovariectomized gilt treated with an estrogen. A = adipose tissue; C = connective tissue sheaths running through the adipose tissue; D = an interlobuular duct that drains sevearl lobules; a TDLU is indicated by the red circle. Histological section of mammary tissue from a gilt.
Another example of a histological section of mammary tissue from an ovariectomized gilt treated with an estrogen. A = adipose tissue; D = interlobular duct; a TDLU is indicated by the arrow. Can you identify the interlobular duct in this TDLU? Histological section of mammary tissue from a gilt.

There is considerable species variation in the extent of mammary development during the postpubertal period. For example acyclic animals, such as the rabbit which remains in estrus for extended periods in the absence of copulation, have extensive duct development during estrus, and even a few alveoli form. On the other hand, cyclic animals have bursts of ductal development. In the rat, ducts proliferate into the fat pad during proestrus and estrus. Mammary duct development increases by about 8% per estrous cycle, then regresses or involutes during metestrus and diestrus. This is similar in cattle, but the post-estrus decline in parenchymal tissue is irregular (see Tucker , J. Dairy Sci. 52:720, 1969). There is a linear relationship between increased udder weight and increasing age of the heifer up to 30 months, about 0.27 kg udder weight per month. This is partly due to increased body wt. and partly due to accumulation of udder fat as heifers put on body conditioning with advancing age.


Hormonal Regulation During the Postpubertal Period

Clearly, the growth during estrus must be related to the ovarian steroid hormones. Estrogen receptors and progesterone receptors both appear in the gland around the time of puberty. However, the exact roles of estrogen and progesterone are still not completely clear. Mammogenic hormones establish the conditions for specific growth patterns in mammary tissue. For example, concurrently elevated blood concentrations of estrogen and progesterone observed in late gestation result in exponential parenchymal growth and in formation of alveoli, whereas the cyclic changes of those hormones associated with estrous cycles result primarily in duct elongation and some lobular tissue formation, but not in formation of alveoli. Mammary development usually is driven by a complex of hormones acting in concert. Effects of many mammogenic hormones are thought to be mediated through stroma-derived growth factors which act in a paracrine manner by eliciting mitogenic responses in the adjacent epithelial structures. Much of our current understanding of how mammogenic hormones and growth factors function arises from research in rodents. However, similarities are being noted in ruminants and understanding of hormone action in rodents can help in understanding similar processes in ruminants.

Estrogen is an important mammogenic factor, particularly in the postpubertal female. Estrogen receptors appear in the gland around the time of puberty, coinciding with the period when the gland becomes exposed to cyclic elevations in estrogen blood concentrations. In rodents, estrogen acts on its receptors in the stromal tissue to stimulate production of growth factors which in turn stimulate ductal development. The evidence available from studies of cattle also indicates that estrogen action on mammary development may be mediated through the stroma.

Progesterone is another ovarian steroid hormone which plays a key role in mammary development. While progesterone receptors have been difficult to identify in mammary fat pad, administration of progesterone can result in proliferation of stromal cells under some physiological conditions. The stimulatory effect of progesterone on DNA synthesis in ductal epithelium is probably mediated indirectly through its effects on stromal cells. The major mammogenic effect of progesterone is mediated through binding to its receptors in epithelial cells and stimulating ductal sidebranching or alveolar bud formation, which are the hallmarks of postpubertal mammary development. Estrogen stimulation of progesterone receptor expression in epithelial cells is required for this progesterone effect. Progesterone, therefore, has a major role in alveolar morphogenesis and a lesser role in ductal morphogenesis. During estrous cycles, duct elongation and expansion of the parenchymal tissue into the fat pad occur in limited bursts associated with the period of elevated estrogen. During the luteal phase of elevated progesterone in ruminants, relatively little further expansion occurs, but formation and maintenance of lobular structures may be stimulated by progesterone, with little ductal regression occurring between cycles.

Synergy between estrogen and progesterone is observed during pregnancy when both hormones are present in high concentrations. Elevated blood concentrations of estrogen and progesterone together establish the conditions required for the exponential cell growth which occurs during pregnancy. Lobuloalveolar development represents the greatest increase in mammary gland tissue mass during pregnancy. In the cow, progesterone is elevated throughout gestation, while estrogen is particularly elevated during the later phase of gestation, coinciding with the period of greatest increase in mammary tissue mass. Estrogen and progesterone have direct effects on the mammary gland, probably mediated by autocrine and paracrine factors produced locally in the tissue. In addition, steroid hormones may have indirect effects via their impact on secretion of prolactin.

