The mammary gland is unique in its requirement to develop in close association with a depot of adipose tissue that is commonly referred to as the mammary fat pad. As discussed throughout this issue, the mammary fat pad represents a complex stromal microenvironment that includes a variety of cell types. In this article we focus on adipocytes as local regulators of epithelial cell growth and their function during lactation. Several important considerations arise from such a discussion. There is a clear and close interrelationship between different stromal tissue types within the mammary fat pad and its adipocytes. Furthermore, these relationships are both stage- and species-dependent, although many questions remain unanswered regarding their roles in these different states. Several lines of evidence also suggest that adipocytes within the mammary fat pad may function differently from those in other fat depots. Finally, past and future technologies present a variety of opportunities to model these complexities in order to more precisely delineate the many potential functions of adipocytes within the mammary glands. A thorough understanding of the role for this cell type in the mammary glands could present numerous opportunities to modify both breast cancer risk and lactation performance.

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy.

This paper resulted from a conference entitled “Lactation and Milk: Defining and refining the critical questions” held at the University of Colorado School of Medicine from January 18–20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond.

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. Published 2001 Wiley-Liss, Inc. †

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.

The growth and morphogenesis of mammary parenchyma varies substantially between species and is regulated by an array of systemic and local factors. Central to this regulation is the mammary fat pad, a matrix of adipose and connective tissue capable of mediating hormone action and synthesizing an array of growth regulatory molecules. In this article we highlight differences between the morphological development of the mammary parenchyma in rodents, humans, and ruminant dairy animals, placing emphasis on differences in the cellular composition and structure of the mammary fat pad. While a great deal remains to be understood about the ability of stroma to locally regulate mammary development, the significance of its contribution is becoming increasingly apparent. The actions of several steroid and peptide hormones appear to be mediated by an array of growth factors, proteases and extracellular matrix components synthesized by constituents of the mammary fat pad. Further, mammary adipose tissue represents a significant store of lipid which, by itself and through its derivatives, could influence the growth of mammary epithelium in diverse ways. This review describes the integral role of the mammary fat pad during mammogenesis, emphasizing the point that species differences must be addressed if local growth and morphogenic mechanisms within the mammary gland are to be resolved.

Targeted deletion of the bZIP transcription factor, CCAAT/enhancer binding protein-beta (C/EBPbeta), was shown previously to result in aberrant ductal morphogenesis and decreased lobuloalveolar development, accompanied by an altered pattern of progesterone receptor (PR) expression. Here, similar changes in the level and pattern of prolactin receptor (PrlR) expression were observed while screening for differentially expressed genes in C/EBPbeta(null) mice. PR patterning was also altered in PrlR(null) mice, as well as in mammary tissue transplants from both PrlR(null) and signal transducer and activator of transcription (Stat) 5a/b-deficient mice, with concomitant defects in hormone-induced proliferation. Down-regulation of PR and activation of Stat5 phosphorylation were seen after estrogen and progesterone treatment in both C/EBPbeta(null) and wild-type mice, indicating that these signaling pathways were functional, despite the failure of steroid hormones to induce proliferation. IGF binding protein-5, IGF-II, and insulin receptor substrate-1 all displayed altered patterns and levels of expression in C/EBPbeta(null) mice, suggestive of a change in the IGF signaling axis. In addition, small proline-rich protein (SPRR2A), a marker of epidermal differentiation, and keratin 6 were misexpressed in the mammary epithelium of C/EBPbeta(null) mice. Together, these data suggest that C/EBPbeta is a master regulator of mammary epithelial cell fate and that the correct spatial pattern of PR and PrlR expression is a critical determinant of hormone-regulated cell proliferation.

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