Impaired Mammary Gland Development Research Articles

TNFAIP3 Reduction-of-Function Drives Female Infertility and CNS Inflammation.

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.

In Utero and Lactational Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants Induces a Premature Development of the Mammary Glands.

In utero and prepubertal development of the mammary glands occurs minimally in a hormone independent manner until puberty where maturation of the hypothalamic-pituitary-gonadal axis drives an extensive remodeling. Nevertheless, because the immature glands contain functional hormone receptors, they are especially vulnerable to the effects of endocrine disruptors, such as brominated flame retardants (BFRs). BFRs are widespread chemicals added to household objects to reduce their flammability, and to which humans are ubiquitously exposed. We previously reported that in utero and lactational exposure to BFRs resulted in an impaired mammary gland development in peripubertal animals. Here, we assessed whether BFR-induced disruption of mammary gland development could manifest earlier in life. Dams were exposed prior to mating until pups' weaning to a BFR mixture (0, 0.06, 20, or 60 mg/kg/day) formulated according to levels found in house dust. The mammary glands of female offspring were collected at weaning. Histo-morphological analyses showed that exposure to 0.06 mg/kg/day accelerates global epithelial development as demonstrated by a significant increase in total epithelial surface area, associated with a tendency to increase of the ductal area and thickness, and of lumen area. Significant increases of the Ki67 cell proliferation index and of the early apoptotic marker cleaved caspase-9 were also observed, as well as an upward trend in the number of thyroid hormone receptor α1 positive cells. These molecular, histologic, and morphometric changes are suggestive of accelerated pubertal development. Thus, our results suggest that exposure to an environmentally relevant mixture of BFRs induces precocious development of the mammary gland.

Got Milk? Identifying and Characterizing Lactation Defects in Genetically-Engineered Mouse Models.

The ability to produce and expel milk is important for the health and survival of all mammals. Nevertheless, our understanding of the molecular events underlying the execution of this process remains incomplete. Whilst impaired mammary gland development and lactational competence remains the subject of focused investigations, defects in these events may also be an unintended consequence of genetic manipulation in rodent models. In this technical report, we outline established and emerging methods to characterize lactation phenotypes in genetically-engineered mouse models. We discuss important considerations of common models, optimized conditions for mating and the importance of litter size and standardization. Methods for quantifying milk production and quality, as well as protocols for wholemount preparation, immunohistochemistry and the preparation of RNA and protein lysates are provided. This review is intended to help guide researchers new to the field of mammary gland biology in the systematic analysis of lactation defects and in the preparation of samples for more focused mechanistic investigations.

Endocrine-disrupting chemicals and breastfeeding duration: a review.

The purpose of this review is to describe epidemiologic and toxicological literature investigating how endocrine-disrupting chemicals (EDCs) affect mammary gland development and function, thereby impacting lactation duration. Perfluoroalkyl and polyfluoroalkyl substances appear to reduce breastfeeding duration through impaired mammary gland development, lactogenesis, and suppressed endocrine signaling. Halogenated aromatic hydrocarbons have differing associations with lactation duration, likely because of the variety of signaling pathways that they affect, pointing to the importance of complex mixtures in epidemiologic studies. Although epidemiologic literature suggests that pesticides and fungicides decrease or have no effect on lactation duration, toxicology literature suggests enhanced mammary gland development through estrogenic and/or antiandrogenic pathways. Toxicological studies suggest that phthalates may affect mammary gland development via estrogenic pathways but no association with lactation duration has been observed. Bisphenol A was associated with decreased duration of breastfeeding, likely through direct and indirect action on estrogenic pathways. EDCs play a role in mammary gland development, function, and lactogenesis, which can affect breastfeeding duration. Further research should explore direct mechanisms of EDCs on lactation, the significance of toxicant mixtures, and transgenerational effects of EDCs on lactation.

