Prenatal synthetic glucocorticoid treatment changes DNA methylation states in male organ systems: multigenerational effects

Abstract

Prenatal synthetic glucocorticoids (sGC) are administered to pregnant women at risk of delivering preterm, approximately 10% of all pregnancies. Animal studies have demonstrated that offspring exposed to elevated glucocorticoids, either by administration of sGC or as a result of maternal stress, are at increased risk of developing behavioral, endocrine, and metabolic abnormalities. DNA methylation is a covalent modification of DNA that plays a critical role in long-lasting programming of gene expression. Here we tested the hypothesis that prenatal sGC treatment has both acute and long-term effects on DNA methylation states in the fetus and offspring and that these effects extend into a subsequent generation. Pregnant guinea pigs were treated with sGC in late gestation, and methylation analysis by luminometric methylation assay was undertaken in organs from fetuses and offspring across two generations. Expression of genes that modify the epigenetic state were measured by quantitative real-time PCR. Results indicate that there are organ-specific developmental trajectories of methylation in the fetus and newborn. Furthermore, these trajectories are substantially modified by intrauterine exposure to sGC. These sGC-induced changes in DNA methylation remain into adulthood and are evident in the next generation. Furthermore, prenatal sGC exposure alters the expression of several genes encoding proteins that modulate the epigenetic state. Several of these changes are long lasting and are also present in the next generation. These data support the hypothesis that prenatal sGC exposure leads to broad changes in critical components of the epigenetic machinery and that these effects can pass to the next generation.

Fig. 1.

The effect of sGC exposure on global states of DNA methylation in the fetus. Genomic DNA methylation was determined in liver (A), adrenal glands (B), kidney (C), and placenta (D) derived from fetuses that had been exposed to sGC (1 mg kg⁻¹; n = 3; solid bars) on d 40, 41, 50, and 51 of gestation and from controls (n = 3; open bars). The level of global methylation was determined at two time points during gestation (GD52 and GD65) using LUMA. Data are presented as mean ± sem. A significant interaction between treatment and age is represented as ###, P < 0.001. A significant difference between control animals at different ages is represented as ++, P < 0.01 and +++, P < 0.001. A significant difference between control and sGC-treated groups at equivalent age is represented as *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

Fig. 2.

Levels of mRNA for genes involved in regulation of epigenetic states in the fetal kidney after sGC treatment (1 mg kg⁻¹; n = 3; solid bars) and nontreated controls (n = 3; open bars) at GD52 and GD65. Dnmt1 (A), Dnmt3a (B), Dnmt3b (C), Mbd2 (D), Mbd3 (E), Mecp2 (F), Gadd45a (G), Crebbp (H), and Tet1 mRNA (I) are shown. All mRNA expression is relative to Gapdh reference gene. Data are presented as mean ± sem. A significant interaction between treatment and age is represented as #, P < 0.05, and ###, P < 0.001. A significant difference between control animals at different ages is represented as ++, P < 0.01, and +++, P < 0.001. A significant difference between control and sGC treated groups at equivalent age is represented as *, P < 0.05, **, P < 0.01, and ***, P < 0.001.

Fig. 3.

Levels of mRNA for genes involved in regulation of epigenetic states in the placenta following sGC treatment (1 mg kg⁻¹; n = 3; solid bars) and nontreated control (n = 3; open bars) at GD52 and GD65. Dnmt1 (A), Dnmt3a (B), Dnmt3b (C), Mbd2 (D), Mbd3 (E), Mecp2 (F), Gadd45a (G), Crebbp (H), and Tet1 mRNA (I) are shown. All mRNA expression is relative to Gapdh reference gene. Data are presented as mean ± sem. A significant interaction between treatment and age is represented as #, P < 0.05, ##, P < 0.01, and ###, P < 0.001. A significant difference between control animals at different ages is represented as +, P < 0.05, ++, P < 0.01, and +++, P < 0.001. A significant difference between control and sGC-treated groups at equivalent age is represented as *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Fig. 4.

The effect of sGC exposure in the F₀ pregnancy on global DNA methylation in F₁ juvenile (n = 4) and adult (n = 4) offspring and F₂ adult offspring (n = 3). Genomic DNA methylation was determined in liver (A), adrenal glands (B), kidney (C), and placenta (D) derived from offspring (F₁ and F₂) of F₀ animals that had been exposed on d 40, 41, 50, 51, 60, and 61 of pregnancy to sGC (1 mg kg⁻¹; solid bars) or vehicle (saline; open bars). Data are presented as mean ± sem. A significant interaction is represented as ##, P < 0.01, and ###, P < 0.001. A significant difference between control and sGC-treated groups at equivalent age is represented as *, P < 0.05, **, P < 0.01, and ***, P < 0.001. A significant difference between F₁ PND10 and F₁ adult control animals is represented as +, P < 0.05, and +++, P < 0.001. A significant difference between F₁ adult and F₂ adult control animals at equivalent age is represented as §, P < 0.05, and §§§, P < 0.001.

