Diverse myeloid cells are recruited to the developing and inflamed mammary gland

The immune system plays fundamental roles in the mammary gland, shaping developmental processes and controlling inflammation during infection and cancer. Here we reveal unanticipated heterogeneity in the myeloid cell compartment during development of virgin, pregnant and involuting mouse mammary glands, and in milk. We investigate the functional consequences of individual and compound chemokine receptor deficiency on cell recruitment. Diverse myeloid cell recruitment was also shown in models of sterile inflammation and bacterial infection. Strikingly, we have shown that inflammation and infection can alter the abundance of terminal end buds, a key developmental structure, within the pubertal mammary gland. This previously unknown effect of inflammatory burden during puberty could have important implications for understanding the control of pubertal timing.

Androgen Receptor Inactivation Resulted in Acceleration in Pubertal Mammary Gland Growth, Upregulation of ERα Expression, and Wnt/β-Catenin Signaling in Female Mice

The androgen receptor (AR) is widely expressed in mammary cells of female mammals including humans and mice, indicating a possible role for AR-mediated androgen actions in breast development, function, and pathology, although the specific mechanisms remain unclear. To elucidate the mechanisms of androgen action in mammary gland physiology and development, we used AR-knockout (ARΔex3KO) female mice with a universally expressed, transcriptionally inactive AR protein harboring an in-frame deletion of its second zinc finger. Although in sexually mature wild-type (WT) and ARex3ΔKO females, the mammary epithelial growth was fully extended to the edge of the fat pad, during puberty, ARex3ΔKO females exhibit significantly accelerated mammary ductal growth and an increased number of terminal end buds compared with WT females. Accelerated ARex3ΔKO female mammary growth was associated with significantly increased mammary epithelial ERα expression and activated Wnt/β-catenin signaling as shown by increased Wnt4 expression and accumulation of nuclear β-catenin. These findings are consistent with increased mammary estrogen exposure although ovarian estradiol content was unchanged compared with WT females. Furthermore, treatment with the potent pure androgen DHT markedly reduced ductal extension and terminal end bud numbers in WT but not in ARΔex3KO females, further supporting the concept that AR-mediated, androgen-induced suppression of murine mammary growth is a physiological characteristic of puberty. In summary, our findings reveal an inhibitory role of AR-mediated androgen actions in pubertal mammary gland development by reducing epithelial cell proliferation and could be mediated by regulation of Wnt/β-catenin signaling.

Lifelong exposure to n-3 PUFA affects pubertal mammary gland development

There is growing evidence that early developmental periods may importantly influence future breast cancer risk. Also, there is great interest in the role of dietary fat in breast cancer risk, but the role of dietary fat during pubertal mammary gland development remains poorly understood. This study investigated the effect of n-3 polyunsaturated fatty acids (PUFA) using complementary dietary and genetic approaches to examine the effect of lifelong exposure of n-3 PUFA or n-6 PUFA (control) on mammary gland development and fatty acid composition. n-3 PUFA from both diet and genetics were enriched in mammary glands as early as 3 weeks of age. Parameters related to mammary gland development, including number of terminal end buds (TEB), percent coverage of ductal tree, and infiltration of TEB, were influenced by n-3 PUFA at 3 and 4 weeks of age. Overall, findings suggest that n-3 PUFA incorporation into the mammary gland early in life plays a role in the morphological development of the mammary gland during puberty.

The Ras-like protein R-Ras2/TC21 is important for proper mammary gland development

R-Ras2/TC21 is a GTPase with high sequence and signaling similarity with Ras subfamily members. Although it has been extensively studied using overexpression studies in cell lines, its physiological role remains poorly characterized. Here we used RRas2-knockout mice expressing β-galactosidase under the regulation of the endogenous RRas2 promoter to investigate the function of this GTPase in vivo. Despite its expression in tissues critical for organismal viability, RRas2−/− mice show no major alterations in viability, growth rates, cardiovascular parameters, or fertility. By contrast, they display a marked and specific defect in the development of the mammary gland during puberty. In the absence of R-Ras2/TC21, this gland forms reduced numbers of terminal end buds (TEBs) and ductal branches, leading to a temporal delay in the extension and arborization of the gland tree in mammary fat pads. This phenotype is linked to cell-autonomous proliferative defects of epithelial cells present in TEBs. These cells also show reduced Erk activation but wild type–like levels of phosphorylated Akt. Using compound RRas2-, HRas-, and NRas-knockout mice, we demonstrate that these GTPases act in a nonsynergistic and nonadditive manner during this morphogenic process.

Prepubertal Daidzein Exposure Enhances Mammary Gland Differentiation and Regulates the Expression of Estrogen Receptor-Alpha and Apoptotic Proteins

Mechanism of chemoprevention by daidzein (500 μg/g bwt) was examined by injecting it subcutaneously at 16th, 18th, and 20th day postpartum, followed by counting of terminal end buds (TEBs), terminal ducts (TDs), and lobules and immunohistochemistry of ER-α, Bcl2, Bax, and caspase-3. DNA fragmentation was also analysed to measure the apoptosis level. Estradiol benzoate (EB) (500 ng/g bwt) and dimethyl sulphoxide (DMSO) were used as reference and vehicle, respectively. Observations show a significant enhancement of mammary gland differentiation at postnatal day 21 (PND21) as well as PND50. There was a significant decrease of ER-α expression at PND21 and increase in its expression at PND50, in daidzein-treated animals. The ratio of expression of Bcl-2 to Bax proteins increased at PND50 the same whereas, it decreased at PND50 due to daidzein. An increased expression of caspase-3 and DNA fragmentation was also seen due to daidzein at PND50. The mammary gland of EB-treated animals showed response a somewhat similar to that of daidzein-treated animals.

The Mammary Gland Microenvironment Directs Progenitor Cell FateIn Vivo

The mammary gland is a unique organ that continually undergoes postnatal developmental changes. In mice, the mammary gland is formed via signals from terminal end buds, which direct ductal growth and elongation. Intriguingly, it is likely that the entire cellular repertoire of the mammary gland is formed from a single antecedent cell. Furthermore, in order to produce progeny of varied lineages (e.g., luminal and myoepithelial cells), signals from the local tissue microenvironment influence mammary stem/progenitor cell fate. Data have shown that cells from the mammary gland microenvironment reprogram adult somatic cells from other organs (testes, nerve) into cells that produce milk and express mammary epithelial cell proteins. Similar results were found for human tumorigenic epithelial carcinoma cells. Presently, it is unclear how the deterministic power of the mammary gland microenvironment controls epithelial cell fate. Regardless, signals generated by the microenvironment have a profound influence on progenitor cell differentiationin vivo.