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Mammary Gland Development and the Prolactin Receptor |
1. INTRODUCTION
In the normal mammary gland, development, growth and differentiation are under the control of a variety of growth factors and hormones. Prolactin is considered a major player. Functional differentiation of the gland as measured by induction of milk protein synthesis both in vivo and in vitro is dependent on prolactin. To summarize our understanding of PRL actions in the development of mammary gland, we will discuss PRL secretion and mammary gland morphogenesis, and will describe recent findings regarding this hormone in the context of mammary development.
2. PITUITARY AND EXTRAPITUITARY PRL SECRETION
Three decades after PRL was identified, the amino acid sequence of sheep PRL (also referred to as lactogenic hormone or luteotropic hormone depending on its biological properties) was determined and shown to be a protein of 199 amino acids. At the end of the 1970's, the nucleotide sequence of PRL cDNAs from several species was identified. As anticipated from earlier structural studies, the primary structure of PRL appeared closely related to that of two other hormones, growth hormone (GH), also of pituitary origin, and placental lactogen (PL), secreted by mammalian placenta. Today, genetic structural, binding and structure- functional studies of these three hormones, as well as the more recently identified somatolactin and prolactin-related proteins, have clearly demonstrated that they all belong to a unique family of proteins.In addition to being synthesized and secreted by lactotrophic cells of the anterior pituitary gland, PRL is also produced by numerous other cells and tissues. The subject of extrapituitary PRL has recently been reviewed and thus will only be briefly discussed. In addition to the anterior pituitary gland, PRL gene expression has been confirmed in various regions of the brain, decidua, myometrium, lacrimal gland, thymus, spleen, circulating lymphocytes and lymphoid cells of bone marrow, and tumors, skin fibroblasts, and sweat glands (reviewed in). PRL can thus be found in several fluid compartments in addition to serum, such as cerebrospinal fluid, amniotic fluid, tears, follicular fluid, and sweat.In addition, the mammary epithelial cell is an important site of PRL synthesis and secretion. PRL is present in significant concentrations in milk and milk PRL is absorbed by the neonatal gut and is thought to cause changes in the maturation of the hypothalamic neuroendocrine system. Interestingly, hypophysectomized rats retain ~ 20% of biologically active PRL in the circulation, which increases to ~ 50% of normal levels with time. Neutralization of circulating PRL with anti-PRL antibodies results in immune dysfunction and death, suggesting that extrapituitary PRL is important, and under some circumstances, can compensate for pituitary PRL.Pituitary PRL acts via a classical endocrine pathway, meaning it is secreted by a gland, transported by the circulatory system and acts on peripheral target cells via specific receptors located on the plasma membrane. The PRL that is produced by different non pituitary/peripheral cell types can act in a more direct fashion, that is as a growth factor, neurotransmitter, or immunomodulator, in an autocrine or paracrine manner. Thus, locally produced PRL can act on adjacent cells (paracrine) or on the PRL-secreting cell itself (autocrine). Using paracrine or autocrine mechanisms, it would thus be possible to activate many of the actions associated with PRL without ever affecting the circulating concentration of the hormone.
3. MAMMARY GLAND ORGANOGENESIS
At birth, mice show rudiments of indistinguishable mammary ductal architecture. Pubertal mammary gland development is controlled by pituitary and gonadal hormones that act directly and indirectly. After growth until the onset of puberty, terminal end buds (TEBs) form and ductal epithelium begins. Proliferation at this stage occurs mainly in the body cells of the terminal end bud. Later during puberty, under the influence of PRL and progesterone, lobular buds branch off from the ductal system. Organogenesis of the mammary gland is completed when the ductal system has grown to the full extent of the fat pad and lobule buds have sprouted at regular intervals along the ducts. Up to midpregnancy, ductal elongation, branching, and the number of lobules increased. Under the influence of PRL, placental lactogens, progesterone, and local growth factors, the lobuloalveolar epithelium undergoes extensive proliferation. At parturition, the lobuloalveolar epithelium is converted to a secretory phenotype and the full complement of milk proteins and lactogenic enzymes are synthesized. Involution of the lobuloalveolar system occurs at the end of the lactation in response to milk stasis, and decrease of systemic lactogens. Thus, PRL and placental lactogens, which bind to the PRL receptor, act during these different stages: lobule budding, lobuloalveolar expansion during pregnancy, and lactational differentiation and maintenance of milk secretion.
