Development of the mammary glands and its regulation: how not all species are equal

Abstract

Species-specific development of the mammary glands (MG) necessitates the use of appropriate animal models in research. The MG of livestock represent a unique model for modeling human breast development. The complexities of MG development reveal unique growth regulatory mechanisms. The MGs are responsive to nutritional intervention that increases their growth and lactational performance. The mammary glands (MGs) produce the “perfect” food as milk. However, they must first assume their anatomical form—in this way the organ is unique, given most of its development occurs after birth. Not surprisingly, this development is tightly coordinated with a female’s developmental and reproductive states, as directed by the species-specific endocrine environment accompanying them (Hovey et al., 2002) (Figure 1). However, not all species undergo the same anatomical or morphological development, which remains an understudied area for applications ranging from lactation to cancer (Rowson et al., 2012). Finally, while there is a range of influences that can impact gland growth, none is as pronounced as the diverse and pleiotropic effect of a female’s nutritional status. Diagrammatic representation of the stages of mammary gland development in the female mouse (left half of each panel) or bovine (right half of each panel) mammary gland. *Puberty and gestational development are associated with periods of allometric growth. 1Allometric mammary growth in ruminants before the onset of puberty. Illustration of developmental changes and features is not to scale. Our goal is to highlight the key anatomical similarities and differences in the MGs across species and during their postnatal development, with an emphasis on the endocrine factors regulating this development. At the same time, we address the role for nutrition during this process and highlight opportunities for further exploration. We anticipate these comparative revelations and the discussion of gaps in knowledge will propagate areas for future research as well as improved animal performance and human health. During embryogenesis the milk “lines” form along the ventral body, thereby dictating symmetrical positioning of the future MGs. In a process of inductive signaling, the mesenchyme (as the future connective tissues of the gland) triggers epidermal cells at the outermost layer of the embryo to yield epithelial cells at locations of the future glands. These epithelial cells continue to cross-talk with the surrounding mesenchyme during sex- and species-specific development. By birth, there is an epithelial rudiment at the future site of the teat/nipple, in close and intimate association with a mixture of stromal cell types, ranging from adipocytes, fibroblasts and immune cells, to primordial cells of the vasculature and lymphatic systems (Hovey et al., 1999). Depending on the species, each gland may have multiple independent epithelial rudiments. The teat or nipple serves as the external apparatus for infant suckling and milk removal from the future epithelial structure (where a teat is defined as the external apparatus drained by a single duct/galactophore, whereas a nipple is drained by at least two of these). As an illustration of this cross-species variation, each MG in mice, cows, and goats has a single galactophore draining each teat, whereas each nipple in pigs externalizes two galactophores from independent epithelial structures, while the nipple of a human breast can have 8–20 galactophores (Russo et al., 2001; Rowson et al., 2012). Developmental programming of the future subcutaneous mammary apparatus is diverse across species. For example, mice and rats have 5 and 6 pairs of MG positioned along the ventral midline, respectively, similar to the positioning in other litter-bearing species. In contrast, ruminants such as sheep, goats, and cattle have an inguinal collection of 2 or 4 glands forming a pendulous udder suspended from the pelvic floor, while elephants have a thoracic udder comprising 2 glands (Turner, 1952). The breasts of humans and nonhuman primates are positioned thoracically, while the 4 glands of macropod marsupials such as kangaroos and wallabies are positioned within the pouch. Glandular positioning is also genetically and epigenetically regulated; while some propose that MG number evolved proportional to the number of offspring, there is also a heritable genetic element to the positioning of teats/nipples, where selective breeding for this trait is commonplace in the pig industry (Felleki and Lundeheim, 2015). Furthermore, while symmetry is the basis for glandular positioning in all species, an uneven number of glands is not unusual, as is frequently the case in pigs. In this way, Alexander Graeme Bell undertook breeding studies in sheep that produced ewes having several more teats than their founders (1899). There is also an epigenetic component to gland positioning in that adults in species ranging from humans to livestock can present with supernumerary teats/nipples (Turner, 1952; Grossl, 2000; Oftedal and Dhouailly, 2013). The mouse has become a mainstay model for defining MG development, although its widespread use ignores the many differences that it holds relative to MGs in other species. Regardless, many of the fundamental processes of MG development are conserved, warranting a review of its MG development here. At birth the simply-branched, canalized single epithelial rudiment is positioned above the teat and lies closely juxtaposed to the adipose-rich stromal microenvironment that will become its supporting matrix for the remainder of its development (Hovey and Aimo, 2010). Prior to puberty, the epithelium grows isometrically, namely at the same rate as the