Fig. 5.

The effect of sGC exposure in the F₀ pregnancy on mRNA expression of genes involved in regulation of epigenetic states in the kidney of F₁ juvenile (n = 4) and adult (n = 4) offspring and F₂ adult offspring (n = 3). Dnmt1 (A), Dnmt3a (B), Dnmt3b (C), Mbd2 (D), Mbd3 (E), Mecp2 (F), Gadd45a (G), Crebbp (H), and Tet1 mRNA (I) were measured in offspring (F₁ and F₂) of F₀ animals that had been exposed to sGC (1 mg kg⁻¹; solid bars) or vehicle (saline; open bars) on d 40, 41, 50, 51, 60, and 61 of pregnancy. All mRNA expression is relative to Gapdh reference gene. Data are presented as mean ± sem. A significant interaction between treatment and age/generation is represented as #, P < 0.05, ##, P < 0.01, and ###, P < 0.001. A significant difference between control and sGC-treated groups at equivalent age is represented as *, P < 0.05, **, P < 0.01, and ***, P < 0.001. A significant difference between F₁ PND10 and F₁ adult control animals is represented as +, P < 0.05, and ++, P < 0.01). A significant difference between F₁ adult and F₂ adult control animals at equivalent age is represented as §, P < 0.05.

Fig. 6.

The effect of sGC exposure in the F₀ pregnancy on mRNA expression of genes involved in regulation of epigenetic states in the cerebellum of F₁ juvenile (n = 4) and adult (n = 4) offspring and F₂ adult offspring (n = 3). Dnmt1 (A), Dnmt3a (B), Dnmt3b (C), Mbd2 (D), Mbd3 (E), Mecp2 (F), Gadd45a (G), Crebbp (H), and Tet1 mRNA (I) were measured in offspring (F₁ and F₂) of F₀ animals that had been exposed to sGC (1 mg kg⁻¹; solid bars) or vehicle (saline; open bars) on days 40, 41, 50, 51, 60, and 61 of pregnancy. All mRNA expression is relative to Gapdh reference gene. Data are presented as mean ± sem. A significant interaction between treatment and age/generation is represented as #, P < 0.05, ##, P < 0.01, and ###, P < 0.001. A significant difference between control and sGC-treated groups at equivalent age is represented as *, P < 0.05, **, P < 0.01, and ***, P < 0.001. A significant difference between F₁ PND10 and F₁ adult control animals is represented as +, P < 0.05, ++, P < 0.01, and +++, P < 0.001. A significant difference between F₁ adult and F₂ adult control animals at equivalent age is represented as §§§, P < 0.001.

Fig. 7.

Correlation analysis of global DNA methylation with mRNA expression of genes involved in epigenetic regulation in the kidney. A correlation analysis was performed between global DNA methylation and mRNA expression of genes that are implicated in the regulation of epigenetic state. The correlation analysis was determined using global methylation and mRNA expression data from the F₁ PND10 animals (n = 4), F₁ adult animals (n = 4), and F₂ adult animals (n = 3). Correlation is shown for Dnmt1 (A), Dnmt3a (B), Dnmt3b (c), Mbd2 (D), Mbd3 (E), Mecp2 (F), Gadd45a (G), Crebbp (H), and Tet1 mRNA (I). The R value and P value for each gene analyzed is presented in the figure.

Fig. 8.

Correlation analysis of global DNA methylation with mRNA expression of genes involved in epigenetic regulation in the cerebellum. A correlation analysis was performed between global DNA methylation and mRNA expression of genes that are implicated in the regulation of epigenetic state. The correlation analysis was determined using global methylation and mRNA expression data from the F₁ PND10 animals (n = 4), F₁ adult animals (n = 4), and F₂ adult animals (n = 3). Correlation is shown for Dnmt1 (A), Dnmt3a (B), Dnmt3b (C), Mbd2 (D), Mbd3 (E), Mecp2 (F), Gadd45a (G), Crebbp (H), and Tet1 mRNA (I). The R value and P value for each gene analyzed is presented in the figure.