Prenatal Exposure to a Androgen Agonist and Antagonist Alters the Ovine Sexually Dimorphic Nucleus in Male But Not Female Lamb Fetuses

The ovine sexually dimorphic nucleus (oSDN), located in the medial preoptic area, is approximately two times larger in rams than in ewes. Differentiation of the oSDN occurs during gestation days (GD) 60 to 90 and the volume of the oSDN can be enlarged by exogenous androgen exposure of female sheep fetuses. This study was designed to test the hypothesis that androgen, acting through the androgen receptor (AR) is largely responsible for gender specific brain differentiation. In experiment 1, pregnant ewes received injections of the non-aromatizable androgen dihydrotestosterone propionate (DHT), flutamide (androgen receptor antagonist) or vehicle during the critical period. Fetuses were delivered at GD 135. Treatments reduced (P<0.05) mean oSDN volume in males but not in females. In experiment 2, to test whether androgen may orchestrate brain masculinization indirectly, we evaluated the effects of hormone treatments on the fetal hypothalamic-pituitary-gonadal (HPG) axis. Pregnant ewes were injected with DHT, flutamide or vehicle from GD 60 to 84 and fetuses were delivered on GD 85. Gene expression data and hormone concentrations were analyzed by two-way ANOVA, with posthoc comparisons made using Fisher’s (LSD) test. Statistical significance for all analyses was defined as P<0.05. Dihydrotestosterone propionate significantly decreased fetal luteinizing hormone (LH) and testosterone, while flutamide significantly increased both hormones in male fetuses. In female fetuses, LH was significantly reduced by DHT exposure but unaffected by flutamide. Dihydrotestosterone propionate significantly decreased both pituitary gonadotropin and hypothalamic kisspeptin expression in males and females. Neuropeptide-Y expression was not altered by treatment but was expressed at a higher level in males. Our results demonstrate a paradoxical effect of exogenous androgen exposure in that oSDN volume was reduced in male fetuses but relatively unchanged in females. This is likely due to the suppression of anterior pituitary LH secretion resulting in reduced testicular testosterone secretion in DHT-treated males. These results demonstrate that AR mediates much or all of the masculinization of the oSDN in males and suggests the effects of DHT on oSDN development are secondary to negative feedback on the fetal HPG axis which may be mediated, in part, by hypothalamic kisspeptin. Collectively, our results support the concept that the HPG axis in the male fetus responds in the expected negative feedback manner to exogenous androgens and that tonic negative feedback by endogenous androgens is disrupted by flutamide. We conclude that, during the critical period of brain sexual differentiation, the fetal reproductive axis in long gestation species, such as sheep, is sufficiently developed to react to perturbations in serum androgen levels and mitigate disruptions in brain masculinization.