Gene expression profile of androgen modulated genes in the murine fetal developing lung

Abstract

Background: Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood.

Methods: To build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17) and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens.

Results: Results revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment.

Conclusion: Our results show clearly that there is a real delay in lung maturation between male and female in this period, the latter pursuing already lung maturation while the proper is not yet fully engaged in the differentiation processes at GD17. In addition, this study provides a list of genes which are under the control of androgens within the lung at the moment of surge of surfactant production in murine fetal lung.

Figure 1

Overview of microarray results obtained from fetal mouse lungs. Gene expression levels were compared at both GD17 and GD18 in males versus females (17 f vs. 17 m and 18 f vs. 18 m) as well as in males versus flutamide-treated males (17 m vs. 17 flut and 18 m vs. 18 flut). The number of genes differentially expressed for each comparison appears in grey boxes. White boxes contain the number of differentially expressed genes shared by comparisons to which they are connected. Flut: flutamide treated males, m: males, f: females.

Figure 2

Microarray validation by quantitative real-time PCR (Q-PCR). Q-PCR were achieved for six genes randomly selected among those for which microarrays reveal a sexual difference and/or flutamide sensitivity: cyclin-dependant kinase 6 (Cdk6), nuclear factor I/B (Nfib), pre B-cell leukemia transcription factor 1 (Pbx1), phosphatase and tensin homolog (Pten), sirtuin 1 (Sirt1), and nuclear receptor subfamily 2, group C, member 2 (Nr2c2). Gene expression data are shown as a relative fold-change of the second experimental group over the first experimental group in each comparison. Genes showing increased expression in male (m) compared to female (f) or in male (m) compared to flutamide treated male (flut) are placed above the axis and those with decreased expression below the axis.

Figure 3

Modulation of fetal pulmonary gene expression by sex and antiandrogen treatment. Grey boxes contain the number of genes highly expressed in male fetal lungs harvested at GD17 or GD18 (17 m and 18 m) when compared with timely corresponding female fetal lungs (17 f and 18 f) or flutamide-treated male fetal lungs (17 flut and 18 flut). Numbers within white boxes represent the number of genes for which the expression is lower in male fetal lungs than in the compared group. Flut: flutamide treated males, m: males, f: females.

Figure 4

Profile of biological processes and molecular functions of genes transcriptionally modulated by sex and antiandrogen treatment. Modulated genes were analyzed on Gene Ontology website www.geneontology.org and results were reported for genes included within categories (GO:category number) related to surfactant production and regulation, pulmonary development and physiology, lipid processing, and androgen receptor signalling. Flut: flutamide treated males, m: males, f: females.

Figure 5

Genes associated to androgen receptor signalling that are modulated by sex or antiandrogen treatment. Genes for which the pulmonary expression level is modulated by sex (A) or antiandrogen treatment (B) at GD17 or GD18. The color gradient within boxes represents the relative magnitude in the differential expression level between compared groups; darker is the box, greater is the difference. Flut: flutamide treated males, m: males, f: females.