Mammogenesis

The term “mammogenesis” can be defined as the growth and development of the mammary gland. Mammogenesis occurs through a series of structural and functional development, differentiation, and involution associated with the growth and reproductive stages of animals and regulated by hormones and growth factors.

25.2.1 Prenatal Development of Mammary Gland

The prenatal development of the mammary gland of bovines starts around 32 days of embryonic life. The fetal ectoderms on either side of inguinal region give rise to mammary band which is the first developmental stage of prenatal mammogenesis. Different stages of mammogenesis are as follows.

Mammary band'. Mammary band developed from the ecto­derm of the inguinal region differentiated from mesenchyma. It is made of a single layer of flattened cuboidal cells that appeared as an abroad band on either side of trunk from upper limb to lower limb. The formation of mammary bands occurs on 32nd day of embryonic life.

Mammary streak: Further layering of ectodermal cells over mammary band develops around 34th day of embryonic life.

Mammary lines: Further the transient mammary line develops from lower ectodermal layer (Malpighian or germinal layer) which is composed of several layers of cells around 35th day of embryonic life.

Mammary crest: Around 37th day of embryonic life, the ectodermal cells of mammary lines are begun to divide into the mesenchymal cell layer leading to the formation of mammary crest.

Mammary hillock: Continual inward growth of the ectoder­mal cells into the mesenchymal layer forms a dome of ectodermal cells into the mesenchyma called mammary hillock occurred around 40th day of embryonic life.

Mammary bud: It is the spherical or globular-shaped ectoder­mal layer into the mesenchymal layer normally seen around the 43rd embryonic day. The ectodermal layer sinks entirely into the mesenchyma with a small depres­sion at the outer pole called mammary pit. Mammogenesis is identical in both sexes up to mammary bud stage. The structure of mammary buds is different in male and female. In females, mammary buds are ovoid and do not form as deep a mammary pit as in the males. In males, mammary buds are spherical. The mammogenesis is faster in females compared to males after the mammary bud stage.

Early teat formation: Rapid growth of the mesenchyme surrounding the mammary buds forces the mammary buds to rise above the epithelium with a slight opening at the distal end of the buds. It starts around the 65th day of embryonic life.

Formation of primary sprout: The solid core of the lower ectodermal layer (Malpighian or germinal layer) invaginates into the mammary buds following the least resistant path which pushes the mesenchymal layer aside and leads to the formation of primary sprouts at the 80th day of embryonic life. It gives rise to gland and teat cistern.

Formation of secondary sprout: At about 90th day of embry­onic life, when the primary sprouts reach its maximum growth (up to 16 cm in cattle), many secondary sprouts emerge as branching of primary sprouts. The upward growth of secondary sprouts in various angles into the mesenchymal layer leads to the formation of tertiary sprouts which in turn convert to the duct system of the udder. However, during fetal mammogenesis very limited growth of primary sprouts is evident.

Canalization of primary and secondary sprout: Formation of lumen in the solid core of epithelial cells in the primary and secondary sprouts is called canalization normally seen around 100th day of embryonic life. It is mediated by the separation of cells in the primary and secondary sprouts.

Formation of gland cistern: Canalization of the inner end of the primary sprout in both directions leads to the forma­tion of the gland cistern at 110th day of embryonic life.

Formation of teat cistern: From 130th day of embryonic life, the progression of the canalization primary sprouts with­out disintegration towards the distal end leads to the formation of teat cistern. During the growth of the teat, the tip of the mammary bud is opened. Initially, it looks like a duct but horizontal movements of the cells increase the cavity of teat cistern and the layers are reduced. At the distal end, streak canal is formed when the duct becomes narrow at its distal end.

The dermis of the skin surrounding the udder is developed from the mesenchymal tissue below the Malpighian layer. The fibrous tissues are formed as threads or bundles perpen­dicular to the base of the udder. The connective tissues appear as whorls (aggregation of cells in the center with a circular periphery). These connective tissues are subsequently replaced by secretory tissues during the development of mammary alveoli.

