Developmental Programming: Gestational Testosterone Treatment Alters Fetal Ovarian Steroidogenic Gene Expression.

Abstract

Gestational administration of testosterone increases ovarian androgen receptor protein expression in the fetus and culminates in hypersecretion of luteinizing hormone (LH), hyperestrogenism, persistent ovarian follicles, and oligo-/anovulation in adults. Since steroidogenic potential of the ovary gets established during fetal life, we hypothesized that maternal testosterone excess also alters steroidogenic enzyme expression within the fetal ovine ovary. Pregnant Suffolk ewes were treated with testosterone proprionate (T, 100 mg, twice weekly) or T + the androgen receptor antagonist, flutamide (15 mg/kg BW daily; T+F,) from days 30 to 90 of gestation. Fetal ovaries were collected on d65 (n=4, 5, and 5 for T, T+F, and control [C] groups, respectively) and d90 (n=5, 7, 4) of gestation and snap frozen. Ovarian RNA was subjected to quantitative RT-PCR to measure expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (Cyp11A1), cytochrome P450 17α-hydroxylase (Cyp17), cytochrome P450 aromatase (Cyp19), 3-β-hydroxysteroid dehydrogenase (3βHSD), 5α-reductase, androgen receptor (AR), estrogen receptors (ER) α and β, and progesterone receptor (PR). Data were analyzed by 2-way ANOVA followed by Bonferroni post-hoc statistical tests. Maternal T had no effect on AR, PR, ERα and ERβ mRNA expression but levels of ERα and AR mRNA were higher in d90 than d65 ovaries. There was no age effect on steroidogenic enzyme expression in control ovaries. Gestational T excess increased (P<0.05) Cyp19 (3.9-fold) and 5α-reductase (1.8-fold) expression in fetal d65 ovaries but decreased (P<0.05) Cyp11A1 expression (3.7 fold) in d90 ovaries. Maternal T excess had no effect on mRNA expression of other steroidogenic enzymes. Co-treatment with the androgen antagonist, flutamide, prevented the T-induced decrease in Cyp11A1 mRNA seen at d90, but not the increase in Cyp19 and 5α-reductase seen in d65 ovaries. Co-treatment with T+F also increased (P<0.05) StAR (2.0-fold) and Cyp11A1 (3.0-fold) expression in d65 ovaries, compared to C and T-treated ovaries. Our findings suggest that the effects of gestational T treatment on Cyp11A1 is mediated by androgenic actions of T and the effects on Cyp19 and 5α-reductase likely via estrogenic actions stemming from aromatization of T to estrogen and that these changes are fetal age dependent. Increased StAR and Cyp11A1 mRNA expression at d65 in the T+F ovaries, but not the T-alone ovaries, indicates that AR is primarily repressing expression of these two transcripts. Additionally, the increased potential for estrogen metabolism (i.e., increased Cyp19) in the d65 T-alone but not T+F ovaries indicates that estrogenic effects can only be manifest if the AR is blocked. Evidence for estrogenic effect is consistent with our recent finding of increased estradiol levels in the fetal blood of day 65 and 90 T-treated fetuses. These studies demonstrate that maternal T treatment alters fetal ovarian steroidogenic gene expression and implicate direct actions of estrogens in addition to androgens in the reprogramming of fetal developmental trajectory leading up to adult reproductive pathologies. Supported by NIH P01 HD44232 (VP). (platform)