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. 2010 Aug 25;151(11):5349–5358. doi: 10.1210/en.2010-0385

Progesterone Treatment Inhibits and Dihydrotestosterone (DHT) Treatment Potentiates Voltage-Gated Calcium Currents in Gonadotropin-Releasing Hormone (GnRH) Neurons

Jianli Sun 1, Suzanne M Moenter 1
PMCID: PMC2954728  PMID: 20739401

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 (ICa) was recorded in the morning or afternoon 8–12 d after surgery using whole-cell voltage-clamp. ICa was increased in afternoon vs. morning in GnRH neurons from OVXE mice but this increase was abolished in cells from OVXEP mice. ICa 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.

Circulating steroid hormones regulate voltage-gated calcium currents in GnRH neurons as a component of the mechanism for steroid feedback.

A pulsatile GnRH signal is required for secretion of the pituitary gonadotropins LH and FSH (1), which drive steroidogenesis and follicular development during the female reproductive cycle (2,3). Variations in GnRH pulse frequency during the cycle are critical for the differential synthesis and release of LH and FSH; low-frequency pulses favor FSH, and high frequencies favor LH (1,4,5). Steroid feedback regulates GnRH pulse frequency. During the luteal phase, progesterone (P)-mediated negative feedback reduces GnRH pulse frequency (6,7,8), favoring FSH synthesis; inhibin from the corpus luteum blocks FSH release at this time (9,10). After the demise of the corpus luteum, low-frequency GnRH release continues, allowing preferential release of FSH in the early follicular phase that is critical for follicular maturation.

In some hyperandrogenemic fertility disorders, including the common disorder polycystic ovary syndrome (PCOS), GnRH pulse frequency remains persistently high, impairing the preferential release of FSH and thus follicular maturation (11,12,13,14,15). Evidence suggests that the high levels of different androgens characteristic of this disorder reduce sensitivity of the hypothalamic-pituitary axis to P-mediated negative feedback (16,17).

The underlying neurobiological mechanisms for these steroid effects cannot be approached in patients. However, mice appear to be a good model with regard to steroid feedback effects. In adult female mice treated with P, GnRH neuron firing activity is suppressed (18). Addition of dihydrotestosterone (DHT) at a dose that is incapable of restoring seminal vesicle mass in castrated male mice (i.e. below normal male levels) (19) countered these effects of P. In the absence of P, DHT treatment increased GnRH neuron activity and LH release; this is important with regard to the typical steroid milieu in women with hyperandrogenemic disorders because P is rarely elevated due to oligoanovulation. In mechanistic studies, these same animal model steroid treatments had similar effects on GABAergic transmission to GnRH neurons, which can be excitatory to these cells (20,21,22), implying engagement of steroid-sensitive afferents in the response to these steroids. The effects of P or an androgen on intrinsic GnRH neuronal properties, however, are unknown.

Voltage-gated calcium channels (VGCCs) mediate Ca2+ influx, thereby regulating Ca2+-dependent cellular processes such as contraction, secretion, excitability, and gene expression (23,24,25,26). VGCCs are classified into low-voltage-activated (LVA) T-type channels and high-voltage-activated (HVA) L-, N-, P/Q-, and R-type channels. GnRH neurons express all five types of VGCCs (27,28,29). VGCCs in GnRH neurons are modified by estradiol feedback, and some of these changes are further dependent on time of day (29,30). In other systems, testosterone, DHT or P treatment can modulate whole-cell calcium currents (31,32,33,34,35). Whether or not GnRH neuron calcium currents are also altered by P and DHT treatment is not known.

To better understand the effects of steroid milieu on GnRH release, we studied how P and DHT treatment modulate HVA VGCCs in GnRH neurons using the whole-cell patch-clamp technique. The data suggest specific subtypes of these channels are targets of differential modulation by P and DHT treatment and are thus poised to be a contributing mechanism to the regulation of GnRH neuronal activity by these steroids.