All these aforesaid developmental stages of mammary gland are completed within the first 6 months of fetal age and no further developments are seen prior to birth.

Prenatal mammogenesis in doe and ewe is similar to cow. However, in goat fetus, hair anlagens are evident on the teat skin. In mouse, prenatal mammogenesis starts around 11th day of embryonic life on each side of the trunk similar to cow. Mammary buds are developed around 12th-14th day of embryonic life. Differentiation of mammary gland in females begins around 15th day which is characterized by sinking of mammary buds into the mesenchyme tissue and leads to the formation of primary mammary cord. The distal end of the primary mammary cord develops lobulo-alveolar system of mouse mammary gland. The prenatal mammogenesis in sow is occurred in the same direction as in mouse but the teat has two ducts.

25.2.2 Postnatal Development of Mammary Gland

25.2.2.1 Mammogenesis from Birth to Puberty

Mammogenesis from birth to puberty is characterized by the appearance of connective tissue and fats in the mammary gland.

25.2.2.2 Mammogenesis During Estrous Cycle

The development of mammary gland during the estrous cycle is characterized by the branching of secondary and tertiary sprouts, the growth of buds from these branches, and the growth of mammary ducts. The growth of mammary gland occurring in each estrous cycle is appeared to be lost by the process of regression in the next cycles. However, a small amount of positive growth takes place during each cycle as the regression is comparatively less compared to the growth.

25.2.2.3 Mammogenesis During Pregnancy

In cattle, exponential growth of mammary gland occurs throughout gestation. It has been reported that 48-94% growth of mammary gland and 60-65% growth of mam­mary parenchyma occur during gestation. A marked increase in the gland cistern occurs during the fifth to sixth months of pregnancy. The proportion of secretory tissues of the mammary gland increases by branching of the ducts and end buds during fourth month of gestation and appears as found during lactation. These secretory tissues replace the fat tissues and form very small lobules. These lobules are joined together to form the lobes. There is a little increase in the duct length (maximum up to 3 cm). The large ducts appear with two layered linings whereas the ductules and the alveoli are having single-layered cuboidal cells. The connective tissues that separate the lobules and lobes contain numerous blood capillaries. The secretory activity of the alveoli begins from ninth month of pregnancy and the alveolar epithelium becomes distended with granu­lar cytoplasm and appearance of fat droplets. The develop­ment of mammary gland in cow during pregnancy is summarized in Table 25.5.

It has also been reported that mammary growth during gestation was proportional to the litter size in sheep, goats, and pigs except in cow.

Table 25.5 Summary of mammary development during pregnancy in cow

25.2.2.4 Mammogenesis During Lactation

Around 10% mammary growth occurs during lactation which is characterized by increased number of secretory tissues due to the mitotic proliferation of cells before and after parturi­tion. Around 65% increase in mammary DNA has been estimated from 10 days before parturition to 10 days post­calving which is maximum at peak lactation. After peak lactation, very little cell proliferation occurs and the destroyed cells are not replaced by newly formed cells. In sows, mammary volume due to hypertrophy and hyperplasia of the secretory cells occur around 28 days of lactation period which was associated with increased DNA content around this period. In sheep and goats, udder volume was exponen­tially increased during up to the last third of pregnancy which gradually declined in lactation.

25.2.3 Hormones and Growth Factors in Mammogenesis

A series of investigations on hypophysectomized and ovari- ectomized animals established the participation of both ovar­ian hormones (estrogen and progesterone) and hypophyseal hormones (prolactin and growth hormone) in mammogenesis. It is now well established that estrogen stimulates duct growth and estrogen and progesterone in combination stimulate lobulo-alveolar growth and the mammogenic action of estrogen and progesterone is mediated only in presence of prolactin and growth hormone. Other mammogenic hormones and growth factors are placen­tal lactogen, glucocorticoids, oxytocin, and insulin like growth factors.