Biological effects of anti-RANKL antibody administration in pregnant mice and their newborns

Denosumab, a fully human monoclonal antibody that neutralizes receptor activator of nuclear factor-κB ligand (RANKL) and blocks osteoclast differentiation, has received approval in Japan for use as an anti-resorptive drug for osteoporosis and skeletal-related events (SREs) in patients with solid cancer. Denosumab is contraindicated during pregnancy, though the effects of blocking RANKL activity on pregnant mothers and their newborns are unclear. We used mice to investigate the effects of an anti-RANKL antibody on maternal and newborn health. Mothers injected with the anti-RANKL antibody had increased bone mass as compared with the controls, while osteoclast number and the level of tartrate-resistant acid phosphatase (TRAP) in serum were increased at the end of pregnancy. Newborn mice exposed to the antibody in utero were normally born, but showed increased bone mass and died within 48 h after birth. None of the newborns were found to have milk in their stomachs, suggesting that they died due to a maternal defect in lactation. Consistent with this, anti-RANKL antibody-injected mothers displayed impaired mammary gland development. However, fostering by healthy surrogate mothers rescued only 33% of the antibody-exposed newborns, suggesting that neonatal mortality was due, at least in part, to an intrinsic defect in the newborns. Our findings show that anti-RANKL antibody administration during pregnancy results in not only an undesirable increase in bone mass, but also has harmful effects on newborn survival.

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2−/−mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2−/− mice.The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.

Serum Dioxin Concentrations and Breast Cancer Risk in the Seveso Women

S-30B2-2 Background/Aims: A widespread environmental contaminant 2,3,7,8-Tetrachlorodibenzo-para-dioxin (TCDD) disrupts multiple endocrine pathways. Impaired mammary gland development has been reported in TCDD-treated mice. Exposure to endocrine-disrupting chemicals, such as TCDD, has been hypothesized to be associated with breast cancer. On 10 July 1976, as a result of a chemical explosion, residents of Seveso, Italy, experienced the highest levels of TCDD in a human population. In 1996, we initiated the Seveso Women Health Study (SWHS), a retrospective cohort study of the reproductive health of the women. We previously reported a significant increased risk for breast cancer incidence with individual serum TCDD level, based on 15 cases, but the cohort averaged only 40 years old in 1996. Herein, we report results from a subsequent follow-up in 2008. Methods: In 1996, we enrolled 981 women who were 0–40 years in 1976, resided in the most contaminated areas, and had adequate archived sera collected soon after the explosion. A total of 833 women (86%) participated in the 2008 follow-up. TCDD concentrations were measured in the archived serum by high-resolution mass spectrometry. Breast cancer cases were identified during interview and confirmed by medical record. We examined the relation of serum TCDD with breast cancer incidence using Cox proportional hazards model. Results: By the 2008 follow-up, 32 (3.3%) women had been diagnosed with breast cancer. Most cases were premenopausal. The average age at diagnosis was 47.4 (±8.3) years, and median (range) serum TCDD level was 60.2 (6.7–1960) ppt, lipid-adjusted. The unadjusted hazard ratio for breast cancer associated with a 10-fold increase in serum TCDD levels (logTCDD) was 1.5 (95% confidence interval: 0.9–2.7). Conclusion: Preliminary analyses suggest individual serum TCDD continues to be positively related with breast cancer incidence in the SWHS cohort, approximately 30 years after exposure. Covariate-adjusted results will be presented.

Isoform-Specific Degradation of PR-B by E6-AP Is Critical for Normal Mammary Gland Development

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-AP(WT)) or ubiquitin-protein ligase-defective E6-AP (E6-AP(C833S)) in the mammary gland. Here we show that overexpression of E6-AP(WT) results in impaired mammary gland development. In contrast, overexpression of E6-AP(C833S) or loss of E6-AP (E6-AP(KO)) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression.

DNA Binding by Estrogen Receptor-α Is Essential for the Transcriptional Response to Estrogen in the Liver and the Uterus

The majority of the biological effects of estrogens in the reproductive tract are mediated by estrogen receptor (ER)alpha, which regulates transcription by several mechanisms. Because the tissue-specific effects of some ERalpha ligands may be caused by tissue-specific transcriptional mechanisms of ERalpha, we aimed to identify the contribution of DNA recognition to these mechanisms in two clinically important target organs, namely uterus and liver. We used a genetic mouse model that dissects DNA binding-dependent vs. independent transcriptional regulation elicited by ERalpha. The EAAE mutant harbors amino acid exchanges at four positions of the DNA-binding domain (DBD) of ERalpha. This construct was knocked in the ERalpha gene locus to produce ERalpha((EAAE/EAAE)) mice devoid of a functional ERalpha DBD. The phenotype of the ERalpha((EAAE/EAAE)) mice resembles the general loss-of-function phenotype of alphaER knockout mutant mice with hypoplastic uteri, hemorrhagic ovaries, and impaired mammary gland development. In agreement with this phenotype, the expression pattern of the ERalpha((EAAE/EAAE)) mutant mice in liver obtained by genome-wide gene expression profiling supports the observation of a near-complete loss of estrogen-dependent gene regulation in comparison with the wild type. Further gene expression analyses to validate the results of the microarray data were performed by quantitative RT-PCR. The analyses indicate that both gene activation and repression by estrogen-bound ERalpha rely on an intact DBD in vivo.

