In Utero Exposure to trans-10, cis-12 Conjugated Linoleic Acid Modifies Postnatal Development of the Mammary Gland and its Hormone Responsiveness.

We previously showed that dietary trans-10, cis-12 conjugated linoleic acid (10,12 CLA) stimulates estrogen-independent mammary growth in young ovariectomized mice. Here we investigated the effects of in utero or postnatal exposure to cis-9, trans-11 (9,11 CLA) and 10,12 CLA on postnatal development of the mammary gland and its responsiveness to ovarian steroids. In the first experiment we fed dams different CLA prior to and during gestation, then cross fostered female pups onto control fed dams prior to assessing the histomorphology of their mammary glands. Pregnant dams in the second experiment were similarly exposed to CLA, after which their female pups were ovariectomized then treated with 17ß-estradiol (E), progesterone (P) or E + P for 5 days. In a third experiment, mature female mice were fed different CLA for 28 days prior to ovariectomy, then treated with E, P or E + P. Our data indicate that 10,12 CLA modifies the responsiveness of the mammary glands to E or E + P when exposure occurs either in utero, or postnatally. These findings underline the sensitivity of the mammary glands to dietary fatty acids and reinforce the potential for maternal nutrition to impact postnatal development of the mammary glands and their risk for developing cancer.

Folate Deficiency Inhibits Development of the Mammary Gland and its Associated Lymphatics in FVB Mice.

BACKGROUND: Folate is essential for DNA synthesis, DNA repair, cell proliferation, development, and morphogenesis. Folic acid (FA) is a nutritional supplement used to fortify human diets. OBJECTIVES: We investigated the effects of dietary FA on early mammary gland (MG) development and hyperplasia. METHODS: Study 1: nulliparous female FVB wild-type (WT) mice were fed control (Con; 2 mg FA/kg), deficient (Def; 0 mg FA/kg), excess (Ex; 5 mg FA/kg), or super excess (S-Ex; 20 mg FA/kg) diets for 8 wk before mating to WT or heterozygous FVB/N-Tg[mouse mammary tumor virus long terminal repeat (MMTV)-polyomavirus middle T antigen (PyVT)]634Mul/J (MMTV-PyMT+/-) transgenic males. Dams were fed these diets until they weaned WT or MMTV-PyMT+/- pups, which were fed the dam's diet from postnatal day (PND) 21 to 42. Tissues were collected from female progeny at PNDs 1, 21, and 42. Study 2: Con or Def diets were fed to WT intact females and males from PND 21 to 56, or to ovariectomized females from PND 21 to 77; tissues were collected at PND 56 or 77. Growth of all offspring, development of MGs, MG hyperplasia, supramammary lymph nodes, thymus and spleen, cell proliferation, and expression of MG growth factors were measured. RESULTS: Study 1: Ex or S-Ex did not affect postnatal MG development or hyperplasia. The rate of isometric MG growth (PND 1-21) was reduced by 69% in Def female progeny (P < 0.0001). Similarly, hyperplastic growth in MGs of Def MMTV-PyMT+/- offspring was 18% of Con (P < 0.05). The Def diet reduced supramammary lymph node size by 20% (P < 0.0001) and increased MG insulin-like growth factor 2 mRNA by 200% (P < 0.05) and protein by 130%-150% (P < 0.05). Study 2: the Def diet did not affect MG growth, but it did reduce supramammary lymph node size (P < 0.05), spleen weight (P < 0.001), and thymic medulla area (P < 0.05). CONCLUSIONS: In utero and postnatal folate deficiency reduced the isometric development of the MGs and early MG hyperplasia. Postnatal folate deficiency reduced the development of lymphatic tissues.

The Transcriptome of Estrogen-Independent Mammary Growth in Female Mice Reveals That Not All Mammary Glands Are Created Equally.

Allometric growth of ducts in the mammary glands (MGs) is widely held to be estrogen dependent. We previously discovered that the dietary fatty acid trans-10, cis-12 conjugated linoleic acid (CLA) stimulates estrogen-independent allometric growth and terminal end bud formation in ovariectomized mice. Given the similar phenotype induced by estrogen and CLA, we investigated the shared and/or divergent mechanisms underlying these changes. We confirmed MG growth induced by CLA is temporally distinct from that elicited by estrogen. We then used RNA sequencing to compare the transcriptome of the MG during similar proliferative and morphological states. Both estrogen and CLA affected the genes involved in proliferation. The transcriptome for estrogen-treated mice included canonical estrogen-induced genes, including Pgr, Areg, and Foxa1. In contrast, their expression was unchanged by CLA. However, CLA, but not estrogen, altered expression of a unique set of inflammation-associated genes, consistent with stromal changes. This CLA-altered signature included increased expression of epidermal growth factor receptor (EGFR) pathway components, consistent with the demonstration that CLA-induced MG growth is EGFR dependent. Our findings highlight a unique role for diet-induced inflammation that underlies estrogen-independent MG development.

Trans-Fatty Acid-Stimulated Mammary Gland Growth in Ovariectomized Mice is Fatty Acid Type and Isomer Specific.