4. STUDIES OF MICE WITH TARGETED GENE DISRUPTIONS
Studies of mice with targeted disruptions of PRL and the PRL receptor (PRLR) genes have demonstrated that this hormone is an obligate regulator of mammary organogenesis, lobuloalveolar growth, and functional differentiation. In nulliparous mice ( PRL-/-) the mammary glands consists of a primary and secondary branched ductal system with numerous terminal end buds along the ducts. Terminal end buds are absent in the mature glands of the normal mice. Thus the complete absence of PRL results in the arrest of mammary organogenesis at an immature pubertal state. Females with one functional allele of the PRL receptor (PRLR+/-) showed almost complete failure of lactation after their first pregnancy. The severity of this phenotype was reduced when the females were mated at 20 weeks and was absent following a second pregnancy. Histological and whole mount analysis of virgin mammary glands are reported elsewhere. In PRLR-/- mice mammary development is reduced, but relatively normal up to puberty. A partial rescue of pregnancy by administration of progesterone allowed analysis of mammary development at day 5.5. Progesterone is required for ductal branching, and the addition of this steroid to maintain the pregnancy is also able to rescue ductal sidebranching in PRLR-/- females, as ductal bifurcation appeared to be normal (Fig 1). To distinguish between the developmental defects intrinsic to the epithelium and those resulting from systemic endocrine alterations, mammary epithelium from PRLR-/- mice was transplanted into mammary fat pads of wild-type mice. In virgin mice, the PRLR-/- epithelial transplants developed normally at puberty; indicating an indirect effect of prolactin on ductal development. During pregnancy, these transplants showed normal side branching and formation of alveolar buds, however without any lobuloalveolar development, These experiments indicate that during pregnancy PRL acts directly on the mammary epithelium to produce lobuloalveolar development.
Fig. 1. Whole mount analysis of mammary development in wild-type and knockout mice at day 5.5 of pregnancy. Whole mounts of mammary glands from mice at 12 weeks of age were prepared from wild-type (A) or knockout (B and C ) animals. Administration of progesterone leads to the maintenance of pregnancy in knockout mice (C). The fourth inguinal mammary gland is shown.
The complete absence of the progesterone receptor gene results in a gland lacking terminal end buds but displaying only some branched ducts. Estradiol receptor α knockout females are infertile, and a lack of ductal growth and differentiation has been reported. In mice deficient in Stat5a, a primary mediator of PRL action, mammary lobuloalveolar outgrowth during pregnancy is curtailed and females are unable to lactate after parturition because of a failure of terminal differetiation. Similarly, mammary gland development is also impaired in Stat5b-/- females and,
although milk protein genes are expressed, there is insufficient milk to feed pups. Interestingly, Stat5b, but not Stat5a, deficient females exhibit severely compromised fertility. Mice homozygous for a germline mutation in A-myb, a nuclear protein regulator of transcription, show a marked underdevelopment of the breast epithelial compartment following pregnancy demonstrating a critical role of A-myb in mammary gland development20. Mice lacking cyclin D1 gene also exhibit a dramatic impairment of mammary gland development leading to inability to lactate their litters.In conclusion, the phenotypes of animals lacking functional genes encoding PRLR, PRL, Stat5a, or Stat5b, confirm the essential role of the lactogenic receptor and its signaling pathways in mammary gland development, whereas estradiol and progesterone receptors are also important but perhaps do not play as central a function as prolactin.
5. MECHANISMS
The role of locally derived growth factors in the mediation of PRL-induced mammary gland development remains unknown. Several growth factors are known to play specific and nonredundant paracrine roles at different stages of mammary development. These include epidermal growth factor (EGF), neuregulin (NRG), Wnt gene products, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF) and insulin-like growth factor- 1 (IGF-1). The interplay between endocrine hormones and epithelial and stromal factors is necessary for normal mammary development. Neuregulin stimulates alveolar development and secretory activity. This member of EGF family is expressed during pregnancy in the stroma so it may be involved in mediating effects which are important during pregnancy. Cyclin D1 knockout mice are devoid of PRL-dependent lobuloalveolar structures in the mature gland , reminiscent of those in progesterone receptor knockout mice. The schematic role of prolactin actions is summarized in Fig 2. 
Fig. 2. Schematic action of PRL on mammary epithelial cells. PRL acts directly on the mammary gland epithelium, and indirectly through the corpus luteum via the signal transducer and activator of transcription Stat5b. In the mammary gland, the major mediator is Stat5a. Growth factor signalling between the mammary epithelium and stroma/adipose compartment induces growth and morphogenesis of the epithelium. Thus, PRL acts through endocrine and autocrine/paracrine pathways.
6. CONCLUSIONS
The technique of gene targeting in mice has been used to develop experimental models where the effects of the complete absence of any lactogen or PRL-mediated effects can be studied. It is clear that there are multiple actions associated with PRL. It will be important to correlate known effects with local production of PRL in some cases in order to distinguish classical endocrine from autocrine/paracrine effects in the mammary gland. The fact that extrapituitary PRL can compensate for pituitary PRL raises the interesting possibility that there may be other effects of PRL than those originally observed in hypophysectomized rats, including a potential role in human breast cancer. The PRL receptor knockout mouse model remains an interesting system to look for effects only activated by PRL or other lactogenic hormones.
In general these model systems will be in the future crucial to facilitate and identify the PRL regulated genes important in the development of the mammary gland.
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Prolactin Receptor