Abstract
Exposure to low doses of environmental estrogens such as bisphenol A and genistein (G) alters mammary gland development. The effects of environmental anti-androgens, such as the fungicide vinclozolin (V), on mammary gland morphogenesis are unknown. We previously reported that perinatal exposure to G, V, and the GV combination causes histological changes in the mammary gland during the peripubertal period, suggesting alterations to the peripubertal hormone response. We now investigate whether perinatal exposure to these compounds alters the gene expression profiles of the developing glands to identify the dysregulated signaling pathways and the underlying mechanisms. G, V, or GV (1 mg/kg body weight per day) was added to diet of Wistar rats, from conception to weaning; female offspring mammary glands were collected at postnatal days (PNDs) 35 and 50. Genes displaying differential expression and belonging to different functional categories were validated by quantitative PCR and immunocytochemistry. At PND35, G had little effect; the slight changes noted were in genes related to morphogenesis. The changes following exposure to V concerned the functional categories associated with development (Cldn1, Krt17, and Sprr1a), carbohydrate metabolism, and steroidogenesis. The GV mixture upregulated genes (Krt17, Pvalb, and Tnni2) involved in muscle development, indicating effects on myoepithelial cells during mammary gland morphogenesis. Importantly, at PND50, cycling females exposed to GV showed an increase in the expression of genes (Csn2, Wap, and Elf5) related to differentiation, consistent with the previously reported abnormal lobuloalveolar development previously described. Thus, perinatal exposure to GV alters the mammary gland hormone response differently at PND35 (puberty) and in animals with established cycles.
Introduction
There is a growing body of evidence that endocrine-disrupting chemicals (EDCs) alter normal mammary gland morphogenesis. EDCs include various families of synthetic compounds, such as plastics (bisphenol A, also called BPA), plasticizers (phthalates), polychlorobiphenyls (PCBs), dioxins, and pesticides. Environmental chemicals with estrogenic activity (xenoestrogens, such as BPA, and the pesticide, dieldrin) and other EDCs not exhibiting estrogenic behavior (such as dioxin and atrazine) may cause abnormal mammary gland development (Markey et al. 2001, Munoz-de-Toro et al. 2005, Fenton 2006, Enoch et al. 2007, Vandenberg et al. 2007) or may increase the risk of breast cancer (Durando et al. 2007, Cameron & Foster 2009). A number of epidemiological studies have suggested that the dietary intake of phytoestrogens decreases the risk of breast cancer in humans (Badger et al. 2005, Warri et al. 2008, Wu et al. 2008). Soy consumption during childhood and adolescence has been consistently linked to a marked reduction in breast cancer risk in Asian and American-Asians (Korde et al. 2009), in agreement with some experimental studies showing that pubertal exposure to genistein (G), a natural phytoestrogen that is prevalent in plants (Wang et al. 2006), reduces mammary cancer risk in rodent models (Warri et al. 2008). Many experimental studies indicate that G which interacts with estrogen receptors (ERs), may affect mammary gland development and the incidence or multiplicity of carcinogen-induced mammary tumors. The precise effects of G mainly depend on the window of exposure (in utero alone, during postnatal days (PNDs) 2–8, or in utero until adult life) (Hilakivi-Clarke et al. 1999a,b, Lamartiniere et al. 2002, Pei et al. 2003, Foster et al. 2004, Warri et al. 2008). The effects of environmental chemicals with anti-androgenic activity (such as fungicides) on the female mammary gland development remain unknown. Also, the implication of an association of G and an anti-androgen EDC in female mammary gland development and mammary tumorigenesis has not been explored.
The window of exposure appears to be a critical factor in the modulation of mammary gland development, as is also reported in epidemiological studies of women born to mothers treated with the synthetic estrogen diethylstilbestrol during pregnancy (Hoover et al. 2011). Adverse health outcomes including an increase in breast cancer risk were recently reported in these women (Hoover et al. 2011). In recent years, the impact of early exposure to EDCs has been increasingly analyzed in animal models (Markey et al. 2001, Munoz-de-Toro et al. 2005, Fenton 2006, Durando et al. 2007, Vandenberg et al. 2007). It was also recently shown that the biological actions of one chemical may be influenced by the presence of another. However, few studies have considered the effects of mixtures of EDCs on mammary glands (Foster et al. 2004, Wang et al. 2006). One way of studying such mixtures involves using combinations of EDCs from the same or different families (Wang et al. 2006, Christiansen et al. 2009, Eustache et al. 2009, Lehraiki et al. 2011), taking into account the hormonal environment of normal development.
Mammary gland development in rats begins 6–7 days before birth, about days 12–14 into embryonic development (Russo & Russo 1978). The primary ducts develop from the epithelial bud and extend into the fat pad in response to signals from the surrounding mesenchyme. Any interference with this process is likely to alter the formation of the mammary gland. Extensive development of the mammary gland then occurs during puberty, when rising levels of ovarian hormones induce the formation of highly proliferative terminal end buds (TEBs) at the tip of the developing ducts (Sternlicht et al. 2006). Estradiol (E2) and GH, and their interaction with the corresponding receptors (ERα and GHR), are required for normal duct growth and morphogenesis (Daniel et al. 1987, Korach et al. 1996). Progesterone induces side branching of the ductal epithelium (Lydon et al. 1995, Atwood et al. 2000). E2 and progesterone are required for the development of alveolar structures at each estrous cycle. However, much less is known about the role of androgens in mammary gland development. Abnormal mammary gland development and growth retardation have been observed in female mice lacking androgen receptor (AR) (reduced duct branching at prepubertal stages and lower levels of lobuloalveolar development in adults) (Yeh et al. 2003). The effects of exposure to anti-androgenic EDCs on the development of the female mammary gland are unknown.
One of the first chemicals to be identified as an anti-androgen was the dicarboximide fungicide vinclozolin (V) (Gray et al. 1994, Kelce et al. 1997). This chemical has been extensively used to treat fruit and vegetables crop and has been recognized as a contaminant of human foods. V metabolism generates two major anti-androgenic compounds, M1 and M2, which may also have non-androgenic functions (Molina-Molina et al. 2006). When administered in utero or during the early postnatal period, V induces malformations, alterations in reproductive function, and sexual differentiation of male rats (Gray et al. 1999, Monosson et al. 1999). Anti-androgens including V have been shown to alter the development of male rat mammary gland. Nipple retention has been identified as a sensitive endpoint for mammary gland development in male rats treated with anti-androgen compounds (Christiansen et al. 2009), suggesting that androgens may play an important role in very early sex differentiation of the mammary gland. Gestational dietary exposure to low-dose V and G has also been shown to have antagonistic effects on rat fertility and on fetal germ cell development (Eustache et al. 2009, Lehraiki et al. 2011). We recently reported that exposure to V alters the peripubertal development of the female rat mammary gland (El Sheikh Saad et al. 2011). Increases in epithelial branching and focal branching defects were observed at puberty (PND35) in the V-exposed group. We also reported that mammary glands from rats exposed to GV or G displayed higher levels of epithelial branching and proliferation, larger TEBs, and ductal hyperplasia than untreated rats (El Sheikh Saad et al. 2011). In animals with an established estrus cycle (PND50), exposure to GV and to a lesser extent V induces the formation of abnormal hyperplasic alveolar structures. We therefore hypothesized that V interferes with endocrine signaling during mammary gland development (El Sheikh Saad et al. 2011). If this proves to be the case, it will be important to identify the role of this compound in hormonal signaling pathways during development. In this study, we aimed to identify the signaling pathways modified by exposure in utero and during lactation to G, V, or GV, at various stages of development. We found that low doses of V and GV induced changes in genes related to development, morphogenesis, differentiation, and metabolism indicating alterations in the development of mammary glands, which depend on the compound or mixture used and the period of development considered. The implications of our findings toward mammary gland tumorigenesis and relevance to breast cancer risk are discussed and require further studies.
Materials and methods
Chemicals
G with a purity of 99% was synthesized (LCOO, Université Bordeaux 1, Talence, France), as previously reported (El Sheikh Saad et al. 2011). V was extracted from the commercial Ronilan (BASF, Levallois-Perret, France) by JP Cravedi's Laboratory (INRA, Toulouse, France) and the final preparation was >95% pure, as described previously (Bursztyka et al. 2008). In addition, the absence of degradation products M1 and M2 was tested by LC–MS as described previously (Bursztyka et al. 2008). Molecules were dissolved in corn oil.
Animals and treatment
Female and male Wistar Han rats (60 females and 60 males) at 8 weeks of age (Harlan France Sarl, Gannat, France) were maintained in an animal facility. Procedures for handling and experimentation followed by ethical guidelines have been described elsewhere (El Sheikh Saad et al. 2011). At arrival, the rats were allowed to acclimatize to the animal facility for 4 weeks before mating under conditions of 22 °C, 55% humidity, and a 12 h light:12 h darkness cycle. Cages and bottles were made of polypropylene to avoid any contamination with BPA or phthalates, and the water was filtered through charcoal to eliminate any pesticide or active endocrine contaminant. The rats were fed a purified diet (phytoestrogen-free, INRA) and water ad libitum, as described previously (Eustache et al. 2009). At gestational day (GD) 1, determined by the presence of intravaginal sperm plug, the dams were randomly divided into groups of 15. From GD1 until weaning (PND21), each pregnant female underwent oral gavage once daily with the appropriate preparations. This study refers to the same pools of animals and to the same chemicals as described in El Sheikh Saad et al. (2011), as they came from the same experiments. Molecules (G or V) were administered at 1 mg/kg body weight per day alone or in combination (GV). They were dissolved in corn oil in order to administrate the one 2 ml/kg-day dose. Control animals received the corn oil vehicle (2 ml/kg body weight per day). Purity was controlled weekly as described previously (El Sheikh Saad et al. 2011). The G dose resembled a soy-based diet intake (El Sheikh Saad et al. 2011). The V dose was higher than human food contamination levels but it was lower than the no observable adverse effect level (NOAEL) combining chronic toxicity, carcinogenicity, and reproductive toxicity in rats (1.2 mg/kg body weight/day; U.S., Environmental Protection Agency (EPA 2003)). The acceptable daily intake (ADI) of V is 600 μg/day per person, corresponding to an exposure of 0.01 mg/kg body weight per day (Eustache et al. 2009). On the day of parturition, pups were sexed and weighed; all litters were standardized in order to contain ten offspring (five males and five females). At weaning, rats were dispatched. Female offspring were randomized (one female per litter), identified by implanted chips, and grouped together in polypropylene cages (five females per cage). Offspring (ten pubertal or ten cycling females per treatment group, or untreated rats) fed the purified diet (phytoestrogen-free) until they were killed (PND35 and PND50) (Fig. 1). Food consummation and body weight were measured twice weekly.