Materials and Methods

Animals

Adult female GnRH-green fluorescent protein mice (36) (2–3 months) were ovariectomized (OVX) under isoflurane (Abbott Laboratories, North Chicago, IL) anesthesia to remove ovarian steroid feedback and received steroid implants as described previously (37). Briefly, some OVX mice received a SILASTIC brand capsule (Dow Corning, Midland, MI) containing 0.625 μg estradiol (OVXE) or 400 μg of the nonaromatizable androgen DHT (OVXD) or both (OVXED) in sesame oil; other OVX mice received 0.625 μg estradiol capsule and 2.5 mg P time-release pellet (Innovative Research of America, Sarasota, FL; OVXEP) or with DHT (OVXEDP). Postoperative analgesia was provided by a long-acting local anesthetic delivered to the surgical sites (0.25% bupivicaine; 7.5 μl/site; Abbott Laboratories). The levels of estradiol and P produced by these implants are physiological (14,38); the DHT implant produces an elevation above female androgen levels but below an effective male dose (19). All hormones were administered in vivo and were not present in any recording solutions. Recordings were made between 8 and 12 d after surgery and steroid replacement. No difference was noted in any parameter as a function of day after surgery, an observation that corresponds well with our previous experience with these models examining GABAergic transmission (37) and GnRH neuron firing rate (18). The treatment duration is longer than the P elevation of the mouse estrous cycle. It was chosen because it is similar to the duration of the P rise during pseudopregnancy in rodents (39) and is also similar to the luteal-phase rise in P that occurs in species that do not exhibit the abbreviated reproductive cycle of small rodents, facilitating comparisons with other species. All procedures were approved by the University of Virginia Animal Care and Use Committee.

Brain slice preparation

Brain slices were prepared as previously described (40,41). Briefly, mice were euthanized at times that corresponded to negative feedback (0900–1030 h, referred to as morning) or surge peak (positive feedback, 1430–1500 h, referred to as afternoon) in mice treated only with estradiol for shorter durations for the purpose of inducing daily surges, which persist for about 5 d after OVXE treatment (42). The brain was rapidly removed and placed in ice-cold high-sucrose saline solution containing the following (in mm): 250 sucrose, 3.5 KCl, 26 NaHCO3, 10 d-glucose, 1.25 Na2HPO4, 1.2 MgSO4, and 2.5 MgCl2. Coronal (300 μm) slices were cut with a Vibratome 3000 (Technical Products International, Inc., St. Louis, MO). Slices were incubated for 30 min at 30–32 C in 50% high-sucrose saline and 50% artificial cerebrospinal fluid (ACSF) solution, containing the following (in mm): 135 NaCl, 3.5 KCl, 26 NaHCO3, 10 d-glucose, 1.25 Na2HPO4, 1.2 MgSO4, and 2.5 CaCl2 (pH 7.4). Slices were then transferred to 100% ACSF solution at room temperature (∼21–23 C) for 0.5–2.5 h. For recording, slices were placed in a recording chamber on the stage of an Olympus BX51WI upright fluorescent microscope and continuously superfused at 5–6 ml/min with oxygenated ACSF at room temperature. Slices were stabilized in the chamber for at least 5 min before recording.

Electrophysiological recording

Green fluorescent protein-GnRH neurons in the preoptic area were identified by brief illumination at 470 nm. Macroscopic Ca2+ currents from GnRH neurons were recorded using the whole-cell configuration of the patch-clamp technique. Patch pipettes (2.5–3.5 mΩ) were drawn from borosilicate glass capillaries (1.65 mm outer diameter, 1.12 mm inner diameter; World Precision Instruments, Sarasota, FL) using a Sutter P97 pipette puller (Sutter Instrument Co., Novato, CA). Electrode capacitance was electronically compensated. Liquid junction potential (<−4 mV) was not corrected (43). Currents were recorded with an Axopatch-700B amplifier (Molecular Devices, Sunnyvale, CA) and filtered at 10 kHz. Voltage command pulses were generated using pCLAMP9.2 software (Molecular Devices). Neuron membrane potential was held at −60 mV between protocols during voltage-clamp recordings. During whole-cell recording, input resistance (Rin), series resistance (Rs), and membrane capacitance (Cm) were continually monitored. Only recordings with Rin higher than 500 mΩ, Rs lower than 20 mΩ, and Cm higher than 10 pF and holding current between 0 and −50 pA were included for analysis. There were no differences among groups in any passive recording properties or series resistance attributable to steroid treatment or time of day. Cells with bad clamping (identified as sluggish, incomplete current response to pulse protocol and/or 10–90% rise time >2.5 msec) were discarded. Recordings were performed from 1–3 h after preparation of brain slices was complete. No more than four cells per animal were recorded. Recorded cells were mapped to an atlas (44) to determine whether any trends based upon anatomical location emerged; no such trends were apparent in these data sets (not shown).