Estrogen: Estrogen induces ductal growth of mammary gland. The effect of estrogen on mammogenesis is species-specific. In laboratory animals (mouse, rat, and rabbit) and cats, physiological dose of estrogen induces duct growth and prolonged administration of a high dose of estrogen causes alveolar growth. In these animals, estrogen has the same mammogenic potential for both male and females. In ruminants and guinea pigs physio­logical dose of estrogen can induce extensive lobulo- alveolar growth including duct growth but the mammogenic potential of estrogen in these animals are more in females compared to males. In the third category animals which include bitches and ferrets, estrogen alone has little or no role in mammary development. The ductal mammogenesis in response to estrogen occurs when the animals attain puberty. Estrogen induces synthesis of IGF- 1 from stromal cells of mammary gland which induce epithelial cell proliferation. In another way estrogen in paracrine manner, induces the release of amphiregulin (AREG), an epidermal growth factor family which binds its stromal cell receptors induce the release of FGFs to stimulate the proliferation of luminal cells. The net effect of estrogen on mammogenesis is the elongation of ducts, side branching, formation of terminal end bud (TEB) together with alveologenesis.

Progesterone: Progesterone in combination with estrogen induces lobule-alveolar growth of mammary gland. Pro­gesterone is responsible to produce a lactation-competent mammary gland by causing alveologenesis and side branching of ducts. Progesterone also promotes the differ­entiation of alveoli during lactogenesis together with pro­lactin. Progesterone induces the synthesis of tumor necrosis factor ligand superfamily, member 11 (TNFSF11), also known as RANKL (receptor activator of NFKB1 ligand) which in turn initiates cell proliferation. Growth hormone (GH): Growth hormone can induce mam­mary growth in hypophysectomized, adrenalectomized, and ovariectomized rats. It was also established that estro­gen and/or progesterone were unable to induce mammary growth without growth hormone or prolactin. According to the modified somatomedin hypothesis, growth hormone acts in two ways in the mammary gland. Firstly, GH directly stimulates the growth and proliferation of mam­mary parenchyma. Secondly, the indirect mammogenic effect of growth hormone is mediated by the secretion of IGF-1 from either liver or mammary stromal cell which acts via autocrine, endocrine, and paracrine mechanisms.

Prolactin: The role of prolactin on mammary growth and differentiation was well established in laboratory species specifically in rabbits where prolactin helps to stimulate lobulo-alveolar system during pregnancy. However, the same effect was questionable in rats and cattle as there was no elevation of prolactin level in these species during pregnancy. Prolactin stimulated the branching of ducts and regression of end buds in virgin animals whereas around pregnancy it stimulates lobulo-alveolar growth. After binding with its specific receptors on the mammary gland epithelium, prolactin induces the expression of whey acidic protein (WAP) gene. WAP is one of the major whey proteins of milk which regulates the prolifer­ation of mammary epithelial cells.

Glucocorticoids: Cortisol causes differentiation of mammary lobulo-alveolar growth in cattle and cortisol-primed dif­ferentiation was essential for the action of prolactin to induce milk protein synthesis during lactogenesis.

Insulin: The mammogenic actions of estrogen and progester­one in hypophysectomized animals are enhanced with the action of exogenous insulin. Higher dose of insulin was proved to be mammogenic when studied in vitro. The action of insulin may mimic the action of IGF-1 as insulin exerts its effect through IGF-1 receptors.

Placental lactogen'. Placental lactogen is mammogenic in rodents but its effect on mammary gland growth and development in cattle is questionable as the concentra­tion of placental lactogen was very low in dam compared to fetus and exogenous administration of pla­cental lactogen had little effect on metabolism in lactating cows.

Insulin like growth factors (IGF): The insulin like growth factor family comprises of ligands (such as IGF-I, IGF-II, and insulin), receptors (IGF-IR, IGF-IIR, and insulin R), and IGF-binding proteins (IGFBPs). The role of IGF on mammogenesis in terms of cell proliferation, migration, and apoptosis has been well established in cattle, goats, sheep, pigs, and mice till puberty. The predominant source of circulating IGFs is liver though some extra­hepatic sources of IGFs have also been identified in uterus and mammary gland as well. The expression of IGF in the mammary gland is low and thus it was postulated that IGF can be transported from liver to mammary gland. The IGF helps in DNA synthesis and stimulates the cells in late G₁ to enter into S phase thus increasing cell proliferation.