Protocol for in utero and lactational exposure to low doses (1 mg/kg per day) of G, V and a mixture of these compounds. Animals were force-fed once daily with the appropriate preparation in a phytoestrogen-free diet (INRA). Details of the diet have been reported elsewhere (El Sheikh Saad et al. 2011) and are detailed in Materials and methods section.
Citation: Journal of Endocrinology 216, 2; 10.1530/JOE-12-0395
All female offspring in these studies were examined daily for vaginal opening to determine pubertal stage. Vaginal smears were collected on the morning on which the animals were killed (PND35 or PND50). The collected vaginal smears were placed on glass slides, air dried, and stained with Hemacolor. At PND35, all animals but one had a vaginal opening. Rats were determined to be in proestrus, estrus, metestrus, or diestrus. We checked whether treated animals were evenly distributed between the various phases of the estrous cycle (not shown).
Mammary gland samples
Female offspring were anaesthetized under isoflurane (2.5% in air). The right fourth mammary glands were harvested to perform whole mount analysis (El Sheikh Saad et al. 2011). The left fourth mammary glands were harvested for mRNA studies (ten animals/group). The lymph nodes were not removed from the mammary gland before RNA isolation. An aliquot of the left fourth mammary gland was also fixed in buffered 10% formalin, dehydrated through graded alcohols, and paraffin embedded. Animals were killed by opening the thorax.
RNA extraction
Total RNA was extracted from the same individual mammary glands obtained from PND35 and PND50 rats as were used for histological studies (El Sheikh Saad et al. 2011). Tissues were homogenized in 1 ml TRIzol Reagent (Invitrogen) per 50 mg tissue, as described previously (El Sheikh Saad et al. 2011). The quantity and purity of each RNA sample were determined from absorbance values at 260 and 280 nm absorbance, and RNA integrity was determined (Bioanalyzer 2100, using Agilent RNA6000 nano chip kit, Agilent Technologies, Massy, France). Only high-quality total RNA samples, with an RNA integrity number (RIN) >8, were considered for further analysis.
Characterization of the genomic profile
RNA samples were used for both genomic analysis and validation experiments. A pooling strategy was initially used for genomic analysis to limit the number of microarray experiments required. Two pools (five rat tissues each) for each treatment (G and V) and untreated animals (control (C)) were analyzed at each period of observation (PND35 and PND50). We thus analyzed 300 ng pooled RNA (each set to 400 ng/μl) coming from five different mammary glands from the same group. Two pools for GV-treated samples were also prepared at PND50, but a specific pool was prepared for GV-treated rats presenting abnormal hyperplastic alveolar structures (GV2, as described in our previous study; El Sheikh Saad et al. 2011). Sixteen RNA samples were thus analyzed for hybridization.
After validation of the RNA quality, 300 ng total RNA was reverse transcribed following the Genechip Whole transcript Sense Target labelling assay kit (Affymetrix, Santa Clara, CA, USA). Briefly, the resulting double-stranded cDNA was used for in vitro transcription with T7 RNA polymerase. After purification, 10 μg cRNA was used for RT with random primers. The cDNA obtained was then purified and fragmented. After control of fragmentation using Bioanalyzer 2100, cDNA was end labeled with biotin using Terminal Transferase (using the whole transcript terminal labelling kit of Affymetrix). cDNA was then hybridized to GeneChip rat gene (Affymetrix) at 45 °C for 17 h. These microarrays have 27342 probe sets representing known rat genes. After overnight hybridization, chips were washed on the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The image was then analyzed with Expression Console software (Affymetrix) to obtain raw data (cell files) and metrics for quality controls. The observations of some of these metrics and the study of the distribution of raw data showed no outlier experiment.
Bioinformatics
Quality assessments, normalization, and statistics
All quality controls, normalization, and statistics were performed using Partek Genomic Suite. Raw data were preprocessing using the Robust Multichip Algorithm (RMA; Irizarry et al. 2003, Nikolsky & Bryant 2009, Mi et al. 2010). We first made a hierarchical clustering for unsupervised analysis (Pearson's dissimilarity and average linkage) with all pooled samples. To find differentially expressed genes, we applied a classical unpaired Student's t-test between compared groups and computed the fold-change for each gene. Then, we used these two statistics to filter and select differentially expressed genes. We selected genes with P value <0.05 and 1.5-fold-changes. All data obtained by microarray analysis have been submitted on GEO Omnibus site with the accession number GSE32432 (private link for reviewers: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=xdafzascymwswrq&acc=GSE32432).
Functional analysis
Function and pathway enrichments of differentially expressed genes lists were carried out through the use of Ingenuity Pathway Analysis (Ingenuity Systems, USA, www.ingenuity.com).
Gene expression analysis by real-time RT-PCR
RT of the RNA (1 μg) from each mammary sample (ten animals/group), prepared as described earlier, was performed with 100 units of Superscript II RNase H-reverse transcriptase (Invitrogen) and 3 μM random hexamers (Pharmacia), as described previously (El Sheikh Saad et al. 2011). The fold-change in gene expression was quantified by real-time quantitative RT-PCR as described previously (Bieche et al. 2004), using the P0 ribosomal protein as an endogenous RNA control. Results are expressed as N-fold differences in target gene expression relative to the expression of the housekeeping gene Po and to untreated samples (set to 1). We selected Po (Rplp0 also known as 36B4) because this gene is widely used as an endogenous control for northern blot analysis. In some experiments, gene expression in mammary gland samples was also normalized with respect to the housekeeping gene Tbp. Tbp was selected as an endogenous control because there are no known Tbp retropseudogenes that might lead to the co-amplification of contaminating genomic DNA, thereby interfering with RT-PCR. We also selected Tbp and Po as endogenous controls because the prevalence of their transcripts is moderate (Ct values between 24 and 26 in the samples) and high (Ct values between 18 and 20) respectively. Finally, Tbp was identified as stably expressed across the estrous cycle (Hvid et al. 2011). The nucleotide sequences of the oligonucleotide primers of the tested genes are shown in Supplementary Table S1, see section on supplementary data given at the end of this article. To avoid amplification of contaminating genomic DNA, one of the two primers bound to the junction between two exons. The specificity of PCR amplicons was checked by agarose gel electrophoresis. PCR was performed with the SYBR Green PCR Core Reagents kit (Perkin–Elmer Applied Biosystems, Foster City, CA, USA). The experiments were performed in duplicate for each data point.
Validation was carried out for each individual RNA (n=10) for each treatment group at PND35 and PND50 and for each selected gene. When change in the expression level for a specific gene in the transcriptomic analysis was correlated with a significant difference in the Q-PCR, the gene was considered as validated.
Immunocytochemistry
The samples were cut into 4 μm sections and processed for hematoxylin and eosin staining. TEBs, terminal ducts, ABs, and lobular epithelial structures were histologically classified, as previously reported (El Sheikh Saad et al. 2011) according to Russo & Russo (1978). Highlights of the previous work are presented in Table 1. After paraffin removal, the sections were subjected to 15-min microwave antigen retrieval in pH 6 citrate buffer and pre-incubated with a blocking solution (5% BSA and 0.5% casein in PBS buffer) to minimize nonspecific binding. Sections were then incubated overnight at 4 °C in a humid chamber with the following antibodies, as described previously (El Sheikh Saad et al. 2011): rabbit polyclonal antibody against β-casein (Csn2) (1:2000 dilution; courtesy of M Glukhova and D Medina, Baylor College of Medicine, Houston, TX, USA) (Durban et al. 1985), against Stat5A (Clone L-20, 1:2000 dilution, sc-1081, Santa Cruz Biotechnology), cytokeratin 17 (Krt17) (Clone V-17; 1:25 dilution, sc-101931, Santa Cruz Biotechnology), claudin 1 (1:50 dilution, Sigma–Aldrich), and monoclonal mouse antibody against smooth muscle actin (Clone 1A4, 1:400, Dako, Carp Carpenteria, CA, USA). Control slides were incubated with normal rabbit or mouse IgG. After endogenous peroxidase quenching with 3% H2O2 in PBS for 10 min, the bound antibodies were revealed with an Impress REAGENT anti-Mouse and anti-Rabbit Ig (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. Aminoethylcarbazole was used as a chromogen. Some slides were counterstained with hematoxylin. The distribution and the intensity of staining were evaluated by conventional light microscopy, as described previously (El Sheikh Saad et al. 2011).
Quantitative modifications and other observations on the mammary gland structures at PND35 and PND50 after gestational/lactational exposure to G, V, and GV at 1 mg/kg body weight per day. This table summarizes the findings of our previous work (El Sheikh Saad et al. 2011)
| Branching (mm) | Proliferation (Ki67 mRNA) | Glandular area (mm2) | Periductal stroma | Other observations | |
|---|---|---|---|---|---|
| PND35 | |||||
| C | 3.8±0.4 | 1 | 183.2±21.8 | 1 | |
| G | 5.6±0.3* | 2.1* | 191.3±10.2 | 1.5* | Large sized TEB; hyperplastic ducts |
| V | 6.0±0.4* | 0.5 | 215.2±30.3 | 0.8 | Focal branching defects; hyperplastic ducts |
| Occasional loss of epithelial cell polarization | |||||
| GV | 5.7±0.4* | 2.4* | 223.0±14.5 | 2.0* | Large sized TEB; hyperplastic ducts |
| PND50 | |||||
| C | 4.5±0.2 | 1 | 447.5±17.8 | 1 | |
| G | 5.7±0.3 | 1.8 | 433.7±15.2 | 1.3* | |
| V | 5.1±0.2 | 2.8* | 430.0±25.5 | 1.3* | Hyperplasic lobuloalveolar structures (20% of animals) |
| GV | 5.7±0.6 | 1.5 | 510.7±13.7* | 1.1 | Hyperplasic lobuloalveolar structures (40% of animals) |
*P<0.05, compared with control.