Drugs and solutions

All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless noted. For voltage-clamp recording of calcium current (ICa), the bath solution consisted of (in mm): 120 NaCl, 10 glucose, 26 NaHCO3, 1.25 Na2HPO4, 1.2 MgSO4, 2.5 CaCl2, 5 4-aminopyridine, 5 CsCl, 10 tetraethylammonium chloride, and 0.0005 tetrodotoxin (pH 7.4) (gassed with 95% O2 and 5% CO2). The pipette solution contained (in mm): 120 Cs-gluconate, 10 HEPES, 10 EGTA, 0.5 CaCl2, 4 Mg-ATP, 0.4 NaGTP, and 20 tetraethylammonium chloride (pH 7.3) (titrated with CsOH, 310 mOsm). Nitrendipine (50 μm), agatoxin IVA (200 nm), and conotoxin GVIA (1 μm) were bath applied; SNX-482 (1 μm) was locally applied by pressure micropipette. Pressure artifact from this local application sometimes delayed the peak of the current but had no effect on amplitude; rise time is thus compared only for total ICa because there is no local drug application during those measurements.

Voltage-clamp protocols

All currents were corrected for leak and capacitive currents online by a P/−6 protocol. To generate Ca2+ channel current-voltage (I-V) activation curves, currents were elicited by a voltage protocol of a 250-msec prepulse at −120 mV to remove inactivation, followed by current measurement at test potentials (250 msec) from −80 mV to +60 mV at 10-mV increments. To determine the steady-state inactivation of ICa, the membrane potential was initially hyperpolarized to −100 mV for 500 msec to remove inactivation, followed by a 1-sec prepulse of −100 to 10 mV in 10-mV increments, then a test pulse of 10 mV for 500 msec; current was quantified during the test pulse. To isolate LVA currents by selective activation, a 1-sec prepulse at −100 mV was followed by a 250-msec test pulse at −50 mV. The voltage protocol for activation of different subtypes of HVA VGCCs is a 250-msec prepulse at −90 mV, followed by current measurement at test potentials (250 msec) from −80 mV to +60 mV at 10-mV increments. Cocktails of specific inhibitors were used to isolate specific HVA subtypes. To isolate L-type channels, agatoxin IVA, conotoxin GVIA, and SNX-482 were used; to isolate N-type channels, nitrendipine, agatoxin IVA, and SNX-482 were used; to isolate P/Q-type channels, nitrendipine, agatoxin IVA, and SNX-482 were used; and to isolate R-type channels, nitrendipine, agatoxin IVA, and conotoxin GVIA were used.

Analysis

The peak current amplitude and sustained current 200 msec after the beginning of test potential were calculated. Current density was calculated by dividing the peak or sustained current amplitude by Cm. Current waveforms were fitted with the Clampfit program (Molecular Devices) or GraphPad Prism program (GraphPad Software, Inc., La Jolla, CA). The voltage dependencies of activation and steady-state inactivation were described with a single Boltzmann distribution: I (V) = Imax/(1 + exp[(V1/2 − V)/k]), where Imax is the maximal current elicited, V1/2 is the half-maximal voltage, and k is the voltage dependence (slope) of the distribution. For all I-V curves and steady-state inactivation curves, fitted values are reported with 95% linear confidence limits. To isolate different HVA subtypes, slices were incubated with other subtype channel blockers for 15 min in the recording chamber before current was recorded. To isolate LVA currents, the protocol was repeated 50 times at 30-sec intervals and averaged to reduce noise. Parametric or nonparametric analyses were performed in Prism as dictated by data distribution. Statistical comparisons of in vivo treatments were done with ANOVA followed by Bonferroni post hoc test. Statistical significance was set at P < 0.05. Data are shown as mean ± sem.