We previously reported that the trans-18:2 fatty acid trans-10, cis-12 conjugated linoleic acid (t10,c12-CLA) stimulates mammary gland development independent of estrogen and its receptor. Given the negative consequences of dietary trans-fatty acids on various aspects of human health, we sought to establish whether other trans-fatty acids could similarly induce ovary-independent mammary gland growth in mice. Prepubertal BALB/cJ mice were ovariectomized at 21 days of age then were fed diets enriched with cis-9, trans-11 CLA (c9,t11-CLA), or mixtures of trans-18:1 fatty acids supplied by partially hydrogenated sunflower, safflower, or linseed oil. The resultant mammary phenotype was evaluated 3 weeks later and compared to the growth response elicited by t10,c12-CLA, or the defined control diet. Whereas partially hydrogenated safflower oil increased mammary gland weight, none of the partially hydrogenated vegetable oils promoted mammary ductal growth. Similarly, the c9,t11-CLA supplemented diet was without effect on mammary development. Taken together, our data emphasize a unique effect of t10,c12-CLA in stimulating estrogen-independent mammary gland growth manifest as increased mammary ductal area and elongation that was not recapitulated by c9,t11-CLA or the partially hydrogenated vegetable oil diets.

A Convenient Method for Evaluating Epithelial Cell Proliferation in the Whole Mammary Glands of Female Mice.

The mammary glands (MG) undergo rapid expansion of the ductal network during puberty in response to endocrine cues including the potent mitogenic effects of estrogen. The proliferation of mammary epithelial cells occurs in a spatially distinctive manner, where terminal end buds located at the ductal termini are the primary site of cell division. Here, we present a relatively high throughput approach to spatially assess epithelial cell proliferation in whole mouse MG using histochemical detection of 5-ethynyl-2'-deoxyuridine in conjunction with a standard curve-based data deconvolution technique to semiquantitatively measure proliferation via wide-field epifluorescent microscopy. This approach was validated against the "gold standard" of counting labeled nuclei from confocal images utilizing computer-assisted image analysis. Our method proved sensitive enough to describe the significant and spatially variable proliferative response to low-dose estrogen after 108 hours. This flexible method presents a timely and economical approach to obtaining spatial information regarding epithelial cell proliferation in the mouse MG.

Mammary gland development--It's not just about estrogen.

The mammary gland (MG) is one of a few organs that undergoes most of its growth after birth. Much of this development occurs concurrently with specific reproductive states, such that the ultimate goal of milk synthesis and secretion is coordinated with the nutritional requirements of the neonate. Central to the reproductive-MG axis is its endocrine regulation, and pivotal to this regulation is the ovarian secretion of estrogen (E). Indeed, it is widely accepted that estrogens are essential for growth of the MG to occur, both for ductal elongation during puberty and for alveolar development during gestation. As the factors regulating MG development continually come to light from the fields of developmental biology, lactation physiology, and breast cancer research, a growing body of evidence serves as a reminder that the MG are not as exclusively dependent on estrogens as might have been thought. The objective of this review is to summarize the state of information regarding our understanding of how estrogen (E) has been implicated as the key regulator of MG development, and to highlight some of the alternative E-independent mechanisms that have been discovered. In particular, we review our findings that dietary trans-10,cis-12 conjugated linoleic acid promotes ductal elongation and that the combination of progesterone (P) and prolactin (PRL) can stimulate branching morphogenesis in the absence of E. Ultimately, these examples stand as a healthy challenge to the question of just how important estrogens are for MG development. Answers to this question, in turn, increase our understanding of MG development across all mammals and the ways in which it can affect milk production.

Alcohol intake stimulates epithelial proliferation in an authentic model of the human breast.

The voluntary consumption of alcohol by humans is a modifiable lifestyle factor that has been consistently linked to a woman's risk of developing breast cancer. We have used an animal model that closely recapitulates breast development in humans to study the effect of alcohol intake on breast growth and morphology. Pubertal female pigs were fed alcohol for 4-5 weeks at 19-21% of total caloric intake, which led to average blood alcohol concentrations of 115-130mg/dL. Alongside increased liver mass, alcohol intake promoted the formation of distended ductules within lobular units in association with increased epithelial proliferation. Alcohol consumption also increased phosphorylation of the transcription factor STAT5 in the mammary epithelium, but did not lead to any evidence of precocious lactogenesis. In conclusion, feeding alcohol to female pigs having a similar physiology and mammary gland morphology to humans during a reproductive state equivalent to human adolescence leads to increased mammary gland proliferation and development of atypical lobular structures. These changes may phenocopy how alcohol intake increases the risk for developing breast cancer in humans.

Diet-induced metabolic change induces estrogen-independent allometric mammary growth.

Lifetime breast cancer risk reflects an unresolved combination of early life factors including diet, body mass index, metabolic syndrome, obesity, and age at first menses. In parallel, the onset of allometric growth by the mammary glands around puberty is widely held to be estrogen (E)-dependent. Here we report that several physiological changes associated with metabolic syndrome in response to a diet supplemented with the trans-10, cis-12 isomer of conjugated linoleic acid lead to ovary-independent allometric growth of the mammary ducts. The E-independence of this diet-induced growth was highlighted by the fact that it occurred both in male mice and with pharmacological inhibition of either E receptor function or E biosynthesis. Reversal of the metabolic phenotype with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone abrogated diet-induced mammary growth. A role for hyperinsulinemia and increased insulin-like growth factor-I receptor (IGF-IR) expression during mammary growth induced by the trans-10, cis-12 isomer of conjugated linoleic acid was confirmed by its reversal upon pharmacological inhibition of IGF-IR function. Diet-stimulated ductal growth also increased mammary tumorigenesis in ovariectomized polyomavirus middle T-antigen mice. Our data demonstrate that diet-induced metabolic dysregulation, independently of ovarian function, stimulates allometric growth within the mammary glands via an IGF-IR-dependent mechanism.