**2. Stages of mammary gland development**

At the embryonic period, the mammary gland is derived from ectoderm cell migration, followed by the formation of disk-shaped placodes. The mammary buds arise as a result of proliferation of the basal cells of the ventral epidermis due to factors secreted by mesenchymal cells present in the mammary bud in a process referred to as branching morphogenesis [4–6]. The mesenchyme is instructive and provides critical information to drive mammary gland development. Two different mesenchymal tissues with different properties are involved in this and, with other cells, become a part of stroma compartment. First type of mammary mesenchyme, termed the fibroblastic mesenchyme, is composed of fibroblastic cells surrounding the epithelial rudiment and the second comprise the fat pad cells, thus is known as the fat pad mesenchyme. A solid cord of epithelial cells extends from the mammary bud and grows through the fibroblastic mesenchymal tissue into the fat pad precursor mesenchyme, which at this stage is a small collection of preadipocytes. In rodents, a single epithelial sprout reaches the fat pad and begins to branch by equal division of the terminal bud. The terminal end buds (TEBs) are created as an outer layer of cap epithelial cells surrounding multilayered body epithelial cells located at the front of the branch that invades into the mammary mesenchyme. The body epithelial cells give rise to mammary epithelial cells and the cap cells are myoepithelial precursors. TEBs move forward through mesenchymal cells leading to formation of a rudimentary ductal system. In rodents, it is composed of 10–15 branches that are generated without hormonal input, and the rudimentary ducts remain largely quiescent until puberty [6, 7]. In humans, several sprouts form, creating multiple mammary trees that unite at the nipple, whereas in ruminants the rudimentary ductal network is connected to a small cisternal cavity that connects to the teat cistern and ultimately communicates with the teat meatus [6–8].

After birth, in the postnatal life until puberty, the gland remains quiescent and exhibits only minimal ductal growth. Interspecies differences occur in the extent of mammary gland development that occurs in neonates. In mice, the mammary tree consists of long, infrequently branching ducts and TEBs. Human mammary gland has a more complex structure composed of approximately 15–20 lobes of glandular tissue, each containing a lactiferous duct that opens onto the breast surface through the mammary pit [9]. In the case of ruminants, the mammary gland consists of terminal ductal units (TDU), which are formed during prenatal development accomplished through the coordinated growth, branching and extension of TDU, as well as growth of the loose connective tissue that surrounds the TDU as it invades the mammary fat pad [8].

basal amniotes-vertebrates. Comparison of mammary-expressed genes between mammalian taxa revealed the sheared presence and high degree of conservation of the genes. Mammary gland fully developed prior to emergence of diverse groups of mammals, and the milk compounds (fat globules, whey proteins, casein micelles, and sugars) are structurally similar

In contrast to most organs that achieve morphological maturity during prenatal development in the process defined as morphogenesis, the majority of mammary gland development leading to its complex morphological maturity occurs mostly during postnatal life of mammals [3]. During embryogenesis, the mammary gland development is driven mostly by mesenchymal cells. In postnatal life, subsequent stages of glandular development: mammogenesis (development of mammary epithelial tissue), lactogenesis (functional differentiation of the mammary epithelium leading to initiation of milk secretion), galactopoiesis (maintenance of milk secretion), and involution (regression of the glandular epithelium), take place under significant regulation of hormones. In parallel, the intraglandular milieu plays also an important role in controlling the progress of events related to mammary gland

At the embryonic period, the mammary gland is derived from ectoderm cell migration, followed by the formation of disk-shaped placodes. The mammary buds arise as a result of proliferation of the basal cells of the ventral epidermis due to factors secreted by mesenchymal cells present in the mammary bud in a process referred to as branching morphogenesis [4–6]. The mesenchyme is instructive and provides critical information to drive mammary gland development. Two different mesenchymal tissues with different properties are involved in this and, with other cells, become a part of stroma compartment. First type of mammary mesenchyme, termed the fibroblastic mesenchyme, is composed of fibroblastic cells surrounding the epithelial rudiment and the second comprise the fat pad cells, thus is known as the fat pad mesenchyme. A solid cord of epithelial cells extends from the mammary bud and grows through the fibroblastic mesenchymal tissue into the fat pad precursor mesenchyme, which at this stage is a small collection of preadipocytes. In rodents, a single epithelial sprout reaches the fat pad and begins to branch by equal division of the terminal bud. The terminal end buds (TEBs) are created as an outer layer of cap epithelial cells surrounding multilayered body epithelial cells located at the front of the branch that invades into the mammary mesenchyme. The body epithelial cells give rise to mammary epithelial cells and the cap cells are myoepithelial precursors. TEBs move forward through mesenchymal cells leading to formation of a rudimentary ductal system. In rodents, it is composed of 10–15 branches that are generated without hormonal input, and the rudimentary ducts remain largely quiescent until puberty [6, 7]. In humans, several sprouts form, creating multiple mammary trees that unite at the nipple, whereas in ruminants the rudimentary ductal network is connected to a small cisternal cavity that connects to the teat cistern and ultimately communicates with the

across all mammalian species [2].

88 Stromal Cells - Structure, Function, and Therapeutic Implications

**2. Stages of mammary gland development**

morphogenesis.

teat meatus [6–8].

With the onset of puberty, a combination of systemic and paracrine hormones induces TEBs to reappear at the ductal tips accompanied by a significant increase in the growth rate. Elongation and branching of the ducts, regulated by proliferation and migration of TEBs cells, rely on both endocrine and local growth regulatory signals, extracellular matrix (ECM) remodeling, and stromal influence. With the beginning of puberty, the epithelium bifurcates and invades into the surrounding stroma creating a tree-like structure of mammary ducts. The majority of mammary ductal morphogenesis occurs with onset of ovarian function because of the cyclic influence of reproductive hormones. Further, with each estrus cycle, the alveoli and ducts undergo cyclic expansion and maturation, followed by a modest regression phase as ovarian hormone levels rise and fall, respectively. These events are under the control of a complex interplay of circulating essential steroids (estrogen and progesterone), polypeptide systemic hormones (e.g., prolactin), metabolic hormones that are responsible for coordinating the body's response to metabolic homeostasis (e.g., growth hormone—GH, glucocorticoids, insulin, leptin), as well as locally acting paracrine hormones and growth factors (e.g., insulin-like growth factor I— IGF-I, hepatocyte growth hormone—HGF, transforming growth factor-β—TGF-β, epidermal growth factor—EGF) [10]. It is worth noting that the hormone acting network regulating the development of the mammary epithelium varies between different species.

The mammary gland is able to undergo its terminal differentiation only in female mammals during pregnancy and lactation. With the onset of gestation period and increased levels of progesterone, alveolar structures give rise to lobuloalveolar structures capable of milk production during lactation. After weaning of the offspring (or termination of milking), the gland undergoes post-lactating regression referred as involution, with loss of most of epithelial components gained during the preceding event. Early involution is evidenced by apoptotic death of alveolar secretory epithelial cells which subsequently are removed by efferocytosis (the process of engulfing and destroying apoptotic cells) [11]. Second phase of the mammary gland involution is defined by degradation of basement membrane and ECM proteins and reduction of lobuloalveolar structures.
