Xenoestrogen-Induced Sex Reversal of Chicken Embryonic Testes Involves Specific CpG Dinucleotide Demethylation at a DNA Hypomethylation Dip in CYP19A1/Aromatase Promoter.

Evidence is accumulating that exposure of mammalian fetuses to environmental endocrine disruptors (EEDs) affects epigenetic regulation of gene expression in germline cells. Such epigenetic changes may be heritable, conveying EED effects trans-generationally. However, mechanistic bases of such EED-induced epigenetic reprogramming largely remain unexplored. Chicken has a long history as a favored homothermic vertebrate model animal for studies in developmental biology due to its excellent accessibility to live embryos at early stages of development. In contrast to mammals whose sex is strictly determined by the XY sex chromosomes, avian phenotypic sex determined by the ZW sex chromosome system [males have homologous (ZZ) and females have heterologous (ZW) sex chromosomes] is readily reversed by fetal exposure to exogenous sex steroids or inhibitors of sex steroid synthesis during early development. Taking advantage of the exogenous estrogen-inducible gonadal feminization of genetically male embryos, in the present study we attempted to establish an in vivo model of EED-induced changes in DNA methylation in germline and somatic cells in gonads. White Leghorn ZZ-male chicken embryos were exposed to 17alpha-ethynylestradiol (EE2) during early stages of development (Day 3-11), and their gonads were collected shortly before hatching (Day 19) for morphological characterization, mRNA expression profiling by microarray and qPCR, and pyrosequencing determination of promoter DNA methylation. Exposure of ZZ-male embryos to EE2 before completion of the morphological testicular differentiation (Day 7.0) induced strong morphological gonadal feminization. This sex reversal was associated with strongly enhanced mRNA expression for CYP19A1/aromatase, which is required for gonadal ovarian differentiation, and FOXL2, a transcription factor that activates CYP19A1 promoter. Expression of ESR1 mRNA encoding estrogen receptor alpha was not affected EE2 or normal sex differentiation of gonads. A CpG dinucleotide in a methylation-dip sequence of CYP19A1 promoter (869 nucleotides upstream of the transcription initiation site) was significantly and specifically hypo-methylated in the feminized ZZ-male gonads (30-35% methylation, n=18) as well as vehicle-exposed ZW-ovaries (20-25% methylation, n=24) compared to vehicle-exposed ZZ-testes (50-55% methylation, n=20). In contrast, neither EE2-induced feminization nor normal sex differentiation affected methylation of this CpG site in the heart, which was relatively hyper-methylated (40-45%, n=8). CpG methylation of ESR1 promoter in gonads was not affected by EE2 or normal sex differentiation. Whereas ESR1 promoter was differentially methylated between MACS-purified gonadal somatic cells and SSEA1-positive germline cells of ZZ-testes (>90% purity), CYP19A1 promoter methylation was nearly between these two cell types. EE2-induced ZZ-gonad feminization resulted in strong hypomethylation of the CYP19A1 promoter CpG(-869) site in both germline and somatic cells. Concomitantly, CYP19A1 mRNA expression was strongly enhanced in both gonadal somatic stromal cells and germline cells. The highly reproducible and sequence-specific DNA hypomethylation of chicken embryonic CYP19A1 promoter induced by exposure to EE2 during early in ovo development may provide unique opportunities to study mechanisms of the epigenetic and trans-generational effects of endocrine disruptors.

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