Russell C. Hovey

Establishing a Framework for the Functional Mammary Gland: From Endocrinology to Morphology

From its embryonic origins, the mammary gland in females undergoes a course of ductal development that supports the establishment of alveolar structures during pregnancy prior to the onset of lactogenesis. This development includes multiple stages of proliferation and morphogenesis that are largely directed by concurrent alterations in key hormones and growth factors across various reproductive states. Ductal elongation is directed by estrogen, growth hormone, insulin-like growth factor-I, and epidermal growth factor, whereas ductal branching and alveolar budding is influenced by additional factors such as progesterone, prolactin, and thyroid hormone. The response by the ductal epithelium to various hormones and growth factors is influenced by epithelial–stromal interactions that differ between species, possibly directing species-specific morphogenesis. Evolving technologies continue to provide the opportunity to further delineate the regulation of ductal development. Defining the hormonal control of ductal development should facilitate a better understanding of the mechanisms underlying mammary gland tumorigenesis.

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.

Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland

Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L‐PRLR) and two of the three short forms (S1‐ and S3‐PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2‐PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L‐PRLR mRNA. By contrast, S1‐PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2‐PRLR became undetectable during puberty. Stromal expression of S3‐PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.

Prolactin-induced expression of vascular endothelial growth factor via Egr-1

Angiogenesis is a dynamic process regulated by both local and systemic factors. Among these is vascular endothelial growth factor (VEGF), a potent effector of angiogenesis and vascular permeability. Previously we showed that VEGF is temporally and spatially regulated in the mouse mammary gland during development and lactation. Given the functions of prolactin (PRL) during these stages and the supporting role of the vasculature, we investigated the regulation of VEGF by PRL. Treatment of HC11 mouse mammary epithelial and Nb2 rat lymphoma cells with PRL induced VEGF expression. Deletion and mutation analysis identified a GC-rich region in the proximal region of the VEGF promoter that constitutively bound Sp1 and PRL-induced Egr-1. These sites conferred PRL-responsiveness leading to increased VEGF transcription. The induction of VEGF by PRL was PRL receptor-, Jak2- and MAP kinase kinase-dependent. Our results indicate that PRL induces VEGF expression through Egr-1, and implicates VEGF as an intermediary of PRL-regulated angiogenesis.

Effects of Neonatal Exposure to Diethylstilbestrol, Tamoxifen, and Toremifene on the BALB/c Mouse Mammary Gland

Abstract In this study, we compared the long-term effects of neonatal exposure to diethylstilbestrol (DES, 0.0125–50 µg), tamoxifen (TAM, 0.0125–50 µg), and toremifene (TOR, 53 µg) on mammary gland development and differentiation. Allometric growth of the mammary ducts was stimulated by neonatal DES exposure (12.5 µg) and impaired by exposure to TAM (25 µg). Neonatal treatment with high doses of DES resulted in mammary ducts that displayed extensive dilatation and precocious lactogenesis in postpubertal, nulliparous females. Initiation of this precocious differentiation coincided with the absence of corpora lutea, increased levels of serum prolactin (PRL), and the induction of Prl mRNA expression within the mammary glands. Neonatal exposure to 1.25 µg TAM increased alveolar development in postpubertal, nulliparous females similar to that recorded in females treated with low doses of DES. Lower doses of TAM did not affect alveolar development, whereas branching morphogenesis and alveolar development were impaired by higher doses. Increased alveolar development in females exposed to 1.25 µg TAM was associated with elevated serum progesterone (P) and increased alveolar development in response to exogenous P. Taken together, our findings demonstrate that neonatal exposure to both DES and TAM exerts long-lasting effects on the proliferation and differentiation of the mammary glands in female BALB/c, primarily as the result of endocrine disruption

Application of In Situ PCR to Studies of the Mammary Gland

The ability to detect low numbers of target nucleic acid sequence provides a powerful tool to investigate the molecular events that underlie development and disease in organs such as the mammary gland. Recent development of the polymerase chain reaction in situ (IS-PCR) now enables the intracellular amplification of DNA by PCR and its subsequent localization in preparations of fixed cells and tissue. This chapter outlines the procedure and materials required for the localization of integrated mouse mammary tumor virus proviral DNA in mouse mammary tissue by an indirect IS-PCR method. Also discussed are several aspects critical to the success of this emerging technique, particularly regarding tissue pretreatment and the importance of appropriate controls. In addition, various potential applications of this technique to studies of the mammary gland are considered.