Statistical analysis
All data were analyzed using SigmaStat software. Data generated for analyzing vaginal opening or by Q-PCR were expressed as mean±s.e.m. and analyzed using one-way ANOVA. Where appropriate, intergroup comparisons were performed using Tukey's multiple comparisons test. Differences were considered significant if the P value was <0.05.
Results
Rats were exposed in utero and during the neonatal period (from conception to weaning) to the phytoestrogen G, the anti-androgen V, or a GV mixture at a dose of 1 mg/kg per day as described in Fig. 1. As described previously (El Sheikh Saad et al. (2011); see Supplementary Figure S1A, see section on supplementary data given at the end of this article), female offspring exposed to the mixture of G and V (GV) exhibited significant early vaginal opening compared with G and V exposed animals (P<0.05; Supplementary Figure S1). However, early vaginal opening was not associated with modifications in body weight (see Supplementary Figure S1B in El Sheikh Saad et al. (2011)) or changes in E2 and progesterone levels (N Lalou, unpublished data). Mammary glands from the different groups were analyzed at PND35 (puberty) and PND50 (virgin cycling females). A previous study by our group showed histological changes in mammary gland development in these conditions (El Sheikh Saad et al. 2011). Highlights of the previous work are presented in Table 1. Briefly, the morphological changes observed at PND35 included increases in TEB size, epithelial branching, and duct proliferation in animals exposed to G- and GV-exposed animals. Focal branching defects, with an occasional loss of epithelial cell polarization, were observed in animals exposed to V. At PND50, animals exposed to G alone displayed no major histological abnormalities. Surprisingly, exposure in utero and during lactation to the binary mixture of G and V compounds resulted in postpubertal hyperplasic lobuloalveolar structures, associated with numerous secretion vacuoles (Table 1).
Mammary gland gene expression profiles at puberty and in adult cycling rats previously exposed to G, V or a mixture of G and V
We compared the gene expression profiles of the mammary glands of pubescent (PND35) and cycling (PND50) female rats exposed to G, V, and GV with those of controls. Two pools (five rat tissues each) for each treatment (G and V) and untreated animals (C) (see Materials and methods section) were analyzed at each period of observation on Affymetrix rat microarray. Nonsupervised hierarchical clustering of the 27342 probe sets (Fig. 2) highlighted cluster of genes modified by each treatment, through comparisons with control rats. The transcriptional effects of the various treatments were clearly different at PND35 and PND50. The criteria for the genes to be analyzed were as follows: up- or downregulation by 1.5-fold (P<0.05). We then removed features that did not correspond to known genes and duplicate probe sets. This process resulted in the identification of genes displaying significant differential expression between the controls and animals treated with the two compounds alone or together at PND35 or PND50 (Supplementary Figure S2, see section on supplementary data given at the end of this article).
Transcriptome alterations in rat mammary gland as a function of exposure to G, V, and GV exposure. Nonsupervised clustering analysis of genes displaying differential expression after exposure to G, V, and the GV mixture relative to C. Two individual chip arrays (n=5 animals/pool) for each treatment were analyzed on the basis of log2 ratios, with the scale shown on the bottom right. The heat map of the expression values for each gene differentially expressed relative to control is shown. Transcriptomic alterations in mammary gland were different between PND35 (puberty) and PND50 (animals with established cycles). At PND50, the samples are GV2 (*) and GV1 (°), as described in Materials and methods section and in El Sheikh Saad et al. (2011). Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-12-0395.
Citation: Journal of Endocrinology 216, 2; 10.1530/JOE-12-0395
At PND35, exposure to G, V, or the GV mixture resulted in changes to the expression of 213 genes: 65 (G), 56 (V), and 113 (GV) genes (Supplementary Figure S2A). Only one gene, Krt17 (encoding the cytokeratin 17), displayed changes in expression in response to all three treatments, G, V, and GV. Most of the genes were upregulated (Supplementary Figure S2A). Twenty genes were regulated by both G and GV (15 up- and five downregulated) (Supplementary Figure S2A and see GEO in Materials and methods section). However, no gene displayed changes in expression in response to both G and V or V and GV (Supplementary Figure S2A).
At PND50, exposure to G, V, or the GV mixture resulted in the overall deregulation of 213 genes: 25 (G), 112 (V), and 119 (GV) genes (Supplementary Figure S2B). Two genes, Pfkb3 and Nr1d1, displayed changes in expression in response to all three treatments, G, V, and GV. Thirty-three genes were common to both V and GV, ten genes displayed changes in expression in response to G and GV, and two genes displayed changes in expression in response to both G and V (Supplementary Figure S2B and see GEO).
Validation of the microarrays by real-time RT-PCR
ERα, progesterone receptor (PR), and AR are present in the developing mammary gland at puberty and in cycling animals. Genes encoding these hormone receptors were analyzed as controls by real-time RT-PCR. Data concerning ERα and AR have been already reported (see Table 2 in El Sheikh Saad et al. (2011)). A significantly higher level of PR expression was observed in the mammary glands of rats exposed to GV and G at PND35, a time point at which estrogens are driving development. No significant change in ERα was detected compared with control (Table 2). For validation of the microarray results, we selected the 16 genes (13 at PND35 and 12 at PND50, nine genes being in common) most strongly overexpressed in the mammary glands of GV- and V-treated rats with respect to untreated rats at PND35 or PND50 (see GEO). These genes were involved in various cellular and molecular functions including muscle development and morphogenesis (Actn2, Casq1, Ckm, Fhl1, Mylk2, Pvalb, Pygm, and Tnni2), development (Cldn1, Elf5, Krt17, Sprr1a, and Tfap2c), and differentiation (Csn2, Fabp3, and Wap). Validation was carried out for each individual RNA (n=10) for each treatment group, according to the criteria described in Materials and methods section. RT-PCR confirmed the results obtained in the microarray experiments for 97% at PND35 and 83% at PND50 using P0 as endogenous control (Tables 2 and 3). The normalization of gene expression with respect to Tbp and Po gave similar results, by real-time RT-PCR, in terms of the effects of the treatments (data not shown). At PND35, the upregulation of Cldn1, Krt17, and Sprr1a expression by exposure to V was validated by RT-PCR (Table 2); the upregulation of Casq1, Krt17, Mylk2, Pygm, Pvalb, and Tnni2 with GV was also validated, as was the induction of Tfap2c and Elf5, which encode transcription factors, by exposure to G (Table 2). The results at PND50 confirmed the upregulation of all genes shown to be upregulated by V and GV in the transcriptomic analysis (Table 3). We also identified five additional genes displaying significant upregulation in the GV-exposed group (Table 3) that were not identified in the transcriptomic assay (Actn2, Csn2, Fabp3, Tnni2, and Wap). The detection of these additional upregulated genes may reflect the higher sensitivity of RT-PCR than of transcriptomic analysis, and/or the heterogeneous values obtained for the animals of the GV group. As we previously observed abnormal development in four GV-treated animals (called GV2) (see El Sheikh Saad et al. (2011)), we compared the expression of these genes in mammary glands from animals of the GV2 and GV1 subgroups. As shown in Table 3, Csn2, Fabp3, and Wap were most strongly upregulated in GV2 than in GV1.
Validation of microarray results by real-time RT-PCR on the mammary glands of rats exposed to G,V or a mixture of the two compounds at PND35. Genes were selected as described in Results section. Data are represented as mean fold changes in expression for each treated group (ten animals/group), as a treated/control group ratio, as detailed in Materials and methods section. Relative expression levels for each gene in the control was set to 1, and the values shown are mean±s.e.m.
| Microarray | RT-PCR | |||||
|---|---|---|---|---|---|---|
| Genes | G | V | GV | G | V | GV |
| Casq1 | NS | NS | 3.3 | 1.8±0.8 | 0.3±0.2 | 4.1±2.5* |
| Ckm | 1.7 | NS | 2.8 | 1.8±0.5 | 0.4±0.1 | 3.5±1.2 |
| Cldn1 | NS | 5.7 | NS | 1.7±0.3 | 6.2±3.6* | 1.6±0.3 |
| Csn2 | NS | NS | −2.0 | 2.0±0.9 | 1.5±0.7 | 0.9±0.4 |
| Elf5 | 1.6 | NS | NS | 4.0±0.5* | 1.4±0.3 | 1.6±0.2 |
| Krt17 | 2.1 | 6.5 | 1.8 | 1.5±0.3 | 8.3±3.6* | 2.0±0.2* |
| Mylk2 | 1.5 | NS | 2.8 | 2.2±0.9* | 0.5±0.2 | 3.2±1.5* |
| Pvalb | NS | NS | 4.0 | 1.8±0.8 | 0.4±0.1 | 5.2±3.0* |
| Pygm | 1.6 | NS | 2.8 | 2.3±1.1* | 0.4±0.2 | 3.6±2.1* |
| Sprr1a | NS | 38.8 | NS | 2.4±0.6 | 55.7±37.1* | 1.0±0.3 |
| Tfap2c | 1.6 | NS | NS | 2.1±0.3* | 1.4±0.2 | 1.3±0.1 |
| Tnni2 | 1.5 | NS | 3.6 | 2.3±0.9 | 0.4±0.2 | 3.6±1.8* |
| Wap | NS | NS | −2.4 | 0.8±0.2 | 1.0±0.3 | 0.4±0.1* |
| Ar | NS | NS | NS | 1.5±0.3 | −1.5±0.1* | 1.3±0.2 |
| Er | NS | NS | NS | 1.3±0.1 | −1.4±0.1 | 1.2±0.1 |
| Pgr | NS | NS | NS | 2.1±0.8 | −1.2±0.4 | 2.0±0.6* |
*Statistically significant difference (P<0.05). Comparisons with results from microarrays are also shown.
