Prolactin Regulation of Mammary Gland Development

Mammary morphogenesis is orchestrated with other reproductive events by pituitary-driven changes to the systemic hormone environment, initiating the formation of a mammary ductal network during puberty and the addition of secretory alveoli during pregnancy. Prolactin is the major driver of development during pregnancy via regulation of ovarian progesterone production (in many species) and direct effects on mammary epithelial cells (in all species). Together these hormones regulate two aspects of development that are the subject of intense interest: (1) a genomic regulatory network that integrates many additional spatial and temporal cues to control gene expression and (2), the activity of a stem and progenitor cell hierarchy. Amalgamation of these two aspects will increase our understanding of cell proliferation and differentiation within the mammary gland, with clear application to our attempts to control breast cancer. Here we focus on providing an over-view of prolactin action during development of the model murine mammary gland. Samantha R. Oakes and Renee L. Rogers contributed equally to this work. Financial Support: Australian Research Council, National Health and Medical Research Council, The Cancer Institute NSW and Cancer Council NSW.     

Regulation of Mammary Gland Development by Tissue Interaction

Development of the mammary glands is initiatedin the embryo but the major part of their developmentoccurs in the adult. While development in puberty andpregnancy is dependent on hormones, prenatal and early postnatal development appear to progressautonomously. Mutual and reciprocalepithelial-mesenchymal interactions are critical forboth phases of development. Specific steps such as theformation of the bud, the first appearance of hormonereceptors, formation of the primary sprout and ductalelongation have been shown to be governed byepithelial-mesenchymal signaling. In recent years, someof the signaling molecules that are required in theseprocesses have been identified through geneinactivation. We discuss the potential role of thesefactors in mediating growth and differentiation. Inaddition we provide evidence that mammary epithelialcells from late embryonic stages are already capable ofsynthesizing milk proteins when subjected to appropriatehormonal stimulation.     

Regulation of Casein Messenger RNA during the Development of the Rat Mammary Gland

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a heterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands. This work was supported by the American Cancer Society Institutional Grant, ACSINS-270, and by a contract, HEW-N01-CP-43385.     

Use of PRKO Mice to Study the Role of Progesterone in Mammary Gland Development

To better understand the distinct physiological roles played by progesterone and estrogen receptors (PR amd ER)⁴ as well as to study directly PR function in an in vivo context, a novel mutant mouse strain, the PR knockout (PRKO) mouse, was generated carrying a germline loss of function mutation at the PR locus. Mouse mammary gland development has been examined in PRKO mice using reciprocal transplantation experiments to investigate the effects of the stromal and epithelial PRs on ductal and lobuloalveolar development. The absence of PR in transplanted donor epithelium, but not in recipient stroma, prevented normal lobuloalveolar development in response to estrogen and progesterone treatment. Conversely, the presence of PR in the transplanted donor epithelium, but not in the recipient stroma, revealed that PR in the stroma may be necessary for ductal development. Stimulation of ductal development by the PR may, therefore, be mediated by an unknown secondary signaling molecule, possibly a growth factor. The continued stimulation of the stromal PR appears to be dependent on reciprocal signal(s) from the epithelium. Thus, the combination of gene knockout and reciprocal transplantation technologies has provided some new insights into the role of stromal-epithelial interactions and steroid hormones in mammary gland development.     

Regulation of Mammary Gland Growth and Morphogenesis by the Mammary Fat Pad: A Species Comparison

The growth and morphogenesis of mammaryparenchyma varies substantially between species and isregulated by an array of systemic and local factors.Central to this regulation is the mammary fat pad, amatrix of adipose and connective tissue capable ofmediating hormone action and synthesizing an array ofgrowth regulatory molecules. In this article wehighlight differences between the morphologicaldevelopment of the mammary parenchyma in rodents, humans,and ruminant dairy animals, placing emphasis ondifferences in the cellular composition and structure ofthe mammary fat pad. While a great deal remains to be understood about the ability of stroma tolocally regulate mammary development, the significanceof its contribution is becoming increasingly apparent.The actions of several steroid and peptide hormones appear to be mediated by an array of growthfactors, proteases and extracellular matrix componentssynthesized by constituents of the mammary fat pad.Further, mammary adipose tissue represents a significant store of lipid which, by itself and through itsderivatives, could influence the growth of mammaryepithelium in diverse ways. This review describes theintegral role of the mammary fat pad duringmammogenesis, emphasizing the point that species differencesmust be addressed if local growth and morphogenicmechanisms within the mammary gland are to beresolved.     

