Sheep exposed to testosterone during a critical period from gestational day (GD) 30 to GD 90 develop masculine genitals and an enlarged male-typical ovine sexually dimorphic nucleus of the preoptic area (oSDN). The present study tested the hypothesis that separate critical periods exist for masculinization of these two anatomical end points. Pregnant ewes were treated with testosterone propionate (TP) either from GD 30 to GD 60 (early TP) or GD 60 to GD 90 (late TP). Control (C) pregnant ewes were treated with corn oil. Fetuses were delivered at GD 135 and the volume of the oSDN was measured. Early TP females possessed a penis and a scrotum devoid of testes, whereas late TP and C females had normal female genitals. Neither period of TP exposure grossly affected the genitals of male fetuses. Despite masculinized genitals, the mean volume of the oSDN in early TP females (0.32 ± 0.06 mm³) was not different from C females (0.24 ± 0.02 mm³) but was significantly enlarged in late TP females (0.49 ± 0.04 mm³; P < 0.05 vs. C) when the genitals appeared normal. In contrast, the volume of the oSDN in late TP males (0.51 ± 0.02 mm³) was not different from C males (0.51 ± 0.04 mm³) but was significantly smaller in the early TP males (0.35 ± 0.04 mm³; P < 0.05 vs. C). These results demonstrate that the prenatal critical period for androgen-dependent differentiation of the oSDN occurs later than, and can be separated temporally from, the period for development of masculine genitals.

The ovine sexually dimorphic nucleus (oSDN) is 2 times larger in rams than in ewes. Sexual differentiation of the oSDN is produced by testosterone exposure during the critical period occurring between gestational day (GD)60 and GD90 (term, 147 d). We tested the hypothesis that testosterone acts through the androgen receptor to control development of the male-typical oSDN. In experiment 1, pregnant ewes received injections of vehicle, androgen receptor antagonist flutamide, or nonaromatizable androgen dihydrotestosterone (DHT) propionate during the critical period. Fetuses were delivered at GD135. Both antagonist and agonist treatments significantly reduced mean oSDN volume in males but had no effects in females. Experiment 2, we analyzed the effect of treatments on the fetal hypothalamic-pituitary-gonadal axis to determine whether compensatory changes in hormone secretion occurred that could explain the effect of DHT. Pregnant ewes were injected with vehicle, flutamide, or DHT propionate from GD60 to GD84, and fetuses were delivered on GD85. Flutamide significantly increased LH and testosterone in males, whereas DHT significantly decreased both hormones. In females, LH was unaffected by flutamide but significantly reduced by DHT exposure. DHT significantly decreased pituitary gonadotropin and hypothalamic kisspeptin mRNA expression in males and females. These results suggest that androgen receptor mediates the effect of testosterone on oSDN masculinization, because this process was blocked by the androgen receptor antagonist flutamide in eugonadal males. In contrast, the reduction of oSDN volume observed after DHT exposure appears to be mediated by a negative feedback mechanism exerted on the hypothalamus to reduce LH and testosterone secretion. The reduced androgen exposure most likely accounted for the decreased oSDN volume. We conclude that, during the critical period, the male reproductive axis in long gestation species, such as sheep, is sufficiently developed to react to perturbations in serum androgens and mitigate disruptions in brain masculinization.

Testosterone plays an essential role in sexual differentiation of the male sheep brain. The ovine sexually dimorphic nucleus (oSDN), is 2 to 3 times larger in males than in females, and this sex difference is under the control of testosterone. The effect of testosterone on oSDN volume may result from enhanced expansion of soma areas and/or dendritic fields. To test this hypothesis, cells derived from the hypothalamus-preoptic area (HPOA) and cerebral cortex (CTX) of lamb fetuses were grown in primary culture to examine the direct morphological effects of testosterone on these cellular components. We found that within two days of plating, neurons derived from both the HPOA and CTX extend neuritic processes and express androgen receptors and aromatase immunoreactivity. Both treated and control neurites continue to grow and branch with increasing time in culture. Treatment with testosterone (10 nM) for 3 days significantly (P < 0.05) increased both total neurite outgrowth (35%) and soma size (8%) in the HPOA and outgrowth (21%) and number of branch points (33%) in the CTX. These findings indicate that testosterone-induced somal enlargement and neurite outgrowth in fetal lamb neurons may contribute to the development of a fully masculine sheep brain.

Prenatal androgens are largely responsible for growth and differentiation of the genital tract and testis and for organization of the control mechanisms regulating male reproductive physiology and behavior. The aim of the present study was to evaluate the impact of inappropriate exposure to excess testosterone (T) during the first trimester of fetal development on the reproductive function, sexual behavior, and fertility potential of rams. We found that biweekly maternal T propionate (100 mg) treatment administered from Day 30-58 of gestation significantly decreased (P < 0.05) postpubertal scrotal circumference and sperm concentration. Prenatal T exposure did not alter ejaculate volume, sperm motility and morphology or testis morphology. There was, however, a trend for more T-exposed rams than controls to be classified as unsatisfactory potential breeders during breeding soundness examinations. Postnatal serum T concentrations were not affected by prenatal T exposure, nor was the expression of key testicular genes essential for spermatogenesis and steroidogenesis. Basal serum LH did not differ between treatment groups, nor did pituitary responsiveness to GnRH. T-exposed rams, like control males, exhibited vigorous libido and were sexually attracted to estrous females. In summary, these results suggest that exposure to exogenous T during the first trimester of gestation can negatively impact spermatogenesis and compromise the reproductive fitness of rams.

Gonadal steroid hormones play important roles during critical periods of development to organize brain structures that control sexually dimorphic neuroendocrine responses and behaviors. Specific receptors for androgens and estrogens must be expressed at appropriate times during development in order to mediate these processes. The present study was performed to test for sex differences in the relative expression of estrogen receptor-α (ERα) and androgen receptor (AR) mRNA during the window of time in gestation that is critical for behavioral masculinization and differentiation of the ovine sexually dimorphic nucleus (oSDN) in the sheep. In addition, we examined whether ERα and AR mRNA expression is localized within the nascent oSDN and could be involved in its development. Using quantitative RT-PCR, we found that females expressed more ERα mRNA than males in medial preoptic area and medial basal hypothalamus during the mid-gestational critical period for brain sexual differentiation. No sex differences were found for AR mRNA in any tissue examined or for ERα in amygdala and frontal cortex. Using radioactive in situ hybridization, we found that the distributions of ERα and AR mRNA overlapped with aromatase mRNA, which delineates the boundaries of the developing oSDN and identifies this nucleus as a target for both androgens and estrogens These data demonstrate that the transcriptional machinery for synthesizing gonadal steroid receptors is functional in the fetal lamb brain during the critical period for sexual differentiation and suggest possible mechanisms for establishing dimorphisms controlled by gonadal steroids may exist at the level of steroid hormone receptor expression.