Validation of microarray results by real-time RT-PCR on mammary gland from rats exposed to G, V, and GV at PND50. Data are represented as mean fold-changes in expression for each treated group vs untreated, as described in Table 2
| Microarray | RT-PCR | |||||||
|---|---|---|---|---|---|---|---|---|
| Genes | G | V | GV | G | V | GV | GV1 | GV2 |
| Actn2 | NS | 4.3 | NS | 2.3±1.2 | 11.9±8.0* | 5.9±1.4* | 3.9±3.3 | 7.2±0.6 |
| Casq1 | NS | 4.6 | NS | 0.8±0.4 | 7.2±3.8* | 2.6±0.8 | 1.7±1.4 | 3.5±1.1 |
| Ckm | NS | 4.1 | 2.1 | 1.0±0.5 | 7.2±3.0* | 5.3±2.8* | 1.6±1.4 | 9.4±5.1 |
| Csn2 | NS | 3.5 | NS | 1.3±0.8 | 7.3±3.7* | 31.4±20.1* | 0.4±0.2 | 62.4±35.3 |
| Elf5 | NS | NS | 1.7 | 2.3±0.6 | 2.9±0.4* | 2.4±0.4* | 1.7±0.3 | 3.1±0.4 |
| Fabp3 | NS | 2.2 | NS | 1.0±0.4 | 3.3±1.3* | 5.8±3.2* | 0.6±0.2 | 10.9±5.4 |
| Fhl1 | NS | 1.6 | NS | 1.5±0.2* | 2.1±0.6* | 1.3±0.1 | 1.3±0.3 | 1.3±0.1 |
| Mylk2 | NS | 1.8 | NS | 1.2±0.5 | 3.9±2.6* | 2.8±0.8 | 0.6±0.3 | 5.1±0.2 |
| Pvalb | NS | 4 | NS | 0.6±0.3 | 4.3±2.4* | 1.7±0.6 | 1.0±0.9 | 2.4±0.7 |
| Pygm | NS | 5 | 2.6 | 1.2±0.5 | 6.0±3.0* | 3.2±1.0* | 1.8±1.4 | 4.6±1.5 |
| Tnni2 | NS | 3.1 | NS | 1.0±0.5 | 8.4±4.5* | 3.5±1.3* | 2.3±2.1 | 4.8±1.6 |
| Wap | NS | 2.6 | NS | 0.8±0.2 | 2.7±0.9* | 32.2±20.6* | 0.8±0.3 | 63.7±36.5 |
| Ar | NS | NS | NS | 2±0.7 | 1.3±0.2 | 1.3±0.2 | 1.7±0.2* | 1±0.3 |
| Er | NS | NS | NS | 1.6±0.3* | 1.5±0.2* | 1.5±0.3* | 2±0.5* | 1±0.4 |
| Pgr | NS | NS | NS | 2.6±1.4 | 1.3±0.4 | 1.7±0.8 | 2±1.1 | 1.4±0.4 |
*Statistically significant difference (P<0.05).
For identification of the biological processes potentially relevant to the observed changes in mammary gland treatment that were altered by the various treatments, we used the Ingenuity Pathway Analysis database to cluster the 213 genes displaying differential expression (on both PND35 and PND50) into common biochemical pathways and physiological processes. Each of the treatments appeared to alter the expression of genes with diverse functions, in a different way at different time points (Supplementary Figure S3, see section on supplementary data given at the end of this article and Table 4, Supplementary Tables S2 and S3, see section on supplementary data given at the end of this article).
Analysis of the functions found to be differentially expressed in the mammary gland of rats after exposure to G, V, and GV at PND35 (A) and PND50 (B)
| Min | Max | Number of genes | |
|---|---|---|---|
| (A) PND35: functions | |||
| G | |||
| Skeletal and muscular system development and function | 6.74×10−5 | 2.49×10−2 | 11 |
| Tissue morphology | 6.74×10−5 | 2.84×10−2 | 10 |
| Carbohydrate metabolism | 3.12×10−4 | 3.54×10−2 | 9 |
| Development | 2.07×10−3 | 4.92×10−2 | 22 |
| Cell cycle | 3.59×10−3 | 2.49×10−2 | 3 |
| Cell death | 3.59×10−3 | 4.92×10−2 | 11 |
| Cell growth and proliferation | 3.59×10−3 | 4.57×10−2 | 8 |
| Lipid metabolism | 3.59×10−3 | 4.92×10−2 | 5 |
| V | |||
| Development | 9.91×10−7 | 1.79×10−2 | 11 |
| Lipid metabolism | 1.23×10−4 | 4.16×10−2 | 4 |
| Tissue morphology | 2.14×10−3 | 2.14×10−3 | 1 |
| Carbohydrate metabolism | 4.28×10−3 | 2.33×10−2 | 1 |
| Cell growth and proliferation | 2.96×10−2 | 2.96×10−2 | 1 |
| GV | |||
| Skeletal and muscular system development and function | 9.97×10−32 | 4.66×10−2 | 41 |
| Tissue morphology | 9.97×10−32 | 3.68×10−2 | 39 |
| Development | 1.95×10−8 | 4.66×10−2 | 27 |
| Carbohydrate metabolism | 1.36×10−3 | 4.66×10−2 | 11 |
| Cell death | 5.94×10−3 | 2.94×10−2 | 3 |
| Lipid metabolism | 5.94×10−3 | 4.66×10−2 | 5 |
| Cell growth and proliferation | 1.03×10−2 | 4.66×10−2 | 2 |
| Cell cycle | 1.19×10−2 | 2.94×10−2 | 3 |
| (B) PND50: functions | |||
| G | |||
| Cell death | 5.79×10−3 | 5.79×10−3 | 1 |
| Carbohydrate metabolism | 1.92×10−2 | 1.92×10−2 | 1 |
| Cell growth and proliferation | 2.68×10−2 | 2.68×10−2 | 1 |
| Development | 2.68×10−2 | 4.64×10−2 | 1 |
| V | |||
| Skeletal and muscular system development and function | 6.18×10−25 | 4.87×10−2 | 41 |
| Tissue morphology | 6.18×10−25 | 4.87×10−2 | 32 |
| Development | 3.17×10−5 | 4.87×10−2 | 22 |
| Carbohydrate metabolism | 7.08×10−5 | 4.87×10−2 | 10 |
| Lipid metabolism | 5.64×10−4 | 4.87×10−2 | 8 |
| Cell cycle | 6.22×10−3 | 4.27×10−2 | 3 |
| Cell growth and proliferation | 6.22×10−3 | 4.29×10−2 | 4 |
| Cell death | 1.85×10−2 | 4.87×10−2 | 6 |
| GV | |||
| Skeletal and muscular system development and function | 2.03×10−5 | 4.66×10−2 | 22 |
| Tissue morphology | 2.03×10−5 | 3.51×10−2 | 19 |
| Carbohydrate metabolism | 1.04×10−4 | 4.09×10−2 | 11 |
| Cell death | 5.16×10−4 | 4.80×10−2 | 22 |
| Cell cycle | 3.83×10−3 | 4.51×10−2 | 8 |
| Lipid metabolism | 5.94×10−3 | 4.73×10−2 | 12 |
| Development | 5.94×10−3 | 4.95×10−2 | 19 |
| Cell growth and proliferation | 1.19×10−2 | 4.66×10−2 | 23 |
The minimum (Min) and the maximum (Max) indicate the most and the least significant P value for genes.
Differentially expressed genes among treatments at PND35 (puberty)
The genes upregulated in response to G (45/65) were generally not strongly upregulated (1.5- to 2-fold, P<0.05) (Supplementary Table S2). The major categories identified related to morphogenesis and development (Supplementary Figure S3 and Table 4), and the genes assigned to these categories included Krt14 and Krt17, Ckm, Pygm, and Mylk2 (Supplementary Table S2). Another functional group altered by G included few genes encoding enzymes involved in metabolism (Supplementary Table S2). The expression of Tfap2c and Elf5, encoding transcription factors, was also modulated by exposure to G (Supplementary Table S2). The 20 downregulated genes (see GEO) included the metabolism-related genes Fkbp5 and Pla2g2d (Supplementary Table S2). The largest functional categories, associated with robust changes (greater than fivefold), following exposure to V concerned development and lipid metabolism (Supplementary Figure S3 and Table 4). Development-related genes displaying changes in expression included Cldn1, Sprra1, numerous Krts, and Klks (kallikrein-related peptidase) (Supplementary Table S2). V also induced the expression of a few genes encoding enzymes involved in steroidogenesis including Cyp17a1 and Cyp2b12 (Cyp2b15) (Supplementary Table S2).
The GV mixture elicited more robust changes in gene expression than G and V alone (two- to four-fold induction, P<0.005, Supplementary Table S2), in terms of development and morphology (Supplementary Figure S3). The functional categories concerned muscle development (e.g. Actn2, Casq1, Ckm, Pvalb, and Tnni2) (Supplementary Table S2 and Supplementary Figure S3) and development (Krt17 and Myh4) (Supplementary Table S2). GV also induced changes in the expression of genes, such as Pvalb, Pfkm, and Prkg3, related to carbohydrate metabolism (Supplementary Figure S3 and Supplementary Table S2). The genes downregulated (<−2.2-fold change) after exposure to the GV mixture included Csn2 and Wap, which are related to cell differentiation (Supplementary Table S2 and see GEO).
Differentially expressed genes among treatments at PND50 (cycling animals)
The G exposed group displayed few significant changes in gene expression (13 genes upregulated and 12 downregulated) (Supplementary Figure S2), and the magnitude of the change was low (fold-change <2) (Supplementary Table S3). The V exposed group displayed significant upregulation of genes (88/112) mostly involved in development and morphology (Supplementary Figure S3), including muscle development (greater than threefold induction, such as Actn2, Casq1, Ckm, Pvalb, Pygm, and Tnni2) (Supplementary Table S3, Supplementary Figure S3 and see GEO). V also induced moderate changes in gene expression related to carbohydrate (Pfkfb3 and Ppp1r3a) and lipid (Fabp3 and Lipa) metabolism (Supplementary Figure S3 and Supplementary Table S3). Twenty-four genes, including Cxcl13 and Lep, were downregulated by exposure to V (Supplementary Table S3).