Dioxin Exposure and Shortened Duration of Lactation in Seveso

ISEE-325 Objective: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant, has been shown to disrupt multiple endocrine systems in animals. Impaired mammary gland development, leading to impaired lactation has been reported in TCDD-treated mice. Higher maternal levels of other endocrine disrupting compounds have been associated with shortened duration of lactation. No epidemiologic studies have examined the potential effects of TCDD exposure on lactation. On July 10, 1976, as a result of a chemical explosion, residents of Seveso, Italy, experienced the highest levels of TCDD in a human population. Twenty years later, we initiated the Seveso Women's Health Study (SWHS), a retrospective cohort study of the reproductive health of the women. We examined the relationship of TCDD with initiation and duration of lactation for the first postexplosion pregnancy. Material and Methods: The SWHS cohort comprises 981 women who were aged 0 to 40 years in 1976 and resided in the most contaminated areas. Individual TCDD exposure was measured in archived sera collected soon after the explosion by high-resolution mass spectrometry. For each live birth after the explosion, women were asked whether they breast-fed and, if yes, how many months. Duration of lactation for the first live birth after the explosion was examined as a continuous dependent variable using multiple linear regression. Results: In total, 252 (64%) women initiated breast-feeding, and the average duration of lactation was 4.6 (±4.3) months. More highly exposed women were nonsignificantly more likely to initiate breast-feeding (OR per 10-fold increase in TCDD = 1.4, 95% CI: 0.9–2.0). However, duration of lactation (in months) was nonsignificantly decreased for each 10-fold increase in TCDD (β = −0.34, 95% CI: −0.96 to 0.27). Conclusions: These results suggest that TCDD exposure, like other endocrine disrupting compounds, may affect women's duration of lactation. Multivariate results will be presented and interpreted in light of study advantages and limitations.

Replacement of connexin43 by connexin26 in transgenic mice leads to dysfunctional reproductive organs and slowed ventricular conduction in the heart

BackgroundIn order to further distinguish unique from general functions of connexin43, we have generated mice in which the coding region of connexin43 was replaced by that of connexin26.ResultsHeterozygous mothers showed impaired mammary gland development responsible for decreased lactation and early postnatal death of the pups which could be partially rescued by wild type foster mothers. Only about 17% of the homozygous connexin43 knock-in connexin26 mice instead of 25% expected according to Mendelian inheritance, were born and only 6% survived to day 21 post partum and longer. Neonatal and adult connexin43 knock-in connexin26 mice exhibited slowed ventricular conduction in their hearts, i.e. similar but delayed electrophysiological abnormalities as connexin43 deficient mice. Furthermore, connexin43 knock-in connexin26 male and female mice were infertile and exhibited hypotrophic gonads. In testes, tubuli seminiferi were developed and spermatogonia as well as some primary spermatocytes were present, but further differentiated stages of spermatogenesis were absent. Ovaries of female connexin43 knock-in connexin26 mice revealed only few follicles and the maturation of follicles was completely impaired.ConclusionThe impaired gametogenesis of homozygous males and females can explain their infertility.