Results

P treatment inhibits ICa in GnRH neurons

To study the effect of P on ICa, we compared the current among GnRH neurons from OVX, OVXE, and OVXEP mice. Because a shorter duration of in vivo estradiol treatment had a time-of-day-dependent effect on GnRH neuron ICa (29), cells were recorded at two times of day, morning and afternoon, to determine whether diurnal changes persist with longer estradiol treatment and in the presence of other gonadal steroids. Representative traces at selected potentials are shown from steroid-treated mice in Fig. 1A; I-V relationships for all groups are shown in Fig. 1, B and C. ICa in GnRH neurons was activated at potentials positive to −40 mV. In OVXE mice, ICa exhibited diurnal changes consistent with previous work examining these currents 2–4 d after estradiol treatment. Specifically, on d 8–12 after estradiol treatment, ICa were not different from those in OVX mice in the morning (n = 28 cells from eight OVX mice; n = 20 cells from five OVXE mice) but were increased in the afternoon (n = 30 cells from eight OVX mice; n = 23 cells from six OVXE mice; Fig. 1, A–C; P < 0.05, OVXE vs. OVX, and OVXEP vs. OVXE). In contrast, in OVXEP mice, there was no difference between ICa in the morning and afternoon (n = 15 cells from four mice). Rather, all measures were similar to morning values from OVXE mice and all values from OVX mice. There were no differences in rise or decay time, activation or steady-state inactivation among OVX, OVXE, and OVXEP mice at different times of day (Fig. 1, D–G; P > 0.6). The membrane potential at which the current was half-activated (V1/2act) was −6.3 ± 0.6 mV in OVX mice (n = 30 cells from eight mice), −6.3 ± 0.5 mV in OVXE mice (n = 43 cells from 11 mice), and −6.5 ± 0.8 mV in OVXEP mice (n = 15 cells from four mice; P > 0.3). The membrane potential at which the current was half-inactivated (V1/2inact) was −26.4 ± 1.0 mV in OVX mice (n = 30), −26.12 ± 1.2 mV in OVXE mice (n = 34), and −26.7 ± 2.1 mV in OVXEP mice (n = 15; P > 0.4). These data suggest the inhibitory action of P on GnRH neuron activity is in part due to its ability to block the estradiol-induced afternoon diurnal increase in ICa without altering current kinetics.

Figure 1.

Figure 1

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P treatment inhibits the diurnal estradiol-mediated increase in ICa in GnRH neurons. A, Representative recordings showing activation of ICa 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.

P treatment inhibits N-type HVA ICa

Next, we examined which subtype of HVA-mediated currents are affected by P in the afternoon. Different subtypes of HVA-mediated current were isolated with combinations of specific HVA blockers as indicated in the methods. Compared with GnRH neurons from OVXE mice, P decreased the amplitude and density of N-type currents in OVXEP mice [Fig. 2C, membrane potentials from −20 to +50 mV; n = 8 cells from three OVXE mice; n = 11 cells from three OVXEP mice; peak P < 0.01; sustained current (SST) P < 0.05] ICa subtype. The I-V relation, activation, and inactivation curves did not change (data not shown), consistent with above results. There was no difference in L-type (Fig. 2A; n = 8 OVXE cells from three mice; n = 8 OVXEP cells from three mice; P > 0.8), P/Q-type (Fig. 2B; n = 9 OVXE cells from three mice; n = 7 OVXEP cells from three mice; P > 0.6), or R-type (Fig. 2D; n = 9 OVXE cells from three mice; n = 8 OVXD cells from three mice; P > 1.1) HVA-mediated currents between OVXE and OVXEP mice.

Figure 2.

Figure 2

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P treatment inhibits specifically N-type currents. A–D, Representative ICa 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.