For the GV exposure group, the largest functional categories (up- and downregulated) were development, morphology, metabolism, proliferation, and cell death (Supplementary Figure S3). The number of genes involved in muscle development and morphology identified was smaller and the level of induction was lower than that observed with V alone (Supplementary Table S3). Cell differentiation-associated genes (Csn2, Fabp3, and Wap) were also significantly upregulated by exposure to V and GV (Supplementary Table S3) in 40% of exposed animals, as shown by real-time RT-PCR (not shown). Other categories related to carbohydrate (such as Pdk4 and Pfkfb3) and lipid metabolism (such as Cyp24a1 and Hmgcs2) were also altered after GV exposure (Supplementary Table S3). Interestingly, the expression of Cyp24A1 was increased in 60% of GV-exposed animals (not shown), compared with controls, G-, and V-exposed animals; twenty-six genes were downregulated by GV exposure, including genes such as Ccnb1 and Cdc20 involved in cell death and the cell cycle (Supplementary Table S3).
Analysis of proteins by immunocytochemistry
Several proteins for which gene expression had been shown to be modulated with P<0.05, and which were considered to be of interest on the basis of the functional categories to which they belonged, were validated by immunocytochemistry. We checked for the presence of cytokeratin 17, claudin 1, and β-casein encoded by genes differentially expressed between treated and normal rat mammary glands. Immunostaining of smooth muscle actin, a marker for myoepithelial cells, was observed in basal cells surrounding the TEB and ducts and in vessels (Fig. 3Aa). Cytokeratin 17 immunostaining was detected in the cells lining the TEBs (cap cells), in some cells in the body (Fig. 3Ab, d, e and f), and in the ductal myoepithelial (not shown). Immunostaining for claudin 1 (Fig. 3Ac, g, h and i), the major component of epithelial tight junctions, was strong at the point of contact between epithelial cells from the TEBs. Both mRNA (Fig. 3C) and protein levels were increased for both cytokeratin 17 and claudin 1 at PND35 (Fig. 3A e and h respectively). Consistent with an increase in the expression of genes encoding milk proteins (Csn2 and Wap) at PND50 in the mammary glands of animals exposed to V and GV (Fig. 3D), β-casein immunostaining (Fig. 3B a, b, c, d and e) was detected in the epithelium of the secreting alveolar structures from GV2 group (Fig. 3Bd), but not in the GV1 group (Fig. 3Be). Activated STAT5A has been detected in the differentiated lactating gland. We therefore also carried out immunostaining for STAT5A on nuclear sections from mammary glands of GV-treated animals at PND50. Nuclear STAT5A immunostaining (corresponding to activated STAT5A) was also observed in the lobuloalveolar units (Fig. 3Bh), consistent with both the increase in the expression of genes (Csn2 and Wap) encoding milk proteins (Fig. 3D) and the secreting alveolar structures observed in the mammary glands of GV-treated animals (El Sheikh Saad et al. 2011).
Immunostaining for specific proteins in the mammary gland. (A) Immunocytochemistry at PND35 (A) and PND50 (B). Mammary sections representing ten animals of each treatment or control group were analyzed. Representative micrographs are presented. (A a, b and c) Paraffin sections of untreated animals stained with antibodies against alpha smooth muscle actin (a), cytokeratin 17 (b), and claudin 1 (c). Positive staining of alpha smooth muscle actin (a) and cytokeratin 17 (b) was shown in the outer myoepithelial cell layer of TEBs. Staining with an antibody against claudin 1 (c), showing positive staining between epithelial cells from a TEB. (d, e, f, g, h and i) Immunostaining cytokeratin 17 (d, e and f) and claudin 1 (g, h and i) in treated animals. Immunostaining for both proteins was increased in V (e and h respectively), compared with G (d and g) or untreated (b and c); GV (f and i). (B) Immunocytochemistry at PND50. (a, b, c, d and e) β-Casein immunostaining of control (a), G (b), V (c), or GV-treated animals (d and e); β-casein immunostaining was increased in the epithelium of the secreting alveolar structures of GV2 (d) and increased compared with control and other treatments; GV1 (e). (f) H&E of lobuloalveolar structures from the GV2 group. (g and h) Stat5A immunostaining. In h, strong STAT5A immunostaining was observed in a lobuloalveolar unit from the GV2 group. Arrow, nuclear STAT5A immunostaining. By contrast, nuclear STAT5A was not observed in the terminal structures from an untreated animal (g). Original magnifications ×40 (a, b, c, d and e), ×20 (f), or ×60 (g and h). (C) Histograms showing the higher levels of mRNA for both claudin 1 and cytokeratin 17 at PND35 in animals exposed to V; (D) significant higher levels of mRNA for Csn2, Fabp3, and Wap in GV (GV2)-exposed animals at PND50. *, Statistically significant difference (P<0.05). Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-12-0395.
Citation: Journal of Endocrinology 216, 2; 10.1530/JOE-12-0395
Changes in gene expression may be dependent on the estrous cycle
Subtle changes in the development of the mammary gland, such as proliferation and differentiation, have been described during the 4–5 days of transition during the rat estrous cycle (Schedin et al. 2000, Hvid et al. 2012). We reanalyzed the major changes in gene expression previously reported by quantitative RT-PCR data for individual RNA expression (as described in Tables 1 and 2), taking into account the stage of the estrous cycle (i.e. proestrus (P), estrus (E), and metestrus-diestrus (M-D)). Gene expression in mammary gland samples was normalized against two housekeeping genes: P0 (Fig. 4) and Tbp (not shown). At PND35 in control rats, several changes were observed in the expression of genes examined (Ki67, Wap, Tcfap2c, and Tnni2 and Ckm) during the estrous cycle (Fig. 4A). Levels of Krt17, Cldn1, and Sprr1a expression were clearly higher with V in E or D stages (Fig. 4A), and Ckm and Tnni2 expression levels were higher with GV in P and E stages (Fig. 4A). Wap expression at D was lower after G, V, and GV treatment than in untreated rats (Fig. 4A). In contrast to these changes observed for a few genes (Cldn1, Krt17, Sprr1a, Ckm, and Tnni2) at PND35 according to the cycle, changes were most obvious during the estrus cycle at PND50. Levels of Csn2, Wap, and Fabp3 expression in P stage were clearly higher with GV treatment (mostly due to GV2 rather than GV1) (Fig. 4B); a smaller effect was observed for these three genes with V in E stage (Fig. 4B), together with an increase in Ckm and Tnni2 expression in P or E stages following treatment with V (Fig. 4B).
Distinctive G-, V-, and GV-associated changes in the mammary gland of treated rats, as a function of position in the estrous cycle. Untreated, G, V, and GV mixture gene profiles (determined by RT-PCR) were shown for PND35 (A) and PND50 (B). The selected genes are derived from Tables 2 and 3. Gene expression (y-axis) was shown for each treatment and each phase of the estrous cycle (x-axis). Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-12-0395.
Citation: Journal of Endocrinology 216, 2; 10.1530/JOE-12-0395
Discussion
Exposure to environmental EDCs with estrogenic activities during fetal development is thought to contribute to abnormal development of the mammary gland. Our data provide the first evidence that exposure to the anti-androgen V and a mixture of G and V in utero and during lactation, modulates mammary gland morphogenesis at the molecular level during puberty and in cycling animals. G administered alone and at low doses had the smallest effect of the treatments tested. Changes in gene expression were observed with V and GV, the precise nature of the changes depending on the period of postnatal development (puberty or postpuberty, i.e. PND35 and PND50) and the compound/or mixture considered. The data obtained are consistent with our previous histological observations (El Sheikh Saad et al. 2011). The signaling pathways deregulated and the underlying mechanisms are discussed.
We investigated the molecular events regulated by exposure to G, V, and GV, by examining the gene expression profiles of whole mammary glands, with validation of the results obtained for the expression of selected genes by RT-PCR and immunocytochemistry. Crucial methodological considerations were taken into account in the interpretation of our data, including the use of a phytoestrogen-free diet, and an analysis of vaginal opening and of vaginal smears on the day on which the rats were killed, for all rats. The RNA used for molecular studies was prepared from the same individual mammary glands obtained from PND35 and PND50 rats for histological studies (El Sheikh Saad et al. 2011), allowing a comparison with previous histological observations. The transcriptomic study was performed with a cut off of 1.5 and a significant threshold of P value <0.05. The largest changes in the expression of the selected genes in our study were normalized to gene expression in mammary samples by RT-PCR on ten samples per treatment, with several housekeeping genes (Po and Tbp), as previously reported (Hvid et al. 2011). We also carefully analyzed our results, taking into account the phase of the estrous cycle for each animal on the day it was killed. Finally, we also validated by immunocytochemistry the detection of several proteins for which commercial antibodies were available. Further studies, including tumorigenic studies, are required to determine the full relevance of these findings.