Autocrine prolactin as a promotor of mammary tumour growth

Prolactin (PRL) plays a key role in normal growth, development and differentiation of the mammary gland. Indeed, strong evidence suggests that the development of alveolar cells requires not only oestradiol and progesterone, but also PRL. In vitro, PRL has mitogenic activity on normal mouse mammary epithelial cells (reviewed in Das & Vonderhaar, 1997). In vivo, PRL also seems to be involved in such proliferative activity, although it is more difficult to distinguish the role of PRL from the influence of the hormonal milieu (Das & Vonderhaar, 1997). This physiological role of PRL in lobular development of the mammary gland is supported by results obtained from mice deficient for PRL (Horseman et al. 1997) or for its receptor (PRLR) (Ormandy et al. 1997). Although the infertility of females homozygous for the deletion of the PRLR gene (PRLR−/−) can be partially reversed by restoring progesterone levels close to normal, their mammary gland fails to differentiate during pregnancy, leading to lactation failure (Binart et al. 2000). In addition, heterozygous mice (PRLR+/−), who have half normal receptor levels, show impaired mammary gland development and fail to lactate following their first pregnancy, clearly indicating that signals mediated by the PRL/PRLR interaction have to achieve a certain level to permit mammary gland differentiation and lactation (Kelly et al. 2002). Since the pioneering work of Topper (Topper, 1970), who observed that PRL was necessary to induce casein synthesis, our understanding of the mechanism of such induction has greatly expanded. PRL appears to be the primary hormone involved in this activity, although other hormones such as insulin and glucocorticoids are also required for lactation.

Development of the mammary gland requires DGAT1 expression in stromal and epithelial tissues.

Mammary gland development is a complex process that is dependent on interactions between the developing mammary epithelium and the surrounding stromal tissues. We show that mice lacking the triglyceride synthesis enzyme acyl CoA:diacylglycerol transferase 1 (DGAT1) have impaired mammary gland development, characterized by decreased epithelial proliferation and alveolar development, and reduced expression of markers of functional differentiation. Transplantation studies demonstrate that the impaired development results from a deficiency of DGAT1 in both the stromal and epithelial tissues. Our findings are the first to link defects in stromal lipid metabolism to impaired mammary gland development.

Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development.

To determine if the production of recombinant human protein C (rHPC) could be increased in milk, we created two lines of mice homozygous for the mouse whey acidic protein (WAP)/human protein C (HPC) transgene. Females of both lines had normal growth, activity and fertility, but failed to lactate normally and were unable to raise litters. Histological analyses of mammary glands from lactating homozygous females showed barely distended alveoli filled with dense-staining milk. Epithelial cells within these alveoli had distinct, centrally located nuclei and contained intracellular lipid droplets. Hemizygous animals derived from these lines were able to lactate and raised normal sized litters. Northern blot analysis showed that the 6.4 homozygous (6.4H) line expressed the transgene at higher levels then corresponding hemizygous (6.4) animals, but the 4.2 homozygous (4.2H) line expressed the transgene at lower levels than the 4.2 hemizygous line. The 6.4H line also had increased rHPC levels in the milk as revealed by western blot analysis. The 4.2H, 6.4, and 6.4H lines showed decreased and/or delayed expression of WAP, beta-casein, and alpha-lactalbumin mRNA's compared to wild type animals during lactogenesis. The 4.2 line showed decreased mRNA expression for beta-casein and alpha-lactalbumin, but normal or higher expression of WAP during lactogenesis. Elevated levels of some proteins were detected in the milk of transgenic mice. From these results, it is concluded that expression of rHPC induced a lactational phenotype that involves abnormal morphological, biochemical, and functional differentiation of mammary epithelial cells. However, the induction of this phenotype does not appear to be directly related to the level of rHPC mRNA expression, thus suggesting that the basis of this phenotype may involve secondary, rather than primary, effects of rHPC on mammary gland development.