Epidermal growth factor (EGF) family: EGF family includes four members including ligands (EGF, ErbB2, ErbB3, and ErbB4) and their receptors. The other growth factors like transforming growth factor (TGF)-α, heparin-binding EGF (HB-EGF), amphiregulin (AR), and neuregulins (NRGs) are also included in this group as they also act through receptor tyrosine kinase (RTK). EGF receptors have been identified in nonpregnant, pregnant, and lactat­ing cow and sheep. But the level of expression was similar in all these stages. EGF family exerts stage-specific effects on the mammary gland. They affect both mammogenesis in terms of ductal growth including alveolar differentia­tion during early puberty (EGFR, ErbB2, and ErbB3) whereas ErbB4 helps in mammogenesis during late preg­nancy and lactation.

Other growth factors: There are several other growth factors either stimulatory or inhibitory to mammogenesis. Plate­let-derived growth factor (PDGF) was found to be stimu­latory to the proliferation of myoepithelial cells by increasing DNA synthesis.

Transforming growth factor-β (TGF-β) has been identified as a mammary gland-derived growth inhibitor having the primary function to induce differentiation. In vitro experi­mentation showed that TGF-β inhibits ductal growth, lobulo-alveolar development, and suppresses casein syn­thesis in pregnant mice.

Vitamin A in mammogenesis: Retinoic acid was involved in mammary epithelium development during embryo­genesis even after birth. The role of retinoic acid dur­ing mammary gland involution was also well established.

25.2.4 Mammary Gland Involution

Involution is a biological process by which the lactating mammary gland undergoes a series of tissue remodeling processes after cessation of milk secretion to restore into a virgin-like state. The mechanisms of involution have been well studied in mice after inducing the involution by weaning at peak lactation and teat sealing but there are notable differences in the mechanisms of mammary involution among species. In ruminants, lactation overlaps pregnancy and at the time of milk stasis, cows are at their last trimester of pregnancy while goats are at their first days to the second trimester of pregnancy. Therefore, the stimuli for mammary involution is opposed by the pregnancy-induced mammogenic and lactogenic stimuli. A brief non-lactating period prior to lactation in those species is required to maxi­mize the production. This non-lactation period with limited involution is termed as dry period.

The mammary gland involution occurs in two phases namely

Reversible phase: In this phase, the events of involution can be stopped if the suckling stimulus is reintroduced and the gland can revert to a state of milk production.

Irreversible phase: The mammary gland is unable to return to a state of milking without being restimulated by mammogenic and lactogenic stimuli.

25.2.4.1 The Events of Mammary Gland Involution

The involution process (both reversible and irreversible) is mediated through a series of events involving apoptosis and tissue remodeling.

Inhibition of milk secretion: The stasis of milk is an essential prerequisite for the initiation of involution. The transition of mammary epithelial cells from secretory columnar epi­thelium to a non-secretory squamous cell is noticed upon milk stasis. Cessation of milking or suckling stimulus facilities the engorgement of mammary alveoli with milk which exerts some pressure to cause distension of udder that favors the release of some local inhibitory factors for milk stasis. One of these factors, “feedback inhibition of lactation,” has been discussed in earlier section (see factors affecting milk yield and composition). The other notable factors are mentioned below (Table 25.6).

Cell death and regression of epithelial cells: The regression of epithelial tissues and their shedding in the alveolar lumen are evident in both reversible and irreversible phases of involution. The regression of epithelial cells is seen in mouse mammary gland as early as 12 h of weaning and peak around day 2 and day 3. During the secretory phase, the epithelial architecture is well maintained by lactogenic factors like prolactin (PRL), glucocorticoids

Table 25.6 Local factors and their role in milk stasis

Table 25.7 The factors responsible for regression and death of epithelial cells

(GC), and IGF-I. But due to the absence of suckling stimuli, the mammary epithelial cells become refractory to these lactogenic hormones and initiate the release of pro-apoptotic factors which mediate the cell regression. The factors responsible for the regression and death of epithelial cells are discussed in Table 25.7.

