A transcriptomics model of estrogen action in the ovine fetal hypothalamus: evidence for estrogenic effects of ICI 182,780

Abstract

Estradiol plays a critical role in stimulating the fetal hypothalamus-pituitary-adrenal axis at the end of gestation. Estradiol action is mediated through nuclear and membrane receptors that can be modulated by ICI 182,780, a pure antiestrogen compound. The objective of this study was to evaluate the transcriptomic profile of estradiol and ICI 182,780, testing the hypothesis that ICI 182,780 antagonizes the action of estradiol in the fetal hypothalamus. Chronically catheterized ovine fetuses were infused for 48 h with: vehicle (Control, n = 6), 17β-estradiol 500 μg/kg/day (Estradiol, n = 4), ICI 182,780 5 μg/kg/day (ICI 5 μg, n = 4) and ICI 182,780 5 mg/kg/day (ICI 5 mg, n = 5). Fetal hypothalami were collected afterward, and gene expression was measured through microarray. Statistical analysis of transcriptomic data was performed with Bioconductor-R and Cytoscape software. Unexpectedly, 35% and 15.5% of the upregulated differentially expressed genes (DEG) by Estradiol significantly overlapped (P < 0.05) with upregulated DEG by ICI 5 mg and ICI 5 μg, respectively. For the downregulated DEG, these percentages were 29.9% and 15.5%, respectively. There was almost no overlap for DEG following opposite directions between Estradiol and ICI ICI 5 mg or ICI 5 μg. Furthermore, most of the genes in the estrogen signaling pathway - after activation of the epidermal growth factor receptor - followed the same direction in Estradiol, ICI 5 μg or ICI 5 mg compared to Control. In conclusion, estradiol and ICI 182,780 have estrogenic genomic effects in the developing brain, suggesting the possibility that the major action of estradiol on the fetal hypothalamus involves another receptor system rather than estrogen receptors.

Keywords: Brain development; estrogen signaling pathway; fetal programming; microarray.

Figure 1

Venn diagrams showing the overlap for the differentially expressed genes (DEG) in fetal hypothalami after 48 h treatment with 17β‐estradiol (500 μg/kg/day; Estradiol, n = 4), low dose of ICI 182,780 (5 μg/kg/day; ICI 5 μg, n = 4) or high dose of ICI 182,780 (5 mg/kg/day; ICI 5 mg, n = 5), compared to controls (n = 6). (A) Upregulated DEG by all the treatments. (B) Downregulated DEG by all the treatments. (C) Downregulated DEG by Estradiol and upregulated DEG by both doses of ICI 182,780. (D) Upregulated DEG by Estradiol and downregulated DEG by both doses of ICI 182,780. (*P < 0.05 by Pearson chi‐square test).

Figure 2

Merged networks of functionally related differentially expressed genes (DEG) in fetal hypothalami after 48 h treatment with 17β‐estradiol (500 μg/kg/day; Estradiol, n = 4), low dose of ICI 182,780 (5 μg/kg/day; ICI 5 μg, n = 4) or high dose of ICI 182,780 (5 mg/kg/day; ICI 5 mg, n = 5), compared to controls (n = 6). (A) Upregulated DEG by all the treatments. (B) Downregulated DEG by all the treatments. (C) Downregulated DEG by Estradiol and upregulated DEG by both doses of ICI 182,780. (D) Upregulated DEG by Estradiol and downregulated DEG by both doses of ICI 182,780. Blue nodes denote the overlapped DEG between the networks.

Figure 3

Subnetworks obtained after testing the “estrogen signaling pathway” network (KEGG pathway: map04915) in differentially expressed genes in fetal hypothalami after 48 h of treatment with (A) 500 μg/kg/day of 17β‐estradiol (n = 4); (B) 5 mg/kg/day of ICI 182,780 (n = 5); and (C) 5 μg/kg/day of ICI 182,780 (n = 4); compared to controls (n = 6). T‐score refers to the t‐statistics obtained after applying individual t‐test to each gene comparing both conditions (treatments vs. control).

Figure 4

Diagram of Estradiol action on the fetal brain. Estradiol binds estrogen receptor alpha (ESR1), inducing its dimerization and nuclear translocation, which can be blocked by ICI 182,780. However, Estradiol and ICI 182,780 bind with high affinity, a transmembrane G‐protein‐coupled receptor (GPR30) which in turn could transactivate epidermal growth factor receptor (EGFR). Green arrow: stimulation, red arrow: inhibition.