IGF Ligand and Receptor Regulation of Mammary Development

The insulin-like growth factors, IGF-I and IGF-II, have endocrine as well as autocrine-paracrine actions on tissue growth. Both IGF ligands are expressed within developing mammary tissue throughout postnatal stages with specific sites of expression in the epithelial and stromal compartments. The elucidation of circulating versus local actions and of epithelial versus stromal actions of IGFs in stimulating mammary epithelial development has been the focus of several laboratories. The recent studies addressing IGF ligand function provide support for the hypotheses that (1) the diverse sites of IGF expression may mediate different cellular outcomes, and (2) IGF-I and IGF-II are distinctly regulated and have diverse functions in mammary development. The mechanisms for IGF function likely are mediated, in part, through diverse IGF signaling receptors. The local actions of the IGF ligands and receptors as revealed through recent publications are the focus of this review. Grant Support: National Institute of Diabetes and Digestive and Kidney Diseases DK60612 and National Cancer Institute CA120850 to TLW     

Developmental and Hormonal Regulation of Protein N-Glycosylation in the Mammary Gland

Glycosylation represents the most commonconjugation of both membrane-bound and secreted proteinsof animal cells. Among the different types ofglycosylation, the N-linked attachment of sugars to thepolypeptide backbone is by far the most abundantmodification. The biosynthesis of the precursorcarbohydrate unit of these proteins is initiated by astepwise assembly ofGlc₃Man₉Glc₂NAc P-P-Dolin the dolichol cycle, its transfer en bloc to the nascent polypeptidein the rough endoplasmic reticulum (RER),³followed by excision of the glucosyl residues byprocessing-specific enzymes, glucosidase I and II, alsoresident in the endoplasmic reticulum. Additionalposttranslational modifications of the carbohydratemoiety in the RER, Golgi, and trans -Golgi network,differ for individual glycoproteins for the completionof final products as high mannose, complex orhybrid glycoproteins en route to their finaldestinations in the secretory pathway. The enzymeGlcNAc-1-P transferase (GPT) catalyzes the first andcommitted step, i.e., the transfer of GlcNAc-1-P fromUDP-GlcNAc to Dol-P to form GlcNAc-P-PDol, in theassembly of the oligosaccharide precursor. GlucosidaseI triggers the maturation phase by clipping the distal 1,2-linked Glc residue on the incipientglycoprotein. The critical juxtaposition of the twoenzymes in the multistep pathway makes them excellentcandidates for the overall regulation of proteinN-glycosylation. The highly elevated needs of glycosylationduring lactation demand regulation of glycosylation inthe gland over and above the levels in the quiescent,virgin and postlactating, regressed gland.     

Early organogenesis of the kidney

The mammalian permanent kidney consists of three cell lineages of different origin: the epithelial cells of the ureter bud, the mesenchymal cells of the nephric blastema and the endothelial cells of the capillaries. Organogenesis is governed by a cascade of morphogenetic interactions between these cell populations, a reciprocal epithelial-mesenchymal interaction between the branching ureter and the metanephric mesenchyme, homotypic interactions between cells of the tubular anlagen, stimulation of angiogenesis by the differentiating blastema and a mesenchymal-endothelial interaction guiding the migration of the capillary endothelial cells. While the biology of these interactive events is well known, as described in this overview, the molecular mechanisms are less well mapped out.     