Prolactin often is associated with initiation of lactation and galactopoeisis, but also has mammogenic effects. Prolactin receptors are present in the fat pad of some species, as well as in the epithelium. Prolactin may act on both epithelial and stromal components of the growing mammary tissue. Inhibition of prolactin secretion inhibits mammary gland development in pregnant goats, pigs and other species. Blood concentrations of prolactin are normally low during pregnancy. Mammary development during pregnancy may not be limited by the normal blood concentrations of prolactin.

Growth hormone (somatotropin) administration to cattle is known to stimulate milk production during lactation. This effect is indirect in that growth hormone stimulates secretion of insulin-like growth factor-I (IGF-I) from the liver, which in turn mediates many of the galactopoeitic effects of growth hormone during lactation. Growth hormone also acts as a mammogenic hormone and can stimulate mammary growth at all stages of development. Direct effects of growth hormone on mammary tissue would require the presence of growth hormone receptors on mammary cells. While this remains a point of controversy, there is evidence for growth hormone receptors in mammary epithelial or stromal cells in various species. Several lines of evidence indicate that growth hormone may act on ruminant mammary tissue by stimulating stromal production of IGF-I which is mitogenic for mammary epithelial cells. The highest level of IGF-I expression in mammary tissue occurs in the fat pad and is greatest during the prepubertal allometric growth phase and during late pregnancy. Mammary expression of IGF-I is regulated by growth hormone, estrogen and positive feedback stimulation from proliferating epithelial cells. The function of IGF-II in mammary growth is less clear then that of IGF-I. Stromal cells probably produce IGF-II, which is regulated by stage of mammary development and hormonal stimulation.

Placental lactogens are secreted from the placenta and may have prolactin- or growth hormone-like activities, depending upon the species. In pregnant goats, placental lactogen in the maternal blood is closely correlated with the number of fetuses present (Hayden et al 1980). This graded concentration of placental lactogen, in combination with other mammogenic hormones, may regulate the extent of mammary development during late pregnancy. In the dairy cow, there is a relationship between placental mass and subsequent milk production. However, the concentration of placental lactogen in maternal blood of the dairy cow is low and the effect of placental mass may result from other placental hormones, including estrogen.

Other hormones also are required for mammary growth, including glucocorticoids, thyroid hormones and insulin. Severely diabetic mice given estrogen and progesterone will develop extensive lobuloalveolar structures. Nevertheless, insulin synergizes with estrogen and progesterone to increase mammary development. Normal blood concentrations of insulin are probably not limiting for normal mammary development.


Autocrine and Paracrine Regulation of Mammary Growth

Autocrine and paracrine factors (local growth factors) play a major role in mammary growth. Many of the effects of the steroid hormones on mammary growth are mediated by local growth factors at the mammary tissue level. These include an interaction between the developing mammary epithelial structures and the mammary fat pad. Mammary epithelial cells will only grow and organize when transplanted into the fat pad. The interaction may involve specific fatty acids from the fat pad which may induce changes in epithelial development. (see Hovey et al 1999).

Mammary stromal cells in the region of the TEB or TDLU may be involved in dissolving the collagen so the structures can expand. The enlarging ducts may promote mesenchyme growth and angiogenesis (development of blood vessels). Epithelial cells in the TDLU and developing ducts probably are interacting with each other to cause synthesis and assembly of the basement membrane.

In addition to the IGF's discussed above in connection with growth hormone, a number of other growth factors have positive or negative effects on mammary gland development. Local production of transforming growth factor-ß (TGF-ß) inhibits mammary growth, such as during the prepubertal period and between estrous cycles. Epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) produced in the mammary tissue stimulate mammary cell proliferation. Both EGF and TGF-a bind to the EGF receptor. The mammogenic action of estrogen and progesterone occurs in part by decreasing local production of the inhibitory TGF-ß, while increasing local production of TGF-a and increasing levels of EGF receptor in epithelium. Stromal EGF receptors also are necessary for normal ductal growth.

Other growth factors produced by stromal cells also are known epithelial cell mitogens and may be involved in mediating the effects of mammogenic hormones. These include hepatocyte growth factor (HGF), and members of the fibroblast growth factor (FGF) family such as acid-FGF and Keratinocyte growth factor (KGF). Basic-FGF also is an epithelial cell mitogen, although the origin of this growth factor in the mammary tissue is uncertain.

Mammary epithelial structures often are growing into a lipid-rich environment of the fat pad. Fatty acids, particularly unsaturated fatty acids, stimulate mammary epithelial cell growth and can substantially enhance the in vitro effects of other growth factors such as IGF-I and EGF. Mammary stromal cells also are involved in dissolving the connective tissue collagen so the epithelial structures can continue to grow. Several proteases involved in tissue remodeling and growth of parenchymal tissue are derived from stromal tissue. Extracellular matrix components, which are important for mammary tissue growth and function, are produced by both epithelial cells and stromal cells.


 
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