DHT treatment potentiates calcium currents in GnRH neuron

To study the effects of androgens on ICa, we compared the current among GnRH neurons from OVX, OVXD, and OVXED mice recorded at different times of day. ICa amplitude and density (Fig. 3, A–C) in cells from OVXD mice was greater than that in cells from OVX mice at most membrane potentials examined regardless of time of day (morning, P < 0.05 at membrane potentials 0 to +30 mV, n = 28 cells from eight OVX mice, n = 38 cells from 10 OVXD mice; afternoon, P < 0.05 at membrane potentials 0 to +40 mV, n = 30 cells from eight OVX mice, n = 40 cells from 11 OVXD mice). No diurnal variation in ICa was observed between cells from OVXD mice recorded in the morning vs. afternoon (Fig. 3, B and C; morning vs. afternoon P > 0.05). ICa in OVXD mice was not different from that in OVXED and OVXE mice in the afternoon (OVXED n = 10 cells from three mice; OVXE n = 23 from six mice), suggesting the augmenting effects of DHT and estradiol in the afternoon are not cumulative. However in the morning, ICa amplitude and density were greater in OVXED mice than in OVXE mice, suggesting DHT overcomes the diurnal suppression of ICa by estradiol. DHT had no effect on the activation or steady-state inactivation curves of ICa in GnRH neurons (Fig. 3, D and E). The V1/2 act was −6.3 ± 0.6 mV in OVX mice (n = 30 cells from eight mice) and −6.4 ± 0.5 mV in OVXD mice (n = 40 cells from 11 mice; P > 0.3). The V1/2 inact was −26.4 ± 1.0 mV in OVX mice (n = 30) and −26.2 ± 0.9 mV in OVXD mice (n = 40; P > 0.4). DHT slightly increased rise time but had no effect on decay time (Fig. 3, F and G; OVX n = 30 cells from eight mice; OVXD n = 40 cells from 11 mice; P < 0.05). These data suggest DHT treatment increases GnRH neuron ICa in a manner that, in contrast to estradiol, does not depend upon time of day and is thus persistent.

Figure 3.

Figure 3

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DHT treatment increases ICa in GnRH neurons. A, Representative recordings showing activation of ICa 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).

DHT treatment potentiates N- and R-type HVA ICa

We next examined which subtypes of HVA-mediated currents are affected by DHT (Fig. 4). Different subtypes were isolated with blockers as above. Compared with GnRH neurons from OVX mice, DHT increased the amplitude and density of N-type (Fig. 4C, membrane potentials from −10 to +30 mV; n = 10 cells from three OVX mice; n = 12 cells from three OVXD mice; P < 0.05) and R-type (Fig. 4D, membrane potentials from 0 to +40 mV; n = 12 cells from three OVX mice; n = 11 cells from three OVXD mice; peak P < 0.01; SST P < 0.05) ICa subtypes. The I-V relation, activation, and inactivation curves did not change (data not shown), consistent with above results. There was no difference in L-type (Fig. 4A; n = 12 OVX cells from three mice; n = 11 OVXD cells from three mice; P > 0.2) or P/Q-type (Fig. 4B; n = 8 OVX cells from three mice; n = 9 OVXD cells from three mice; P > 0.9) HVA-mediated currents between OVX and OVXD mice.

Figure 4.

Figure 4

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DHT treatment increases specifically N-type and R-type currents. A–D, Representative ICa 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.

P treatment blocks the augmentation of N-type current by DHT treatment

In women with PCOS (16,17) and in previous work with these animal models (18,37), androgens interfere with the efficacy of P negative feedback. To determine how progestin and DHT treatments interact to regulate ICa in GnRH neurons, we examined currents in cells from OVXEDP mice. Whole-cell ICa amplitude and density in OVXEDP mice was less than in OVXD or OVXED mice and was not different from OVXEP mice (Fig. 5A; n = 10 cells from three mice; OVXED vs. OVXEDP P < 0.05; OVXD vs. OVXEDP P < 0.05). P cotreatment blocked the augmentation of N-type calcium channels by DHT treatment (Fig. 5B, membrane potentials from −10 to +40 mV; n = 12 cells from three OVXD mice; n = 8 cells from three OVXEDP mice; peak P < 0.01; SST P < 0.05).

Figure 5.

Figure 5

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Cotreatment with P blocks the DHT-induced augmentation of ICa in GnRH neurons. A (top), Representative traces showing activation of ICa 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.