Regulation of p53 and Its Targets During Involution of the Mammary Gland

Post-lactational involution of the mammary glandprovides a system in which to study the expression andfunction of genes that regulate apoptosis in the contextof a normal tissue. The functions of the p53 tumor suppressor gene have been extensivelystudied as a mediator of apoptosis in response to DNAdamage, but its regulation in normal physiologicprocesses has been poorly characterized. Expression of p53 mRNA was shown to be among the firstgenes to be induced in mammary tissue following weaningof neonates. Although involution proceeds in the absenceof a functional p53 gene, it is delayed compared to normal individuals. Therefore, involutioncan be viewed as biphasic with initial responses beingsensitive to p53, whereas secondary responses beingp53-independent. These observations can be exploited to determine the subset of genes that arep53-responsive and that mediate the effects of p53 innormal mammary tissue.     

Mammary Gland Growth and Development from the Postnatal Period to Postmenopause: Ovarian Steroid Receptor Ontogeny and Regulation in the Mouse

Ovarian steroid hormones play a critical role inregulating mammary gland growth and development. Themammary gland sequentially acquires and cyclicallyexhibits proliferative responses to estrogen and/or progesterone from birth to postmenopause. Thefocus of this review is to presentour currentunderstanding of estrogen and progesterone receptordistribution in epithelial and stromal cells and theirfunctions in relation to mammary gland development.Insights gained from the study of the normal mammarygland are relevant to our understanding of theconditions which may predispose women to the developmentof breast cancer as well as to alterations inhormonal regulation that occur in breastcancer.     

Role of Prolactin and Vasoinhibins in the Regulation of Vascular Function in Mammary Gland

The formation of new blood vessels has become a major focus of mammary gland research stimulated by the therapeutic opportunities of controlling angiogenesis in breast cancer. Normal growth and involution of the mammary gland are profoundly affected by the expansion and regression of blood vessels, whereas dysregulation of angiogenesis is characteristic of breast cancer growth and metastasis. Prolactin stimulates the growth and differentiation of the mammary gland under normal conditions, but its role in breast cancer is controversial. Its action is complicated by the fact that prolactin itself is angiogenic, but proteases cleave prolactin to generate vasoinhibins, a family of peptides that act on endothelial cells to suppress angiogenesis and vasodilation and to promote apoptosis-mediated vascular regression. This review summarizes our current knowledge about the vascular effects of prolactin and the generation and action of vasoinhibins, and discusses their possible contribution to the regulation of blood vessels in the normal and malignant mammary gland.     

Exploring of the Unpredicted Effects of Olfactory Network Injuries on Mammary Gland Degeneration: A Preliminary Experimental Study

ABSTRACT

Purpose of the study: Hypofunctioning breasts are typically considered a dysfunction of higher brain centers that regulate hormonal feedback, and olfactory information has been proposed as a triggering factor for lactation in the maternal body. However, there are no substantive studies regarding whether olfaction disorders and/or loss of olfactory sense may result in breast gland atrophy by causing diminished olfactory stimulation. To fill this gap in the literature, we studied the histologic features of breast glands as a sample model in animals that had undergone an olfactory bulb lesion (OBL). Materials and methods: This study was conducted on 22 rats. Six, eight, and six of them were used as control, SHAM, and OBL groups, respectively. After 10 weeks, the animals were decapitated. Olfactory bulbs and breast glands were stained with Hematoxylin-eosin and tunnel dye. Specimens were analyzed stereologically to evaluate the loss in volume of the olfactory bulbs, total breast follicle volume (TBFV) and Meissner's corpuscles per cubic centimeter, and these two senior metrics were compared with each other statistically. Results: Olfactory bulb volume loss and breast gland atrophy were both detected in study group. Mean TBFV and OB volumes were measured as: (296 ± 89) × 10⁶ µm³/cm³ and 4.43 ± 0.98 mm³ in control (Group I); (264 ± 63) × 10⁶ µm³/cm³ and 3.86 ± 0.81 mm³ in SHAM (Group II) and (194 ± 52) × 10⁶ µm³/cm³ and 1.52 ± 0.36 mm³ in OBL group (Group III). It was noted that the TBFV was significantly diminished, with apoptotic degradation in the olfactory bulbs and breast glands of OBL-applied animals (p < 0.001). Conclusion: It seems that diminished milk secretion is attributable to the degradation of breast glands that results from olfaction loss in OBL animals.     

