Sexual Differentiation of the LH Surge Response to Estradiol in Sheep Treated Prenatally with Testosterone Plus an Antiandrogen.

Abstract

The steroid feedback mechanisms regulating GnRH secretion are sexually differentiated by testicular steroids during prenatal development. In sheep, prenatal testosterone (T) treatment of developing females results in disruption of the GnRH surge response to estradiol (E) positive feedback. Defeminization of the surge mechanism is considered to be defeminized by the estrogenic metabolites of testosterone because the surge mechanism is functional in females treated prenatally with the non-aromatizable androgen, dihydrotestosterone (DHT). However, it is difficult to confirm this premise by studying the organizing actions of prenatal estrogens because of the disruptive effects of exogenous estrogens on the maternal uterus. We developed an approach to study the consequences of excess estrogens in the developing female by co-administering T and the antiandrogen flutamide, and used this approach to test the hypothesis that prenatal estrogens defeminize the GnRH surge response to E. We predicted that the surge would be defeminized by prenatal T and T+flutamide treatments, but not by prenatal DHT. Lambs born to untreated females or females treated with T, DHT, or T+flutamide from Days 30-90 of gestation were neonatally gonadectomized, and steroid levels were standardized with a chronic E implant (30-mm sc). At 8 months of age, the chronic implants were removed and females and T+flutamide males were treated with new implants to maintain luteal phase concentrations of progesterone (P) and E for 9 days. After 9 days, P implants were removed and additional E implants (4 x 30-mm) were inserted to maintain follicular phase concentrations of E. Plasma concentrations of LH were measured in venous blood samples collected every 2 hours from -4 to 72 h from E administration. An LH surge was produced in all untreated, control (C, n=6) and DHT-treated females (n=7), 1 of 9 T-treated females, 4 of 6 T+flutamide-treated females, and 3 of 7 T+flutamide males. The onset of the surge relative to E administration did not differ between C (18.7 ± 1.6 h) and DHT (16.9 ± 0.9 h) females, but it was delayed (p < 0.02) in the T+flutamide groups (females: 40.5 ± 6.9 h; males: 66.7 ± 1.8 h). The peak amplitude of the LH surge was not different between C (175.9 ± 13.7 ng/ml) and DHT (209.6 ± 18.0 ng/ml) females, but was lower in T+flutamide females (84.2 ± 15.3 ng/ml) and males (48.0 ± 14.9 ng/ml). The one LH surge produced by a T-treated female had an onset of 44 h and a peak amplitude of 30.3 ng/ml. The LH surge response in some of the T+flutamide females and males contradicts our prediction, and fails to support our hypothesis that prenatal estrogens, made available to the developing fetus by the aromatization of T, defeminize the positive feedback actions of estradiol. However, the LH surges in the T+flutamide animals were abnormal, particularly in the males. This suggests that endogenous androgens not blocked by flutamide treatment or having organizational actions outside of the treatment period contributed to differentiation of the surge mechanism. Alternatively, individual differences in neural aromatase activity could result in different levels of estradiol exposure during the treatment period. We conclude that prenatal T+flutamide treatment defeminizes the neural circuitry underlying the GnRH surge mechanism, but that both androgens and estrogens may both be necessary for complete ablation of the surge response. Supported by P01 HD-44232.