DHT treatment has no effect on T-type calcium current

LVA T-type ICa are present in GnRH neurons but in our experience are not adequately revealed by the above protocol (29). Because activation of LVA currents can lead to action potential initiation and DHT is associated with increased GnRH neuron activity, we hypothesized that androgens increase LVA currents. We examined the effects of DHT on LVA currents using a prolonged (1 sec) prepulse at −100 mV to provide a strong signal to remove inactivation, followed by depolarization of the membrane potential to −50 mV to activate specifically LVA currents; this protocol was repeated 50 times at 30-sec intervals and the resulting current traces averaged (29). Consistent with previous work, approximately 42% of GnRH neurons exhibited a small-amplitude LVA-mediated current that could be blocked by 100 μm Ni2+ (Fig. 6A). Contrary to our hypothesis, neither the percentage of cells with LVA nor the peak amplitude of LVA current were different in OVX and OVXD mice (Fig. 6, B and C; n = 11 OVX cells from four animals; n = 8 OVXD cells from three animals; P > 0.5). These data suggest the DHT treatment used in these studies has no effect on T-type ICa in GnRH neurons.

Figure 6.

Figure 6

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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).

Discussion

Steroid feedback regulates GnRH pulse frequency, and alterations in the efficacy of this feedback are associated with infertility. Here we show that one mechanism by which steroids may alter GnRH neuron physiology is by inducing functional changes in the VGCCs in these cells. Specifically, P treatment reduces VGCC function in these cells, whereas DHT treatment increases VGCC function. In previous work with these same animal models, DHT treatment increased GnRH neuron firing, but P treatment inhibited GnRH neurons. The present changes in VGCC function are consistent with the previous changes in activity. However, P appears to be a dominant influence on VGCC function in these cells because it abolished the ability of DHT to increase ICa. Of note, in women with hyperandrogenemic fertility disorders, a steroid milieu that is androgen rich but P poor is typical due to oligoanovulation and lack of corpora lutea; thus, the activating effects of androgens on ICa may be expected to be predominant.

During the normal reproductive cycle, the primary action of P is to provide negative feedback that reduces the frequency of episodic GnRH release. P receptors are estrogen dependent (45); thus, we tested the effects of P in the presence of estradiol to ensure expression of these receptors. Because a shorter duration of estradiol treatment alters VGCCs in a time-of-day-dependent manner (29), we examined VGCCs at two times of day. The longer duration of estradiol (8–12 d) used in the present study had similar effects to those observed previously; specifically, estradiol increased VGCCs during the afternoon hours, which corresponds to the time of positive feedback actions of this steroid. Although the diurnal changes in LH induced by estradiol are more marked 2–4 d after OVXE treatment, there is still an approximately 6-fold increase in LH during the afternoon hours on d 10 and 12 after OVX plus estradiol treatment (42). The present data suggest estradiol-induced increases in GnRH neuron VGCCs during the afternoon may help mediate this. The addition of a physiological dose of P obliterated the afternoon estradiol-induced increases in GnRH neuron VGCCs. Progesterone specifically inhibited N-type calcium currents, which is one of the subtypes augmented by in vivo estradiol treatment (29). Interestingly, P did not inhibit L-type currents, which were also increased by shorter-duration estradiol treatment. It is possible that the effect of estradiol on L-type current is transient. The ability of P to block an estradiol-induced surge is one basis for daily oral steroid contraceptives (46,47). The present data suggest that one mechanism for this may be through P inhibition of estradiol-induced changes in VGCCs in GnRH neurons.

The reduction of GnRH pulse frequency that occurs as a result of P-mediated negative feedback is critical for setting the stage for the early follicular phase of the subsequent cycle. Specifically, after regression of the corpora lutea, the low-frequency GnRH signal preferentially stimulates FSH synthesis and release, allowing relative domination of this gonadotropin during the early follicular phase when follicular development is essential (48,49). In some forms of hyperandrogenemic infertility, such as the common disorder of PCOS (12), there is persistent high-frequency LH (and presumably GnRH) release, leading to a suppression of the FSH to LH ratio, which contributes to the oligoanovulation characteristic of that disorder (50). Elevated androgen levels reduce sensitivity to P feedback in women with PCOS (12). Here we show that an androgen, in the form of DHT, increases HVA VGCCs in GnRH neurons. Specifically, current via N- and R-type subunits was increased in DHT-treated mice. This effect was observed at both times of day examined and was similar in the presence and absence of estradiol, suggesting it is androgen receptor mediated.

