Developmental programming: gestational bisphenol-A treatment alters trajectory of fetal ovarian gene expression

Abstract

Bisphenol-A (BPA), a ubiquitous environmental endocrine disrupting chemical, is a component of polycarbonate plastic and epoxy resins. Because of its estrogenic properties, there is increasing concern relative to risks from exposures during critical periods of early organ differentiation. Prenatal BPA treatment in sheep results in low birth weight, hypergonadotropism, and ovarian cycle disruptions. This study tested the hypothesis that gestational exposure to bisphenol A, at an environmentally relevant dose, induces early perturbations in the ovarian transcriptome (mRNA and microRNA). Pregnant Suffolk ewes were treated with bisphenol A (0.5 mg/kg, sc, daily, produced ∼2.6 ng/mL of unconjugated BPA in umbilical arterial samples of BPA treated fetuses approaching median levels of BPA measured in maternal circulation) from days 30 to 90 of gestation. Expression of steroidogenic enzymes, steroid/gonadotropin receptors, key ovarian regulators, and microRNA biogenesis components were measured by RT-PCR using RNA derived from fetal ovaries collected on gestational days 65 and 90. An age-dependent effect was evident in most steroidogenic enzymes, steroid receptors, and key ovarian regulators. Prenatal BPA increased Cyp19 and 5α-reductase expression in day 65, but not day 90, ovaries. Fetal ovarian microRNA expression was altered by prenatal BPA with 45 down-regulated (>1.5-fold) at day 65 and 11 down-regulated at day 90 of gestation. These included microRNAs targeting Sry-related high-mobility-group box (SOX) family genes, kit ligand, and insulin-related genes. The results of this study demonstrate that exposure to BPA at an environmentally relevant dose alters fetal ovarian steroidogenic gene and microRNA expression of relevance to gonadal differentiation, folliculogenesis, and insulin homeostasis.

Figure 1.

Free-BPA concentrations (nanograms per milliliter) in umbilical arterial plasma of female fetuses at gestational day 90. Controls (C; n = 3) are shown as open bars and BPA treated (n = 4) as closed bars. Asterisk above mean ± SEM denotes significant (P < .01) difference between treatment groups.

Figure 2.

Age-dependent gene expression changes in fetal ovarian steroidogenic enzymes (top 3 panels) between day 65 (left graph) and day 90 (right graph) of gestation in control (C; open bars) and BPA-treated (closed bars) females. Results for 3β1HSD, 3β2HSD, and 17βHSD of control and BPA-treated females were combined for testing age effects when there was no treatment effect within age. Treatment effects were observed only with Cyp19 and 5α-reductase expression (2 bottom panels). Asterisks represent statistical differences between ages (P < .005) or treatment groups. Control (n = 4 and 5, respectively, at day 65 and day 90) and prenatal BPA-treated (n = 5, both ages) groups.

Figure 3.

Age-dependent changes in fetal ovarian steroid receptors, growth factors, and insulin-related gene expression between day 65 (left graph) and day 90 (right graph) of gestation in control (open bars) and BPA-treated (closed bars) females. Results of control and BPA-treated females were combined for testing age effects when there was no treatment effect within age. Asterisks represent statistical differences between ages (P < .005). Control (n = 4 and 5, respectively, at day 65 and day 90) and prenatal BPA-treated (n = 5, both ages) groups.

Figure 4.

Impact of gestational BPA treatment on the expression of the miRNA processing enzymes Ago2, Dicer, DGCR8, Drosha, and Ran GTPase from fetal day 65 (left panels) and day 90 (right panels) control (C; open bars) and BPA-treated (closed bars) females. Results are expressed as mean ± SEM. Control (n = 4 and 5, respectively, at day 65 and day 90) and prenatal BPA-treated (n = 5, both ages) groups.