G and GV increase the expression of genes encoding cytoskeletal elements, involved in ductal branching, during puberty (PND35)
During puberty, E2 directs ductal elongation and branching (Sternlicht et al. 2006). We have previously reported that both an increase in ductal branching and a proliferation within the TEBs at PND35 following exposure to G and GV in utero and during lactation (El Sheikh Saad et al. 2011). In this study, we provide the first evidence that in utero exposure to GV and G strongly alters gene transcription at puberty and has effects on developmental genes, consistent with the changes to mammary gland morphogenesis observed in our previous study (El Sheikh Saad et al. 2011). The largest category of genes concerned encodes cytoskeletal elements, such as actin, actin-associated proteins, and cytokeratins. The expression of several genes encoding actin-associated proteins including Pvalb, Pygm, and Tnni2, and the expression of Ckm and Mylk2 was increased by the combination of G with V (GV), and G alone to a lesser extent, as shown in our transcriptomic study and validated by RT-PCR (see Table 2). The modulation of these genes has been observed following treatment with BPA, benzyl butyl phthalate (BBP), and transforming growth factor β (TGFβ) in the rodent mammary gland (Xie et al. 2003, Moral et al. 2007, 2008). Creatine kinase (Ckm) has also been detected in the mammary gland (Raymond et al. 2011). Recently, methylated CKM has been correlated with clinical prognosis in breast cancer patients (Jeschke et al. 2012). The expression of the gene encoding cytokeratin 17 was increased by G and GV in myoepithelial cells, as shown by RT-PCR and immunocytochemistry. Cytokeratin 17 was observed in basal cells in TEB, i.e. in the cells that expressed cytokeratin 14 (a marker of basal cells in TEBs). These results suggest that major changes to the actomyosin cytoskeletal architecture of cells, including myoepithelial cells (Moumen et al. 2011), occur during mammary gland morphogenesis at puberty. Moreover, the significant increase in expression of Tcfap2c, which encodes the transcription factor AP2γ (Table 2) in response to G treatment, is also consistent with the increases in epithelial cell proliferation in TEBs and ductal branching observed at PND35 following exposure to G (El Sheikh Saad et al. 2011). AP2γ is strongly expressed in the cap cells lining the TEBs (smooth muscle actin +) and in ductal contractile myoepithelial (cytokeratin 14+) cells in the developing rodent mammary gland (Friedrichs et al. 2007, Jager et al. 2010). The loss of AP2γ/Tfap2c has been shown to reduce cellular proliferation within TEBs and to impair ductal branching and elongation (Jager et al. 2010). Interestingly, V, when administered alone, did not appear to modulate these genes, except for cytokeratin 17, suggesting that G may be the driving force behind regulation of these genes by GV at PND35.
V alters gene expression related to the development of the mammary gland at PND35
V alone had a different effect on gene expression profile from G and GV in the pubertal mammary gland. V increases the expression of Krt17, Cldn1, and Sprr1a, whereas G did not, as shown by RT-PCR (Table 2). Claudin 1 was detected in epithelial cells by immunocytochemistry (see Fig. 3). The increase in Cldn1 expression induced by V in mammary glands is consistent with the modulation of expression of this gene by androgens previously reported in mouse immature Sertoli cells (Gye 2003). Changes in the expression of the gene encoding claudin 1 (Cldn1), the major component of the epithelial tight junctions, have also been reported to be associated with tumorigenesis in human breast (Myal et al. 2010). We observed, for the first time, the presence of KRT17 in myoepithelial cells (Fig. 3) and its significant increase by V (also upregulated by GV, see Table 2). Together, these findings suggest a role of these two V-regulated proteins, KRT17 and CLDN1, in three-dimensional organization and cell–cell contacts. The significant increase in Sprr1a expression observed after V treatment may be consistent with the increase in ductal branching that we have previously reported with V (El Sheikh Saad et al. 2011). SPRR1A is mostly found in the TEBs and ducts (epithelial cells) of the mouse mammary gland (Morris et al. 2006). This protein has also been described as an axonal growth and guidance protein, suggesting a possible role in ductal growth and morphogenesis in the developing mammary gland (Morris et al. 2006). Interestingly, at PND35, the modulation of claudin 1 (epithelial cells) and cytokeratin 17 (myoepithelial cells) was mostly observed in estrus (Fig. 4), suggesting a coordinated regulation; in addition, the modulation of Sprr1a was observed mostly in diestrus. Further studies are required to clarify these findings. The expression of Cyp17A encoding 17α-hydroxylase/17,20-lyase has been shown to be modulated in fetal testis by V and prochoraz (another fungicide with anti-androgen activity), resulting in changes to androgen metabolism (Laier et al. 2006). The modulation of genes involved in steroidogenesis by V at PND35 (see Supplementary Table S2) may reflect changes in estrogen and androgen balance during fetal development.
V and GV increase the expression of genes involved in ductal side branching and lobuloalveolar development in virgin cycling animals (PND50)
In the developing mammary gland of cycling animals, short ‘tertiary’ side branches of the ductal epithelium form in response to progesterone, and lobuloalveolar structures develop at the end of the tertiary branches (Atwood et al. 2000, Haslam et al. 2008, El Sheikh Saad et al. 2011). We found striking differences in gene expression in the mammary gland between PND35 and PDN50 following exposure to V or GV, consistent with the major histological modifications observed in postpubertal mammary glands exposed to V and GV (El Sheikh Saad et al. 2011). Indeed, increased glandular area and lobuloalveolar development were observed after exposure to GV and V at PND50 (El Sheikh Saad et al. 2011). First, a subset of genes (Actn2, Casq1, Mylk2, Tnni2, Pvalb, and Pygm) for which expression levels were strongly upregulated by V and GV (as shown using RT-PCR) in cycling animals was related to muscle development (Tables 3 and Supplementary Table S3). Our results again suggest that changes in the actomyosin cytoskeletal architecture of myoepithelial cells may occur at PND50, depending on the treatment. The postpubertal significant increase in Wap, Csn2, and Fabp3 by V, and GV is consistent with the presence of secretion vacuoles in the alveolar structures observed at PND50 from animals exposed to V and GV (El Sheikh Saad et al. 2011), suggesting an early terminal differentiation of alveolar cells after V and GV exposure. The increase in STAT5A activation (as shown by nuclear immunostaining in Fig. 3), which is mainly lactation dependent (Liu et al. 1997, Siegel & Muller 2010), also agrees with the increases in milk protein levels. Finally, at PND50, we found a significant increase in Elf5 expression with V and GV treatment (Table 3) in 80–90% animals (not shown). ELF5 is a transcription factor that mediates prolactin signaling (Brisken & Rajaram 2006). It has been reported that overexpression of Elf5 in an inducible transgenic model causes alveolar differentiation and milk secretion in virgin mice and disrupt ductal morphogenesis (Oakes et al. 2008). Altogether, differences in gene expression in the mammary gland observed at PND50 following in utero exposure to V or GV are consistent with the major histological modifications observed in postpubertal mammary glands (increased glandular area and lobuloalveolar development, and possibly focal branching defects). In line of our findings, Wang et al. (2006) have observed marked feminization of the adult male mammary gland following in utero and onward dietary exposure to the mixture of G and methoxychlor (a pesticide having estrogenic and anti-androgenic activities); prominent proliferation of both ducts and alveoli; secretory material was seen in alveolar lumen, which is normally absent in untreated male mammary glands (Wang et al. 2006). Further studies are required to analyze the complex mechanism of action of endocrine-active chemicals that may act as agonists or antagonists through one or more hormone receptors.
There are few studies on the effect of the estrous cycle on mammary gland development. These studies have concerned the effects of the estrous cycle on proliferation in rats, and conflicting results have been observed (Schedin et al. 2000, Hvid et al. 2011, 2012). This study also examined the influence of the estrous cycle on a number of important markers involved in mammary gland morphogenesis in young virgin female rats (5 and 7 weeks respectively). Changes were observed for a few genes (Cldn1, Krt17, and Sprr1a) at PND35 according to the cycle (as mentioned earlier); most of the genes showed changes during the estrus cycle at PND50, such as those concerning Csn2, Wap, and Fabp3, demonstrating the importance of evaluating within a stage for other studies. β-Casein (Csn2) and whey acidic protein (Wap) are classical markers of mammary gland differentiation and widely used as indicators of reduced breast cancer risk. The higher mRNA expression of these two genes at PND50 in the mammary gland of GV group relative to G or V alone may suggest a protective role of maternal exposure to G in the presence of V. This is also supported by lower Ki67 expression during estrous in GV-exposed animals. Experiments using DMBA-induced carcinogenesis are ongoing.
Mechanisms underlying the effects of V
The mechanisms underlying the patterns of gene regulation observed at PND35 and PND50 after exposure to G, V, and GV during gestation and lactation remain to be explored. V is an anti-androgenic compound that can be metabolized to generate two ER agonists, M1 and M2 (Molina-Molina et al. 2006). Several fungicides have been shown to have anti-androgenic activity through interaction with ARs. Some xenoestrogens found in food contaminants present in the human diet may also have anti-androgenic actions, especially at low doses (Sohoni & Sumpter 1998, Andersen et al. 2002, Lemaire et al. 2004), which might modify normal mammary gland development and growth. The action of androgens and their antagonists in normal mammary gland morphogenesis remains poorly understood. It has been shown that growth retardation (reduced duct branching at prepubertal stages and lower levels of lobuloalveolar development in adults) is observed in female mice lacking AR (Yeh et al. 2003). Retained nipples in male rats treated with V have suggested that androgens may play an important role in early sex differentiation of the mammary gland (Gray et al. 1994, 1999). Cooperation of signaling pathways between epithelium and mesenchyme in embryonic or in the postnatal mammary gland development have been addressed in several reviews (Robinson 2007, Su et al. 2011). The AR is abundantly expressed in normal mammary epithelium, but not detected in mammary stroma or myoepithelium (Somboonporn & Davis (2004) and data not shown). The gene expression profile was determined in the whole mammary gland. Thus, the changes in gene expression observed in our study can be related to modifications in the biology of the epithelial or stromal component (such as adipocytes, fibroblasts, and immune cells). Changes in lipid metabolism observed from the V-exposed group (Supplementary Table S2) may occur in the adipose tissue and changes in the immune-related genes (see GEO) may be a reflection of modifications in the number or function of infiltrated immune cells. All these cell types have an essential role in the development of the mammary gland. These results suggest that V exposure may alter epithelium–stroma interaction during mammary gland development. Androgens also inhibit estrogen-induced mammary epithelial proliferation and breast growth (Somboonporn & Davis 2004, Dimitrakakis & Bondy 2009), which can be reversed by anti-androgens (flutamide), suggesting that AR-dependent mechanisms are involved. An increase in cellular proliferation and branching in response to flutamide treatment has been recently reported throughout the mammary gland of postpubertal mice (12 weeks) (Peters et al. 2011), whereas a decrease in mammary epithelial cell proliferation and gland development has been reported in prepubertal AR knockout mice (Yeh et al. 2003). These findings indicate differential effects of an anti-AR antagonist in the peri- and postpubertal murine mammary gland; in mature glands, flutamide promotes the proliferation of breast epithelial cells, by allowing estrogen signaling to predominate, through interactions between AR and ER (i.e. between estrogen and androgen signaling) (Peters et al. 2009). In our study, exposure during gestation/lactation to a mixture of GV results in both significant alterations of mammary gland development in female offspring at peri-puberty and lobuloalveolar hyperplasia in cycling rats, consistent with differences in gene profiles at PND35 and PND50. These findings also suggest that the molecular mechanisms underlying the effects of V in the mammary gland are highly dependent on the stage of mammary gland development (peri- or postpuberty), possibly with an anti-androgen effect of V on selected genes at PND35 and a mixed anti-androgen/estrogenic effect of GV in cycling animals (i.e. in the presence of significant levels of E2).