rest of the body (Hovey et al., 2002). With the onset of puberty, the gland enters an allometric growth phase when the ductal epithelium proliferates at a rate faster than the rest of the body. Morphologically, the ductal tips have enlarged monopodial termini, or terminal end buds (TEBs), that are filled with abundant mitotic epithelial cells arising from the pluripotent leading cap cells (Figure 2) (Paine and Lewis, 2017). Of course, one might expect that this concentrated zone of epithelial proliferation would amass a solid collection of epithelial cells, thereby precluding the formation of a canalized ductal system required for future drainage of milk to the teats. Instead, a proportion of newly formed epithelial cells almost immediately undergo apoptosis to clear a hollow luminal cavity within the center of the TEB (Paine and Lewis, 2017). The uniqueness of these TEB is even more pronounced when considering that the leading cap cells concurrently yield a unique population of contractile myoepithelial cells that arrange longitudinally on the basal edge around the subtending tubular ducts. Finally, a singular advancing TEB will bifurcate as the ducts advance, creating a branched ductal network that fills the mammary fat pad after several rounds of estrous cycling. Importantly, TEB never encroach closer than 0.25 mm on surrounding ducts, ensuring the ductal network retains an unrestricted yet volume-optimized ductal network, all as the result of local signaling and control mechanisms that are poorly understood (Hovey et al., 2002). Ductal elongation is complete once the mammary fat pad is completely filled by the ductal tree; in mice, the mammary fat pad is mostly composed of white adipocytes, and concurrently undergoes neovascularization and lymphangiogenesis as the ductal epithelium advances (Hovey and Aimo, 2010). Fluorescent labeling of mitosis in terminal end buds in the mammary glands of ovariectomized mice after treatment with exogenous estrogen for 156 hours. Proliferating epithelial cells were detected by 5-ethynyl-2′-deoxyuridine (EdU) histochemistry (green) overlaid with DAPI (blue). A) Entire ductal network showing the abundance of EdU-positive cells in the terminal end buds. B) Higher magnification of EdU-positive terminal end buds. A range of inherent factors, besides the endocrine environment, influences development of the ductal network in mice, which informs our understanding of related processes in other species. For example, the rate and extent of ductal elongation and also branching morphogenesis in sexually-mature female mice is genetically-influenced, as highlighted by the range of phenotypes present in the MGs of various strains (Hadsell et al., 2015). In the same way, despite their similarity to mice, female rats develop a ductal network that is much more sympodially branched (Russo and Russo, 1978). The influence of genetics is also evident in male mice from different strains, where they may or may not survive androgen-induced destruction of the ductal rudiment in utero, or may develop a ductal network at various positions. Several environmental factors also determine the ductal phenotype. For example, the supportive extracellular environment of white adipose tissue cannot be replaced by any other tissue matrix (Hovey and Aimo, 2010), albeit that the ducts can (and do, in the case of the thoracic MGs) also grow into brown adipose tissue (Hovey and Aimo, 2010). Anatomic positioning of the glands also influences the progress and advancement of the ductal epithelium, where anterior glands in mice are smaller. Once the ductal network has filled the mammary fat pad, it resumes an isometric rate of growth, undergoing low levels of mitosis in its lateral branches with each recurrent estrous cycle. While growth of the ductal network during puberty is the of epithelial cells in the gland during is At the morphological of from lateral branches the ductal epithelial cells, with each draining to its subtending the hollow lateral on which it of are as and these are into (Turner, 1952). is most and at the their ductal develop with a single layer of epithelial cells a hollow and their basal is a network of myoepithelial cells in a as to milk in to The of epithelial cells into this structure not on the formation of lateral epithelial cells also their to a mixture of extracellular matrix and As the network its the surrounding and lymphatic and of the tissue at which there can also be cell the of milk during As the and morphological development of the MGs in mice is from that in other species (Rowson et al., 2012). Our is not to an of mammary development across various species, to highlight the key control for and this of development from that in of the mammary epithelium in more closely that in the human breast et al., its development the ductal network in mice, and the and branched growth in ruminants (Rowson et al., 2012). birth, the ductal network in pigs with ductal elongation directed by At the same time, there are branched at the of ducts that can be defined as terminal ductal these have that the number of on each The development of these in pigs has defined the same as that to in humans et al., The ductal network also with connective as occurs in the human breast (Hovey et al., 1999). As the ductal network grows into the surrounding white adipose its also become in connective tissue that the supporting (Hovey et al., et al., in other species, the glands of undergo of epithelial during where the anterior glands are while the glands are and The MGs of ruminants are unique by of their as independent glands within the udder The udder 2 glandular in sheep and goats, or 4 glandular in (Turner, 1952). As an example, mammary growth in after the mammary around of basal and luminal epithelial cells are evident in the