Puny Pin1 > –/– Mice

Pin1 is a prolyl isomerase that regulates the activity of specific proteins by promoting the isomerization of the phosphorylated Ser-Pro or phosphorylated Thr-Pro bond. Liou et al . analyzed the phenotypes of the Pin1 –/– mice and found that adult animals exhibited several characteristics of cell proliferative defects: decreased body weight, testicular and retinal atrophy, and impaired mammary gland development during pregnancy. Because these phenotypes are also characteristic of mice deficient in cyclin D1, Liou et al . examined cyclin D1 in the Pin1 –/– mice and found that cyclin D1 protein levels were reduced in several tissues that are normally proliferating, including the Pin1 –/– -affected tissues. Analysis of cyclin D1 in Pin1 –/– mouse embryonic fibroblasts (MEFs) showed that in addition to decreasing cyclin D1 transcription, loss of Pin1 also resulted in decreased stability of cyclin D1 and redistribution of cyclin D1 to the cytoplasm compared with the nuclear localization typical of wild-type MEFs. The effects of Pin1 and binding of Pin1 to cyclin D in glutathione S -transferase pull-down experiments were dependent on the presence of a Thr that can be phosphorylated and is located in a Pin1 consensus motif. Thus, Pin1 appears to regulate cyclin D1 at both the transcriptional and posttranscriptional level to control cell proliferation. Y.-C. Liou, A. Ryo, H.-K. Huang, P.-J. Lu, R. Bronson, F. Fujimore, T. Uchida, T. Hunter, K. P. Lu, Loss of Pin1 function in the mouse causes phenotypes resembling cyclin D1-null phenotypes. Proc. Natl. Acad. Sci. U.S.A. 99 , 1335-1340 (2002). [Abstract] [Full Text]

Targeted expression of the only zinc finger gene in transgenic mice is associated with impaired mammary development.

The only zinc finger (OZF) gene encodes a protein consisting mainly of 10 zinc finger motifs of the Krüppel type of yet unknown function. To potentially assess its in vivo role, mammary targeted deregulation of the expression of the murine gene was performed in transgenic mice using a goat beta-casein-based transgene. Mammary expression of the transgene was observed in the 11 lines obtained. In three expressing lines, this expression was tissue-specific and developmentally regulated. Further analysis of mice from two expressing lines revealed that transgene-homozygous females could not sustain full growth of their pups. This phenotype was associated with an impaired mammary gland development noticeable only after mid-gestation. It was characterised by an increase of the adipocyte to acini ratio and low or absence of fat globules within these acini compared to non-transgenic control animals. These transgenic observations strongly suggest that OZF is active in the mammary gland, interfering with the lactation process and thus that the described transgenic mice could be useful models to search for the cellular partner(s) of this protein.

Impaired mammary gland and lymphoid development caused by inducible expression of Axin in transgenic mice.

Axin is a component of the canonical Wnt pathway that negatively regulates signal transduction by promoting degradation of β-catenin. To study the role of Axin in development, we developed strains of transgenic mice in which its expression can be manipulated by the administration of doxycycline (Dox). Animals carrying both mouse mammary tumor virus (MMTV)–reverse tetracycline transactivator and tetracycline response element (TRE)2–Axin–green fluorescent protein (GFP) transgenes exhibited Dox-dependent Axin expression and, when induced from birth, displayed abnormalities in the development of mammary glands and lymphoid tissues, both sites in which the MMTV promoter is active. The transgenic mammary glands underwent normal ductal elongation and side branching during sexual maturation and early pregnancy, but failed to develop lobulo-alveoli, resulting in a defect in lactation. Axin attenuated the expression of cyclin D1, a Wnt target that promotes the growth and differentiation of mammary lobulo-alveoli. Increased apoptosis occurred in the mammary epithelia, consistent with the inhibition of a Wnt/cyclin D1 survival signal by Axin. High levels of programmed cell death also occurred in the thymus and spleen. Immature thymocytes underwent massive apoptosis, indicating that the overexpression of Axin blocks the normal development of T lymphocytes. Our data imply that the Axin tumor suppressor controls cell survival, growth, and differentiation through the regulation of an apoptotic signaling pathway.

Mammary gland specific hEGF receptor transgene expression induces neoplasia and inhibits differentiation.

The epidermal growth factor receptor (EGFR) is overexpressed in about 48% of human breast cancer tissues. To analyse the role of the EGFR in mammary tumor development we generated transgenic mice expressing the human EGFR under the control of either the MMTV-LTR (MHERc) or the beta-lactoglobulin promoter (BLGHERc). The BLGHERc-transgene was expressed exclusively in the female mammary gland, whereas the MHERc transgene was expressed more promiscuously in other organs, such as ovary, salivary gland and testis. Female virgin and lactating transgenic mice of both strains have impaired mammary gland development. Virgin EGFR transgenic mice developed mammary epithelial hyperplasias, whereas in lactating animals progression to dysplasias and tubular adenocarcinomas was observed. In both strains the number of dysplasias increased after multiple pregnancies. The transgene expression pattern was heterogeneous, but generally restricted to regions of impaired mammary gland development. Highest EGFR transgene expression was observed in adenocarcinomas. By using a whole mount organ culture system to study the differentiation potential of the mammary epithelium, we observed a reduced number of fully developed alveoli and a decrease in whey acidic protein expression. Taken together, EGFR overexpression results in a dramatic effect of impaired mammary gland development in vitro as well as in vivo, reducing the differentiation potential of the mammary epithelium and inducing epithelial cell transformation.