Conclusion
Early hormonal disturbances (in utero and/or during lactation) with effects on the mammary gland of the offspring have been described for the combination of G and the fungicide (anti-androgen) V. Detailed gene profiling analysis identified new pathways involved in the development of the mammary gland after exposure to this mixture of G and V in utero and during lactation, extending our previous results (El Sheikh Saad et al. 2011). The effects of the anti-androgen V on the mammary gland depend on the period considered (peri- or postpubertal). These studies, based on exposure during gestation and lactation, demonstrate for the first time that V and GV may be active on the mammary fetal anlage in females and that these compounds may modify mammary gland development under hormonal action. Our results suggest a continuum of development from fetal to pubertal stages. Finally, this study highlights the health risks associated with the presence of products in the environment, even at low doses, more particularly the potentially adverse effects of these compounds on the mammary gland during embryonic development. Further studies are required to assess the potential effect of these products on human breast cancer.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-12-0395.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by INSERM, CNRS, and Region Ile-de-France. H El S S and A T received a fellowship from Region Ile-de-France (France). The funding source had no involvement in any step in the conduct of the research, preparation of the article, in analysis and interpretation of data, and in the decision to submit the paper. The authors declare they have no competing financial interest.
Author contribution statement
M P-A conceived and designed the experiments. H El S S performed the experiments. H El S S, A T, S V, and I B analyzed the data. M P-A wrote the paper. H El S S and I B involved in critical reading of the manuscript.
Acknowledgements
The authors thank F Dumont and F Letourneur for the microarray hybridization (platform analysis, Hôpital Cochin, Paris), R Bergès for technical teams at INRA Dijon, especially. They also thank MC Canivenc-Lavier (INRA, Dijon, France), D Vaiman, J Auger, and F Mondon (INSERM U 567, Hôpital Cochin, Paris) for their advice (PNRPE 2006), and N Peyri (INSERM U965, Paris). They thank Dr D Medina, Baylor College of Medecine, Houston TX, USA, for the gift of rabbit polyclonal antibody against β-casein (Durban et al. 1985).
References
Andersen HR, Vinggaard AM, Rasmussen TH, Gjermandsen IM & Bonefeld-Jorgensen EC 2002 Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and Applied Pharmacology 179 1–12. (doi:10.1006/taap.2001.9347)
Atwood CS, Hovey RC, Glover JP, Chepko G, Ginsburg E, Robison WG & Vonderhaar BK 2000 Progesterone induces side-branching of the ductal epithelium in the mammary glands of peripubertal mice. Journal of Endocrinology 167 39–52. (doi:10.1677/joe.0.1670039)
Badger TM, Ronis MJ, Simmen RC & Simmen FA 2005 Soy protein isolate and protection against cancer. Journal of the American College of Nutrition 24 146S–149S.
Bieche I, Tozlu S, Girault I & Lidereau R 2004 Identification of a three-gene expression signature of poor-prognosis breast carcinoma. Molecular Cancer 3 37. (doi:10.1186/1476-4598-3-37)
Brisken C & Rajaram RD 2006 Alveolar and lactogenic differentiation. Journal of Mammary Gland Biology & Neoplasia 11 239–248. (doi:10.1007/s10911-006-9026-0)
Bursztyka J, Debrauwer L, Perdu E, Jouanin I, Jaeg JP & Cravedi JP 2008 Biotransformation of vinclozolin in rat precision-cut liver slices: comparison with in vivo metabolic pattern. Journal of Agricultural and Food Chemistry 56 4832–4839. (doi:10.1021/jf0728045)
Cameron HL & Foster WG 2009 Developmental and lactational exposure to dieldrin alters mammary tumorigenesis in Her2/neu transgenic mice. PLoS ONE 4 e4303. (doi:10.1371/journal.pone.0004303)
Christiansen S, Scholze M, Dalgaard M, Vinggaard AM, Axelstad M, Kortenkamp A & Hass U 2009 Synergistic disruption of external male sex organ development by a mixture of four antiandrogens. Environmental Health Perspectives 117 1839–1846.
Daniel CW, Silberstein GB & Strickland P 1987 Direct action of 17β-estradiol on mouse mammary ducts analyzed by sustained release implants and steroid autoradiography. Cancer Research 47 6052–6057.
Dimitrakakis C & Bondy C 2009 Androgens and the breast. Breast Cancer Research 11 212. (doi:10.1186/bcr2413)
Durando M, Kass L, Piva J, Sonnenschein C, Soto AM, Luque EH & Munoz-de-Toro M 2007 Prenatal bisphenol A exposure induces preneoplastic lesions in the mammary gland in Wistar rats. Environmental Health Perspectives 115 80–86. (doi:10.1289/ehp.9282)
Durban EM, Medina D & Butel JS 1985 Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo. Developmental Biology 109 288–298. (doi:10.1016/0012-1606(85)90456-7)
El Sheikh Saad H, Meduri G, Phrakonkham P, Berges R, Vacher S, Djallali M, Auger J, Canivenc-Lavier MC & Perrot-Applanat M 2011 Abnormal peripubertal development of the rat mammary gland following exposure in utero and during lactation to a mixture of genistein and the food contaminant vinclozolin. Reproductive Toxicology 32 15–25. (doi:10.1016/j.reprotox.2011.03.001)
Enoch RR, Stanko JP, Greiner SN, Youngblood GL, Rayner JL & Fenton SE 2007 Mammary gland development as a sensitive end point after acute prenatal exposure to an atrazine metabolite mixture in female Long–Evans rats. Environmental Health Perspectives 115 541–547. (doi:10.1289/ehp.9612)
Eustache F, Mondon F, Canivenc-Lavier MC, Lesaffre C, Fulla Y, Berges R, Cravedi JP, Vaiman D & Auger J 2009 Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility. Environmental Health Perspectives 117 1272–1279.
Fenton SE 2006 Endocrine-disrupting compounds and mammary gland development: early exposure and later life consequences. Endocrinology 147 S18–S24. (doi:10.1210/en.2005-1131)
Foster WG, Younglai EV, Boutross-Tadross O, Hughes CL & Wade MG 2004 Mammary gland morphology in Sprague–Dawley rats following treatment with an organochlorine mixture in utero and neonatal genistein. Toxicological Sciences 77 91–100. (doi:10.1093/toxsci/kfg247)
Friedrichs N, Steiner S, Buettner R & Knoepfle G 2007 Immunohistochemical expression patterns of AP2alpha and AP2gamma in the developing fetal human breast. Histopathology 51 814–823. (doi:10.1111/j.1365-2559.2007.02887.x)
Gray LE Jr, Ostby JS & Kelce WR 1994 Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicology and Applied Pharmacology 129 46–52. (doi:10.1006/taap.1994.1227)
Gray LE Jr, Ostby J, Monosson E & Kelce WR 1999 Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicology and Industrial Health 15 48–64. (doi:10.1191/074823399678846646)
Gye MC 2003 Expression of claudin-1 in mouse testis. Archives of Andrology 49 271–279. (doi:10.1080/713828170)
Haslam SZ, Drolet A, Smith K, Tan M & Aupperlee M 2008 Progestin-regulated luminal cell and myoepithelial cell-specific responses in mammary organoid culture. Endocrinology 149 2098–2107. (doi:10.1210/en.2007-1398)
Hilakivi-Clarke L, Cho E, Onojafe I, Raygada M & Clarke R 1999a Maternal exposure to genistein during pregnancy increases carcinogen-induced mammary tumorigenesis in female rat offspring. Oncology Reports 6 1089–1095.
Hilakivi-Clarke L, Onojafe I, Raygada M, Cho E, Skaar T, Russo I & Clarke R 1999b Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis. British Journal of Cancer 80 1682–1688. (doi:10.1038/sj.bjc.6690584)
Hoover RN, Hyer M, Pfeiffer RM, Adam E, Bond B, Cheville AL, Colton T, Hartge P, Hatch EE & Herbst AL et al. 2011 Adverse health outcomes in women exposed in utero to diethylstilbestrol. New England Journal of Medicine 365 1304–1314. (doi:10.1056/NEJMoa1013961)
Hvid H, Ekstrom CT, Vienberg S, Oleksiewicz MB & Klopfleisch R 2011 Identification of stable and oestrus cycle-independent housekeeping genes in the rat mammary gland and other tissues. Veterinary Journal 190 103–108. (doi:10.1016/j.tvjl.2010.09.002)
Hvid H, Thorup I, Sjogren I, Oleksiewicz MB & Jensen HE 2012 Mammary gland proliferation in female rats: effects of the estrous cycle, pseudo-pregnancy and age. Experimental and Toxicologic Pathology 64 321–332. (doi:10.1016/j.etp.2010.09.005)
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U & Speed TP 2003 Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4 249–264. (doi:10.1093/biostatistics/4.2.249)
Jager R, Schafer S, Hau-Liersch M & Schorle H 2010 Loss of transcription factor AP-2gamma/TFAP2C impairs branching morphogenesis of the murine mammary gland. Developmental Dynamics 239 1027–1033. (doi:10.1002/dvdy.22239)
Jeschke J, Van Neste L, Glockner SC, Dhir M, Calmon MF, Deregowski V, Van Criekinge W, Vlassenbroeck I, Koch A & Chan TA et al. 2012 Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen. Epigenetics 7 701–709. (doi:10.4161/epi.20445)
Kelce WR, Lambright CR, Gray LE Jr & Roberts KP 1997 Vinclozolin and p,p′-DDE alter androgen-dependent gene expression: in vivo confirmation of an androgen receptor-mediated mechanism. Toxicology and Applied Pharmacology 142 192–200. (doi:10.1006/taap.1996.7966)
Korach KS, Couse JF, Curtis SW, Washburn TF, Lindzey J, Kimbro KS, Eddy EM, Migliaccio S, Snedeker SM & Lubahn DB et al. 1996 Estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes. Recent Progress in Hormone Research 51 159–186.(discussion 186–158)
Korde LA, Wu AH, Fears T, Nomura AM, West DW, Kolonel LN, Pike MC, Hoover RN & Ziegler RG 2009 Childhood soy intake and breast cancer risk in Asian American women. Cancer Epidemiology, Biomarkers & Prevention 18 1050–1059. (doi:10.1158/1055-9965.EPI-08-0405)
Laier P, Metzdorff SB, Borch J, Hagen ML, Hass U, Christiansen S, Axelstad M, Kledal T, Dalgaard M & McKinnell C et al. 2006 Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz. Toxicology and Applied Pharmacology 213 160–171. (doi:10.1016/j.taap.2005.10.013)
Lamartiniere CA, Cotroneo MS, Fritz WA, Wang J, Mentor-Marcel R & Elgavish A 2002 Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. Journal of Nutrition 132 552S–558S.
