Prenatal androgens alter GABAergic drive to gonadotropin-releasing hormone neurons: implications for a common fertility disorder

Abstract

Polycystic ovary syndrome, a fertility disorder affecting approximately 7% of women, is characterized by elevated androgens, disrupted reproductive cycles, and high luteinizing hormone, the latter reflecting increased gonadotropin-releasing hormone (GnRH) release. In animal models, a similar reproductive endocrine phenotype occurs after prenatal androgen exposure. To study the effects of in utero androgen exposure directly on GnRH neurons, the central regulators of fertility, we prenatally androgenized (PNA) transgenic mice that express GFP in these cells. Pregnant females were injected with dihydrotestosterone, and their female offspring were studied as adults. PNA mice had irregular estrous cycles and elevated testosterone and luteinizing hormone levels, suggesting altered hypothalamo-pituitary-gonadal axis function. GnRH neurons receive a major input from gamma-aminobutyric acid (GABA)ergic neurons, and GABA type A receptor activation may play a role in their regulation by steroids. We tested whether PNA alters GABAergic drive to GnRH neurons by comparing frequency and size of GABAergic postsynaptic currents in GnRH neurons from PNA and control females. Both postsynaptic current frequency and size were increased in PNA mice; these effects were reversed by in vivo treatment with the androgen receptor antagonist flutamide, suggesting that androgens mediated these effects. Changes in postsynaptic current frequency and size were action potential-independent, suggesting the possibility that PNA increased connectivity between GABAergic and GnRH neurons. The ability of prenatal steroid exposure to initiate changes that alter functional inputs to GnRH neurons in adults has important implications for understanding the regulation of normal reproduction as well as the hypothalamic abnormalities of fertility disorders.

Fig. 1.

PNA disrupts estrous cyclicity in adulthood. (A) Representative estrous cycles in control (Upper), PNA, and PNA plus flutamide mice (Lower). Arrow indicates time flutamide treatment was begun. (B) Percent of days spent in each estrous cycle stage (E, estrus; D, diestrus; P, proestrus) in PNA, control, and PNA plus flutamide mice. *, P < 0.05 versus control.

Fig. 2.

PNA elevates LH and testosterone (T) levels in adult diestrus mice (n = 6 control; n = 6 PNA). *, P < 0.05 versus control.

Fig. 3.

PNA increased afferent GABAergic drive to GnRH neurons. (A) Recordings of GABAergic PSCs from a representative GnRH neuron from a control female (Top), a PNA female (Middle), and a PNA female treated in vivo with flutamide (Bottom). (B) Mean ± SEM PSC frequency in GnRH neurons from control (n = 6 cells), PNA (n = 17), and PNA plus flutamide (n = 8) adult females. *, P < 0.05 versus control (Con).

Fig. 4.

PNA increased the efficacy of GABA_A receptor activation in GnRH neurons. (A) Averaged (Left) and normalized averaged (Right) PSC traces from representative GnRH neurons from control, PNA, and PNA + flutamide females show differences in PSC amplitude and decay time, respectively, among the groups. (B) Percent change of mean values of PSC rate of rise, amplitude, and decay time in GnRH neurons in PNA and PNA + flutamide females from control values. (C) Probability distributions of PSC rate of rise (Left), amplitude (Center), and decay time (Right), created by using all PSC events from all cells in each treatment group, show among-group comparisons for these PSC properties. *, P < 0.05 versus control.

Fig. 5.

Effects of PNA on PSC size are action potential-independent. (A–C) Probability distributions of PSC rate of rise (A), amplitude (B), and decay time (C) show comparisons for these PSC properties in GnRH neurons from PNA females before and after in vitro treatment with TTX (0.5 μM, PNA + TTX). Distributions for each PSC parameter are also shown for GnRH neurons from control mice for comparison. (D) Percent change from baseline after in vitro treatment with TTX in PSC rate of rise, amplitude, and decay time in GnRH neurons from PNA females. *, P < 0.05 versus control (A–C) or from baseline (D).