Previous work in a similar mouse model indicates that DHT treatment increases GnRH neuron activity and LH release and also increases GABAergic transmission, which can be excitatory to GnRH neurons (18,20,37). In contrast, P reduced both GnRH neuronal activity and GABAergic transmission to these cells. Collectively, these data were interpreted as possible mechanisms for the apparent opposing actions of DHT and P in the control of GnRH neurons and also as possible mechanisms for independent excitatory effects of androgens in hyperandrogenic, nonovulating (i.e. low P) women. The present data support and extend these findings to a new mechanism, specifically a change in the intrinsic properties of GnRH neurons. Increased HVA calcium current in the presence of elevated androgens and absence of P, the typical steroid milieu of women with PCOS, may be associated with greater vesicle release per action potential; thus, these changes could increase output of this neuroendocrine system.

LVA calcium channels play an important role in generation of repetitive activity in several cell types, including cardiac pacemaker cells (51) and thalamic neurons (52). Hence we hypothesized that one mechanism by which DHT treatment increases GnRH neuron activity is to increase LVA-mediated current. Consistent with this hypothesis, the expression of T-type subunits and LVA-mediated currents in GnRH neurons appears to depend on developmental stage and species (28,29,30,53). No changes were observed, however, in the size of this current or in the percentage of cells expressing LVA-mediated current with and without DHT treatment, suggesting changes in this current do not underlie DHT-mediated increases in GnRH neuron activity.

The present data and previous work provide clear evidence for the regulation of GnRH neuron VGCCs by all three classes of sex steroids. Of interest in this regard, steroid receptor expression by mammalian GnRH neurons appears quite limited. Androgen receptor has not been detected in mammalian GnRH neurons (54,55). Likewise, no colocalization of P receptor and GnRH was detected in ewes (56) or monkeys (57), whereas approximately 20% of guinea pig GnRH neurons (58) express P receptor. Finally, GnRH neurons in vivo appear to express only the β-isoform of the estradiol receptor (59). The above observations suggest there are intermediate steroid-sensitive neurons that release neurotransmitters and/or neuromodulators to mediate the feedback of DHT and P on GnRH neuron intrinsic properties. In this regard, kisspeptin expression is testosterone regulated (60,61), as is GnRH release from medial basal hypothalamic fragments in response to neuropeptide Y (62). DHT also regulates GABAergic transmission to GnRH neurons (37); this could alter gene expression via effects on the GABAB receptor. Likewise, kisspeptin (63), opioid, GABAergic (64), dopamine, and neuropeptide Y neurons (65) have been proposed to mediate the effects of P on these cells. These neuromodulators have been demonstrated to regulate HVA ICa in other systems (66,67,68,69,70,71,72). Changes in macroscopic current can be via several mechanisms including changes in subunit gene expression, posttranslational modification of channel proteins, and trafficking in and out of the membrane (73,74,75). Together these data suggest DHT and P may indirectly alter GnRH neuron HVA-mediated calcium currents through these neuromodulators.

In conclusion, the present study shows a novel mechanism by which androgens and P feed back to modulate the function of GnRH neurons. The androgen DHT and P can change GnRH neuron intrinsic properties in addition to synaptic transmission to these cells to regulate GnRH neuron activity. Determining the underlying neural circuits engaged by DHT and P to modulate GnRH neuron VGCCs will be important to understand the central neural control of reproduction.

Acknowledgments

We thank Debra Fisher for excellent technical assistance and Justyna Pielecka-Fortuna, Jessica Kennett, Katarzyna Glanowska, and Pei-San Tsai for useful editorial comments.

Footnotes

This work was supported by NIH U54HD 28934.

Portions of this work were presented in abstract form at the 2009 Society for Neuroscience Meeting.

Disclosure Summary: J.S. and S.M.M. have nothing to disclose.

First Published Online August 25, 2010

Abbreviations: ACSF, Artificial cerebrospinal fluid; DHT, dihydrotestosterone; HVA, high-voltage activated; ICa, calcium current; I-V, current-voltage; LVA, low-voltage activated; OVX, ovariectomized; OVXD, OVX with DHT; OVXE, OVX with estradiol; P, progesterone; PCOS, polycystic ovary syndrome; SST, sustained current; VGCC; voltage-gated calcium channels.

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