A Role for Fibroblast Growth Factor Signaling in the Lobuloalveolar Development of the Mammary Gland

The inappropriate expression of growth factors, or activating mutations of their receptors, have been implicated as causative factors in mouse and human mammary cancer. For example, it has been known for some time that three members of the fibroblast growth factor (FGF)³ family behave like oncogenes in virally induced mammary cancer of mice. In normal circumstances, signaling via FGF receptors is known to mediate growth, differentiation, and patterning, during embryogenesis and fetal development. A powerful approach to dissecting the roles for these signaling pathways is to determine the developmental consequences of abrogating their function in transgenic mice. In this review, we describe the use of dominant negative FGF receptors to evaluate the contribution of specific FGF signals in normal mammary gland development. These studies have revealed that normal lobuloalveolar development requires FGF signaling to the mammary epithelium, a function that is presumably usurped by MMTV in mouse mammary tumorigenesis.     

The Mammary Bud as a Skin Appendage: Unique and Shared Aspects of Development

Like other skin appendages, the embryonic mammary gland develops via extensive epithelial–mesenchymal interactions. Early stages in embryonic mammary development strikingly resemble analogous steps in the development of hair follicles and teeth. In each case the first morphological sign of development is a localized thickening in the surface epithelium that subsequently invaginates to form a mammary, hair follicle or tooth bud. Similar sets of intersecting signaling pathways are involved in patterning the mammary, hair follicle and dental epithelium, directing placode formation, and controlling bud invagination. Despite these similarities, subsequent events in the formation of these appendages are diverse. The mammary bud extends to form a sprout that begins to branch upon contact with the mammary fat pad. Hair follicles also extend into the underlying mesenchyme, but instead of branching, hair follicle epithelium folds around a condensation of dermal cells. In contrast, teeth undergo a more complex folding morphogenesis. Here, we review what is known of the molecular and cellular mechanisms controlling early steps in the development of these organs, attempt to unravel both common themes and unique aspects that can begin to explain the diversity of appendage formation, and discuss human genetic diseases that affect appendage morphogenesis.     

The Mammary Gland as a Bioreactor: Expression, Processing, and Production of Recombinant Proteins

A variety of transgenic animal species are beingused to produce recombinant proteins. The generalapproach is to target the expression of the desiredprotein to the mammary gland using regulatory elements derived from a milk protein gene and thencollect and purify the product from milk. Promotersequences from a number of different milk protein geneshave been used to target expression to the mammarygland, although significant problems remain withregard to achieving transgene expression levelsconsistent with commercial exploitation. The mammarygland appears to be capable of carrying out the complexposttranslational modifications, such as glycosylation and-carboxylation required for the biologicalactivity and stability of specific proteins. Effectivepurification protocols have been established andproducts produced by this route have now enteredclinical trials.     

Interactions of Prolactin and Growth Hormone (GH) in the Regulation of Mammary Gland Function and Epithelial Cell Survival

The relative importance of GH³ and prolactin in mammary gland function varies between species with prolactin playing a major role in rodents and GH taking lead role in ruminants. In rodents, however, GH appears to play a vital role in maintaining a high-fat/low volume milk in the absence of prolactin and a similar finding has been demonstrated in goats where prolactin deficiency causes a more modest (15%) decrease in milk yield. Surprisingly GH-deficiency in goats induced no further decline in milk yield whereas exogenous GH or prolactin both stimulated milk output considerably. Although direct effects of prolactin on mammary epithelial cells are well-documented effects of GH are believed to be mediated indirectly via IGF-1 production from the liver. We have been unable to confirm this hypothesis in rats and believe this to be because it is too simplistic. By considering prolactin and GH to be survival factors for the mammary gland we now propose a mechanism by which they interact through the IGF system. Involution of the mammary gland involves apoptosis and, in rats, it is induced by prolactin-deficiency or milk accumulation. Coincidentally with this process mammary epithelial cells synthesize and secrete an IGF binding protein, IGFBP-5. We hypothesize that GH stimulates IGF-1 production, possibly from the mammary parenchyma. IGF-1 then acts as a survival factor for the mammary gland. Prolactin plays an essential role since it suppresses the secretion of IGFBP-5 which would otherwise inhibit IGF-1 action and lead to the induction of cell death.