Detection of Expressional Changes Induced by Intrauterine Growth Restriction in the Developing Rat Mammary Gland via Exploratory Pathways Analysis

<div><p>Background</p><p>Intrauterine growth restriction (IUGR) is thought to lead to fetal programming that in turn contributes to developmental changes of many organs postnatally. There is evidence that IUGR is a risk factor for the development of metabolic and cardiovascular disease later in life. A higher incidence of breast cancer was also observed after IUGR. This could be due to changes in mammary gland developmental pathways. We sought to characterise IUGR-induced alterations of the complex pathways of mammary development at the level of the transcriptome in a rat model of IUGR, using pathways analysis bioinformatics.</p><p>Methodology/Principal Findings</p><p>We analysed the mammary glands of Wistar rats with IUGR induced by maternal low protein (LP) diet at the beginning (d21) and the end (d28) of pubertal ductal morphogenesis. Mammary glands of the LP group were smaller in size at d28, however did not show morphologic changes. We identified multiple differentially expressed genes in the mammary gland using Agilent SurePrint arrays at d21 and d28. In silico analysis was carried out using Ingenuity Pathways Analysis. In mammary gland tissue of LP rats at d21 of life a prominent upregulation of WT1 and CDKN1A (p21) expression was observed. Differentially regulated genes were associated with the extracellular regulated kinase (ERK)-1/-2 pathway. Western Blot analysis showed reduced levels of phosphorylated ERK-1/-2 in the mammary glands of the LP group at d21. To identify possible changes in circulating steroid levels, serum LC-Tandem mass-spectrometry was performed. LP rats showed higher serum progesterone levels and an increased corticosterone/dehydrocorticosterone-ratio at d28.</p><p>Conclusions/Significance</p><p>Our data obtained from gene array analysis support the hypothesis that IUGR influences pubertal development of the rat mammary gland. We identified prominent differential regulation of genes and pathways for factors regulating cell cycle and growth. Moreover, we detected new pathways which appear to be programmed by IUGR.</p></div

Primers and probes.

<p>Primers and probes.</p

Auxologic data.

<p>Body weight (A), body length (B) and tail length (C) of the LP (white bars/squares) and NP (black bars/triangles) group during postnatal development (left), at day 1 (middle) and day 21 vs. day 28 (right). At d1 - d21 weight data was recorded from n = 40 LP and n = 47 NP animals, at d21-d28 from n = 20 LP and n = 24 NP rats. Length and tail length measurements came from n = 20 LP and n = 26 NP pups at d1 – d21, and from n = 20 LP and n = 24 NP rats at d21 – d28. LP =  low protein (white bars); NP =  normal protein (black bars); d =  day; * =  p<0.05, ** =  p<0.01; *** =  p<0.001; ns =  not significant.</p

BioVenn diagram analysis of functional gene clusters: Displayed are the results of the comparative analysis of differentially regulated genes using IPA Ingenuity software and consecutive Venn diagram transformation.

<p>The upper section lists differentially regulated genes of the generic networks mammary gland development, reactive oxygen species (ROS), insulin resistance and early mammary adenocarcinoma at day 21 and day 28 as heatmaps. The lower section displays overlapping genes of these generic networks as Venn Diagrams at day 21 (on the left) and day 28 (on the right). The quantity of regulated genes of each network cluster is indicated in the respective small Venn diagrams by number. In the upper section, common genes that are differentially regulated at both day 21 and day 28 are represented by the amount of overlap of the circles and listed in the small table next to the diagram. In contrast, the tables in the lower section of this figure display overlapping genes of three selected pathways as indicated. Red =  up-regulated gene; green =  down-regulated gene; white =  fold-change value of 0.</p

Analysis of Wilms tumor suppressor gene 1 (WT1) expression in lumbar mammary glands at day 21 and day 28.

<p>WT1 stained positive (black arrow head) in epithelial cells (A, WT1-negative cells are indicated by arrows), with a significant increase of WT1 positive cells per area at day 28 in the LP group. RT-PCR indicates a significant induction of WT1 and CDKN1a (p21) expression at day 21 (B). LP =  low protein (white bars); NP =  normal protein (black bars); d =  day; * =  p<0.05 versus NP of the same time point.</p

Serum steroid levels detected by Liquid Chromatography Tandem Mass Spectrometry [LC-Tandem MS]: Serum corticosterone [B] –to- dehydrocorticosterone [DHB] ratio (A) and serum progesterone levels (B) were determined at day 21 (LP n = 21, NP n = 22) and day 28 (LP n = 20, NP n = 24).

<p>Further data regarding LC-Tandem MS intensity profiles of (dehydro-) corticosterone and progesterone (e.g. multiple reaction monitoring, retention time etc.) and a detailed description of the method can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100504#pone.0100504.s006" target="_blank">Method S1</a>. LP =  low protein (white bars); NP =  normal protein (black bars); d =  day; * =  p<0.05, ** =  p<0.01; *** =  p<0.001; ns =  not significant.</p

IPA gene network analysis: Top differentially regulated genes at day 21 and day 28 associated with mammary gland (patho-)physiology.

<p>IPA gene network analysis: Top differentially regulated genes at day 21 and day 28 associated with mammary gland (patho-)physiology.</p

Analysis of extracellular signal-regulated kinase (ERK) -1 and -2 activities via Western blot.

<p>(A) Tissue lysates from lumbar mammary glands at day 21 and day 28 were probed with an antibody recognizing phosphorylated (activated) and total (phosphorylated and non-phosphorylated) ERK1 (also known as p44, upper band 44kDa) and ERK2 (also known as p42, lower band 42kDa). Amidoblack (Ambl) served as control. (B+C) Densitometric analysis presented as the ratio of activated ERK-1/-2 to total ERK at day 21 (B) and day 28 (C). LP =  low protein (white bars); NP =  normal protein (black bars); d =  day; * =  p<0.05, ** =  p<0.01.</p

Analysis of ductal morphogenesis.

<p>A+B) Exemplary transmitted light microscope images of lumbar mammary gland whole mount preparations at day 28 of LP (A) and NP (B). Magnification is indicated by the black bar (10 mm). The percentage of lumbar mammary fat pad occupied by ducto-alveolar structures (“area proportion” in %, C), as well as the area (mm²) occupied by ducto-alveolar structures itself (D) were examined at day 21 (LP n = 13, NP n = 18) and day 28 (LP n = 12, NP n = 10). Furthermore the rate of proliferation was determined immunohistochemically in terminal end buds (TEB) via PCNA-stain (E, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100504#pone.0100504.s001" target="_blank">Figure S1</a>), with LP d21 (n = 6), LP d28 (n = 10), and NP d21 (n = 7), NP d28 (n = 9). LP =  low protein (white bars); NP =  normal protein (black bars); d =  day; * =  p<0.05, ** =  p<0.01; *** =  p<0.001; ns =  not significant.</p