Impaired Mammary Gland Development in Cyl-1−/− Mice during Pregnancy and Lactation Is Epithelial Cell Autonomous

A specific defect of mice lacking cyclin D1 (Cyl-1−/−) is impaired development of the mammary gland during pregnancy. Here we show that when tissue from Cyl-1−/− mammary gland was transplanted into empty mammary fat pad of wild-type mice, the abnormal phenotype was maintained, indicating that it is epithelial cell autonomous. Nevertheless, in pregnancy the early proliferative response, which is characterized by extensive side branching, still occurs in the absence of cyclin D1. However, the response is atypical due to a marked reduction in the formation of accompanying alveoli. This reduction and delay in alveolar development persists throughout pregnancy. Moreover, although prolactin synthesis and release appear to be normal, lactogenesis is severely compromised. Consistent with the appearance of numerous side branches, progesterone receptor expression was readily detected in the mammary tissue of pregnant Cyl-1−/− mice, although there was a significant change in the ratio of the two (A and B) receptor isoforms. In Cyl-1−/− mammary glands during late pregnancy there was a decrease in the abundance of total and phosphorylated Stat5a, as well as delayed onset and substantial diminution of milk protein expression. The biochemical analysis suggests that there is a cumulative delay in growth and differentiation of the mammary gland during pregnancy that results in a severely compromised gland when, at parturition, further development is curtailed by the abrupt change in hormonal milieu.

Think globally, act locally: the making of a mouse mammary gland.

On a phylogenetic scale of organ development the mammary gland is a recent acquisition. It was introduced 200 million years ago with the appearance of mammals to provide nourishment to the newborn in the form of milk. The mammary gland is characterized by a unique dependence on hormonal signals for terminal differentiation, which is attained only after pregnancy. At the time of birth, the anlage consists of a few rudimentary ducts in the vicinity of the nipple. Pronounced ductal outgrowth and branching commences at puberty, and in pregnancy an expanded lobulo-alveolar compartment develops. Functional differentiation of the secretory epithelium coincides with parturition and large amounts of milk are produced and secreted during lactation. After weaning of the young, the entire alveolar epithelial compartment is remodeled to resemble a virgin-like state. With each pregnancy, a new round of lobulo-alveolar development occurs. During the past 100 years, intensive efforts have been made to understand the endocrine control of mammopoiesis and lactogenesis. Classical research on endocrine ablated animals firmly established that ovarian steroids and pituitary peptide hormones are mandatory and sufficient for breast development and lactation. In 1900, Halban first established that mammary growth is controlled by the ovary (Halban 1900). He demonstrated that ovariectomy caused mammary regression, and that transplanted ovaries prevented the castration atrophy of mammary glands. Twenty-eight years later, Stricker and Grueter induced mammary development and milk secretion artificially in castrated virgin rabbits by injection of pituitary extract (Stricker and Grueter 1928). In 1933, Riddle, Bates, and Dykshorn purified the respective pituitary hormone (Riddle et al. 1933) and named it prolactin(PRL). In the last several years, the ability to delete genes from the mouse genome has allowed us to identify genetic components of mammary gland development. Molecular insight into the underlying genetic framework and signaling networks of the developing tissue has been gained through experimental manipulations of tissues from wild-type and knockout mice. Two distinct, yet braided, developmental concepts have unfolded. First, discrete signaling networks activated by systemic endocrine hormones induce mammopoiesis. Secondly, some of these signals are relayed through reciprocal interactions between the epithelium and the stroma. Table 1 contains those genes whose elimination from the mouse genome results in impaired mammary gland development. Among these genes are some of the ‘‘usual suspects’’ but also some previously unidentified players. Each mutation affects specific and distinct aspects of mammary development. These knockout mice not only confirmed the involvement of hormonal signaling but also provided tools to identify the tissue compartment that receives and executes these signals.