Progesterone treatment inhibits and dihydrotestosterone (DHT) treatment potentiates voltage-gated calcium currents in gonadotropin-releasing hormone (GnRH) neurons

Abstract

GnRH neurons are central regulators of fertility, and their activity is modulated by steroid feedback. In normal females, GnRH secretion is regulated by estradiol and progesterone (P). Excess androgens present in hyperandrogenemic fertility disorders may disrupt communication of negative feedback signals from P and/or independently stimulate GnRH release. Voltage-gated calcium channels (VGCCs) are important in regulating excitability and hormone release. Estradiol alters VGCCs in a time-of-day-dependent manner. To further elucidate ovarian steroid modulation of GnRH neuron VGCCs, we studied the effects of dihydrotestosterone (DHT) and P. Adult mice were ovariectomized (OVX) or OVX and treated with implants containing DHT (OVXD), estradiol (OVXE), estradiol and DHT (OVXED), estradiol and P (OVXEP), or estradiol, DHT, and P (OVXEDP). Macroscopic calcium current (I(Ca)) was recorded in the morning or afternoon 8-12 d after surgery using whole-cell voltage-clamp. I(Ca) was increased in afternoon vs. morning in GnRH neurons from OVXE mice but this increase was abolished in cells from OVXEP mice. I(Ca) in cells from OVXD mice was increased regardless of time of day; there was no additional effect in OVXED mice. P reduced N-type and DHT potentiated N- and R-type VGCCs; P blocked the DHT potentiation of N-type-mediated current. These data suggest P and DHT have opposing actions on VGCCs in GnRH neurons, but in the presence of both steroids, P dominates. VGCCs are targets of ovarian steroid feedback modulation of GnRH neuron activity and, more specifically, a potential mechanism whereby androgens could activate GnRH neuronal function.

Figure 1

P treatment inhibits the diurnal estradiol-mediated increase in I_Ca in GnRH neurons. A, Representative recordings showing activation of I_Ca in GnRH neurons from OVXE (left) and OVXEP (right) mice in the morning (top) and afternoon (bottom). For clarity, only four voltage steps during the test pulse (−80, −10, 0, and 10 mV) are shown (bottom left). B and C, Average I-V curves of peak current from GnRH neurons in the morning (B) and afternoon (C). *, P < 0.05, OVXE vs. OVXEP, and OVX vs. OVXE. D and E, Activation (D) and steady-state inactivation (E) curves in the morning and afternoon. F and G, Rise and decay time.

Figure 2

P treatment inhibits specifically N-type currents. A–D, Representative I_Ca of each subtype of HVA in response to a step from −90 to +10 mV (top) and average I-V plot for that subtype (below). The notch on the rising phase of the current in B and C is due to pressure artifact from application of SNX-482; amplitude is not affected by this artifact, and this cell was most representative of mean current amplitude and density. *, P < 0.05 SST; #, P < 0.01 peak OVXEP vs. OVXE.

Figure 3

DHT treatment increases I_Ca in GnRH neurons. A, Representative recordings showing activation of I_Ca in GnRH neurons from OVX (left), OVXD (middle), and OVXED (right) mice. For clarity, only four voltage steps during the test pulse (−80, −10, 0, and 10 mV) are shown (bottom left). B and C, Average I-V curves of peak current from GnRH neurons in the morning (B) and afternoon; data from OVXE mice are repeated for comparison (C). D and E, Activation (D) and steady-state inactivation (E) curves. F and G, Rise and decay time. *, P < 0.05, OVX vs. OVXD, and OVX vs. OVXED).

Figure 4

DHT treatment increases specifically N-type and R-type currents. A–D, Representative I_Ca of each subtype of HVA in response to a step from −90 to +10 mV (top) and average I-V plot for that subtype (below). *, P < 0.05 SST; #, P < 0.05 peak in C; #, P < 0.01 peak in D, OVX vs. OVXD.

Figure 5

Cotreatment with P blocks the DHT-induced augmentation of I_Ca in GnRH neurons. A (top), Representative traces showing activation of I_Ca in GnRH neurons from OVXD (left) and OVXEDP (right) mice. For clarity, only four voltage steps during the test pulse (−80, −10, 0, and 10 mV) are shown; bottom, average I-V plot. B, Representative N-type current in response to a step from −90 to +10 mV (top) and average I-V plot (bottom). Data from OVXD and OVXED mice are repeated for comparison. *, P < 0.05 SST; #, P < 0.01 peak, OVXEP vs. OVXE.

Figure 6

DHT treatment does not alter LVA-mediated current. A, Representative average of 50 repeats of a voltage protocol (bottom) to reveal LVA current (top trace) and its blockade by Ni²⁺ (middle trace); a cell without LVA is also shown (bottom trace). B and C, Percentage of GnRH neurons with LVA (B) and the peak current amplitude of LVA (C).