Lehraiki A, Messiaen S, Berges R, Canivenc-Lavier MC, Auger J, Habert R & Levacher C 2011 Antagonistic effects of gestational dietary exposure to low-dose vinclozolin and genistein on rat fetal germ cell development. Reproductive Toxicology 31 424–430. (doi:10.1016/j.reprotox.2010.12.005)
Lemaire G, Terouanne B, Mauvais P, Michel S & Rahmani R 2004 Effect of organochlorine pesticides on human androgen receptor activation in vitro. Toxicology and Applied Pharmacology 196 235–246. (doi:10.1016/j.taap.2003.12.011)
Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A & Hennighausen L 1997 Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes and Development 11 179–186. (doi:10.1101/gad.11.2.179)
Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CA Jr, Shyamala G, Conneely OM & O'Malley BW 1995 Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes and Development 9 2266–2278. (doi:10.1101/gad.9.18.2266)
Markey CM, Luque EH, Munoz De Toro M, Sonnenschein C & Soto AM 2001 In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland. Biology of Reproduction 65 1215–1223.
Mi H, Dong Q, Muruganujan A, Gaudet P, Lewis S & Thomas PD 2010 PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the Gene Ontology Consortium. Nucleic Acids Research 38 D204–D210. (doi:10.1093/nar/gkp1019)
Molina-Molina JM, Hillenweck A, Jouanin I, Zalko D, Cravedi JP, Fernandez MF, Pillon A, Nicolas JC, Olea N & Balaguer P 2006 Steroid receptor profiling of vinclozolin and its primary metabolites. Toxicology and Applied Pharmacology 216 44–54. (doi:10.1016/j.taap.2006.04.005)
Monosson E, Kelce WR, Lambright C, Ostby J & Gray LE Jr 1999 Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicology and Industrial Health 15 65–79.
Moral R, Wang R, Russo IH, Mailo DA, Lamartiniere CA & Russo J 2007 The plasticizer butyl benzyl phthalate induces genomic changes in rat mammary gland after neonatal/prepubertal exposure. BMC Genomics 8 453. (doi:10.1186/1471-2164-8-453)
Moral R, Wang R, Russo IH, Lamartiniere CA, Pereira J & Russo J 2008 Effect of prenatal exposure to the endocrine disruptor bisphenol A on mammary gland morphology and gene expression signature. Journal of Endocrinology 196 101–112. (doi:10.1677/JOE-07-0056)
Morris JS, Stein T, Pringle MA, Davies CR, Weber-Hall S, Ferrier RK, Bell AK, Heath VJ & Gusterson BA 2006 Involvement of axonal guidance proteins and their signaling partners in the developing mouse mammary gland. Journal of Cellular Physiology 206 16–24. (doi:10.1002/jcp.20427)
Moumen M, Chiche A, Cagnet S, Petit V, Raymond K, Faraldo MM, Deugnier MA & Glukhova MA 2011 The mammary myoepithelial cell. International Journal of Developmental Biology 55 763–771. (doi:10.1387/ijdb.113385mm)
Munoz-de-Toro M, Markey CM, Wadia PR, Luque EH, Rubin BS, Sonnenschein C & Soto AM 2005 Perinatal exposure to bisphenol-A alters peripubertal mammary gland development in mice. Endocrinology 146 4138–4147. (doi:10.1210/en.2005-0340)
Myal Y, Leygue E & Blanchard AA 2010 Claudin 1 in breast tumorigenesis: revelation of a possible novel "claudin high" subset of breast cancers. Journal of Biomedicine & Biotechnology 2010 956897. (doi:10.1155/2010/956897)
Nikolsky Y & Bryant J 2009 Protein networks and pathway analysis. Preface. Methods in Molecular Biology 563 v–vii.
Oakes SR, Naylor MJ, Asselin-Labat ML, Blazek KD, Gardiner-Garden M, Hilton HN, Kazlauskas M, Pritchard MA, Chodosh LA & Pfeffer OL et al. 2008 The Ets transcription factor Elf5 specifies mammary alveolar cell fate. Genes and Development 22 581–586. (doi:10.1101/gad.1614608)
Pei RJ, Sato M, Yuri T, Danbara N, Nikaido Y & Tsubura A 2003 Effect of prenatal and prepubertal genistein exposure on N-methyl-N-nitrosourea-induced mammary tumorigenesis in female Sprague–Dawley rats. In Vivo 17 349–357.
Peters AA, Buchanan G, Ricciardelli C, Bianco-Miotto T, Centenera MM, Harris JM, Jindal S, Segara D, Jia L & Moore NL et al. 2009 Androgen receptor inhibits estrogen receptor-alpha activity and is prognostic in breast cancer. Cancer Research 69 6131–6140. (doi:10.1158/0008-5472.CAN-09-0452)
Peters AA, Ingman WV, Tilley WD & Butler LM 2011 Differential effects of exogenous androgen and an androgen receptor antagonist in the peri- and postpubertal murine mammary gland. Endocrinology 152 3728–3737. (doi:10.1210/en.2011-1133)
Raymond K, Cagnet S, Kreft M, Janssen H, Sonnenberg A & Glukhova MA 2011 Control of mammary myoepithelial cell contractile function by alpha3beta1 integrin signalling. EMBO Journal 30 1896–1906. (doi:10.1038/emboj.2011.113)
Robinson GW 2007 Cooperation of signalling pathways in embryonic mammary gland development. Nature Reviews. Genetics 8 963–972. (doi:10.1038/nrg2227)
Russo IH & Russo J 1978 Developmental stage of the rat mammary gland as determinant of its susceptibility to 7,12-dimethylbenz[a]anthracene. Journal of the National Cancer Institute 61 1439–1449.
Schedin P, Mitrenga T & Kaeck M 2000 Estrous cycle regulation of mammary epithelial cell proliferation, differentiation, and death in the Sprague–Dawley rat: a model for investigating the role of estrous cycling in mammary carcinogenesis. Journal of Mammary Gland Biology & Neoplasia 5 211–225. (doi:10.1023/A:1026447506666)
Siegel PM & Muller WJ 2010 Transcription factor regulatory networks in mammary epithelial development and tumorigenesis. Oncogene 29 2753–2759. (doi:10.1038/onc.2010.43)
Sohoni P & Sumpter JP 1998 Several environmental oestrogens are also anti-androgens. Journal of Endocrinology 158 327–339. (doi:10.1677/joe.0.1580327)
Somboonporn W & Davis SR 2004 Testosterone effects on the breast: implications for testosterone therapy for women. Endocrine Reviews 25 374–388. (doi:10.1210/er.2003-0016)
Sternlicht MD, Kouros-Mehr H, Lu P & Werb Z 2006 Hormonal and local control of mammary branching morphogenesis. Differentiation 74 365–381. (doi:10.1111/j.1432-0436.2006.00105.x)
Su Y, Shankar K, Rahal O & Simmen RC 2011 Bidirectional signaling of mammary epithelium and stroma: implications for breast cancer – preventive actions of dietary factors. Journal of Nutritional Biochemistry 22 605–611. (doi:10.1016/j.jnutbio.2010.09.008)
Vandenberg LN, Maffini MV, Wadia PR, Sonnenschein C, Rubin BS & Soto AM 2007 Exposure to environmentally relevant doses of the xenoestrogen bisphenol-A alters development of the fetal mouse mammary gland. Endocrinology 148 116–127. (doi:10.1210/en.2006-0561)
Wang XJ, Bartolucci-Page E, Fenton SE & You L 2006 Altered mammary gland development in male rats exposed to genistein and methoxychlor. Toxicological Sciences 91 93–103. (doi:10.1093/toxsci/kfj120)
Warri A, Saarinen NM, Makela S & Hilakivi-Clarke L 2008 The role of early life genistein exposures in modifying breast cancer risk. British Journal of Cancer 98 1485–1493. (doi:10.1038/sj.bjc.6604321)
Wu AH, Yu MC, Tseng CC & Pike MC 2008 Epidemiology of soy exposures and breast cancer risk. British Journal of Cancer 98 9–14. (doi:10.1038/sj.bjc.6604145)
Xie L, Law BK, Aakre ME, Edgerton M, Shyr Y, Bhowmick NA & Moses HL 2003 Transforming growth factor beta-regulated gene expression in a mouse mammary gland epithelial cell line. Breast Cancer Research 5 R187–R198. (doi:10.1186/bcr640)
Yeh S, Hu YC, Wang PH, Xie C, Xu Q, Tsai MY, Dong Z, Wang RS, Lee TH & Chang C 2003 Abnormal mammary gland development and growth retardation in female mice and MCF7 breast cancer cells lacking androgen receptor. Journal of Experimental Medicine 198 1899–1908. (doi:10.1084/jem.20031233)