Involution-associated Immune response and clearance of cell debris: The stasis of milk in the mammary gland results in the accumulation of residual milk and cell death results in shedding of epithelial cells along with cellular debris. The removal of these waste materials is crucial for maintaining the normal health of the mammary gland. The induction of immune response followed by clearance of cell debris are thus two important mechanisms of involution.

Activation of neutrophils: Increased leukocyte counts during the time of mammary involution along with higher expressions of pro-inflammatory cytokines and their receptors (e.g., IL-1α, IL-I β, and IL-13) in mice suggests the elucidation of immune responses in the mammary gland. Involvement of 5-HT as an immune mediator dur­ing early involution may induce immune response either directly by activating the immune cells or indirectly after the release of pro-inflammatory cytokines.

Activation of acute phase response (APR): Around 12 acute phase proteins for activation of macrophage (e.g., CD68) and B cell chemokine (e.g., CXCL14) have been identified during different phages of involution in mouse mammary gland. Induction of APR also initiates the phagocytic ability of non-preferential phagocytes like mammary epithelial cells.

B lymphocyte activation: Activation of B lymphocytes are responsible for the local synthesis of immunoglobulins (IgA, IgM, and IgG) underlying the epithelium in both mouse and ruminant mammary gland.

Induction of an immune response recruits both preferential (neutrophils and macrophages) and non-preferential (epi­thelial cells) phagocytes to clear cell and tissue debris. Mammary epithelial cells usually respond during the early phase of involution and mostly ingest apoptotic cells, casein micelles, and milk fat globules. In contrast, professional phagocytes (macrophages) respond late and their role in clearing cell debris is limited compared to epithelial cells.

Disruption of epithelial tight junctions (TJ): Mammary TJs provide an apical seal and holds the mammary epithelial cells together. It also provides a barrier for the paracellular transport of fluids and ions between the lumen and the interstitial space. Disruption of mammary epithelial TJs facilitates the progression of mammary involution. The disruption of TJs are highly reversible and can be restored by the initiation of suckling stimulus within 18-24 h in goats and cows by positive actions of PRL and GC. But the continual absence of suckling decreases the permissive actions of PRL and GC on TGs along with the release of only locally produced disrupting factor 5-HT.

Remodeling of extracellular matrix (ECM) and basement membrane (BM): The stroma of the mammary gland is composed of a variety of tissues like adipose tissue, con­nective tissue, fibroblast, vascular components, and ECM.

Table 25.8 The components of the plasminogen system and matrix metalloproteinase (MMP) system in mammary gland ECM remodeling

Table 25.9 Factors regulating vascular remodeling during mammary gland involution

ECM or basement membrane (BM) is a cementing sub­stance composed of laminin, collagen, fibronectin, and integrins synthesized from myoepithelial cells. The main function of ECM is to anchor epithelial and myoepithelial cells. The breakdown of BM via proteolysis to become thick, folded, and discontinuous are the characteristic features of ECM remodeling during mammary gland invo­lution. This part of involution is irreversible.

The remodeling of ECM is mediated by two systems namely plasminogen system and matrix metalloproteinase (MMP) system. The components of these two systems and their mechanism are depicted in Table 25.8.

Vascular remodeling: Vascular remodeling consists of vas­cular regression and angiogenesis. The mammary gland vasculature is composed of a capillary network spreading as a honeycomb structure surrounding each alveolus during lactation. The alveolar engorgement due to milk stasis leads to an increase in the perivascular capillary volume seen in the initiation of involution. The vascular regression begins around day 6 of involution in mouse mammary gland characterized by clusters of capillaries and the vascular network becomes similar to the virgin gland by day 10 of involution. The angiogenesis occurs simultaneously with vascular regression in adipose tis­sue. There are several local and systemic factors that regulate vascular remodeling during mammary gland involution as summarized in Table 25.9.

25.3