mammary which on to yield the future and ducts and By birth, the epithelial is as a of ducts at the of the teat, with several it from that in there is of the that male and female have mammary above the teat, drained by a single the future gland is as an enlarged cavity juxtaposed to the teat et al., 2002) (Figure a layer of stromal connective tissue the branched epithelial and it from of the mammary fat Finally, myoepithelial cells are not around the epithelial et al., 2002). developmental features and changes have also for the MGs of (Hovey et al., of the mammary gland from a a of The the above the The female is around of when a of allometric growth to of mammary growth to be an of the future lactational performance of However, studies have this to the first of as a of mammary growth. the MG increases in by than during this time, while body and (Figure gland development in at of that were a fat milk or a milk and fat in from et al., and et al., The epithelial a of epithelial that are et al., 2002). in mice, the advances into the surrounding adipose-rich mammary fat pad In the adipose environment of the mammary fat pad is also with a of connective tissue that become the and the (Hovey et al., 1999). we that these connective tissue for the pendulous udder (Hovey et al., 1999). Our is to highlight similarities and the MG of humans and other species. A key of the human breast is the 8–20 independent draining each nipple that are present in and (Russo et al., the mammary epithelium of humans can milk by a to as With the onset of puberty, the 8–20 ductal undergo a where ducts into the surrounding adipose tissue as by enlarged ductal termini, of TEB in (Russo et al., However, the of these in humans poorly The ductal network also a having branched and at the ductal these develop and in to most an of terminal or and are present in the stages of postnatal breast development. These can assume a more and branched as they progress to and during The and have as having an of and (Russo et al., The morphological of these is by branching of the terminal The most structures, develop during as a of further and of in pigs and each ductal network in the human breast undergoes development within a of connective each has a connective tissue while the advancing with as it the surrounding adipose tissue (Hovey and Aimo, 2010). In these and connective tissues with that to the suspended breast to the and signaling the of on the MGs that have (Hovey et al., 2002). a key is that that are as by of epithelial cells in the MGs are the result of of that same is the role of various cell within the mammary they be adipocytes, connective tissue various immune cell types, or the vasculature and where each of these cell has as a local of endocrine (Hovey et al., 1999). each of these cell can and and growth the epidermal growth factors, the growth factors, and site factors this these local regulatory are not to growth factors also from (Hovey and Aimo, 2010), or their and In a similar local signaling on the of several different to yield local growth such as and these highlight the of MG growth and is at the of and While this these of are for ranging from the to a to an to of the of the such as for of the to of cells, or to several of these to and of of the mammary a not all epithelial cells are or in the same way to different Furthermore, signaling different cells in the epithelium is for the of such as estrogen and For example, in the case of signaling, several of that epithelial cells to cells that et al., In the same way, there is of and and in luminal epithelial cells (Hovey et al., in with their to these and growth signaling such as growth and et al., 2000; et al., The extent of local cross-talk of epithelial cells occurs after will to given the and several of have given to the population of epithelial cells that can also signaling to the luminal For example, the to the of the epithelium to the of various growth factors et al., epithelial proliferation in ovariectomized as to myoepithelial cell et al., there is a of and regulatory that can the of a range of endocrine at during MG development. While for many of these mechanisms have in mouse the these are also in glands having different and growth that be for humans or The and their and are for postnatal ductal development, as highlighted by the that or in allometric growth of the ductal network in and humans (Turner, 1952; et al., 2002). this knowledge a the of can also to mammary in humans and to be unique in this given that not allometric growth of the glands et al., phase of allometric growth is to the of given that can ductal growth in all the species (Hovey et al., Rowson et al., 2012). branching and development of the mammary epithelium during recurrent estrous on the and coordinated to and In this way, epithelial of the which is a across species (Hovey et al., 2002). proliferation within a of the mammary epithelium to branching morphogenesis and While epithelial cells are responsive to in there are several that this in also the local of a range of growth from the or from epithelial cells et al., As the to is also by growth While the are for MG development, their also a gland as the of et al., an of in it clear that during this endocrine on of the mammary fat pad to a local of which as a to epithelial proliferation and In at this TEB by ductal elongation and In ruminants it is that the is given that the MG at the same there is the onset of allometric growth to puberty (Hovey et al., et al., a is with the that and local can growth of the mammary epithelium of in et al., Not surprisingly, these of in with the of that concurrently of by the et al., and in the of stromal that further this local and not its and during also a role during gland growth. 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