Androgens Increase Gonadotropin-Releasing Hormone Neuron Firing Activity in Females and Interfere with Progesterone Negative Feedb
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《内分泌学杂志》
Departments of Internal Medicine and Cell Biology, University of Virginia, Charlottesville, Virginia 22908
Abstract
GnRH neurons are the central regulators of fertility, and their activity is modulated by steroid feedback. In women with hyperandrogenemic infertility and in animal models of these disorders, elevated androgen levels interfere with progesterone (P) negative feedback. Our previous work showed that steroids altered the frequency and amplitude of -aminobutyric acid (GABA) transmission to GnRH neurons. Specifically, P inhibited GABA transmission, which can excite GnRH neurons, whereas dihydrotestosterone (DHT) increased GABA transmission. In this study the GnRH neuron firing rate was examined in the same animal models. Adult (>2 months) female mice were ovariectomized and treated for 8–12 d with implants containing estradiol (E), E and P, E and DHT, or E, P, and DHT. Targeted extracellular recordings were used to examine the long-term firing activity of green fluorescent protein-identified GnRH neurons in brain slices from these mice. In comparing E alone to E plus P animals, P increased the percentage of time that GnRH neurons were quiescent and reduced the area under the curve of the firing rate and the instantaneous firing frequency, suggesting that P provides additional negative feedback over E alone. The addition of DHT markedly increased GnRH neuron activity in both the presence and absence of P. DHT also altered the firing pattern of GnRH neurons, such that peaks in the firing rate detected by the Cluster8 algorithm were approximately doubled in frequency and amplitude. These data support and extend our previous findings and are consistent with the hypothesis that the changes in GABAergic transmission observed in these animal models impact upon the activity of GnRH neurons, and central androgen action probably stimulates GnRH release.
Introduction
GnRH NEURONS FORM the final common pathway regulating reproductive function. The secretion of GnRH occurs in discrete bursts or pulses, with essentially no secretion between bursts of release (1, 2, 3), except during the preovulatory period in females when a GnRH surge occurs (4, 5, 6). In normal females, pulsatile and surge modes of GnRH secretion are regulated by the ovarian steroids estradiol (E) and progesterone (P). P, produced during the luteal phase of the cycle, provides negative feedback to lower GnRH/LH pulse frequency (7, 8, 9). Low frequency GnRH pulses continue into the early follicular phase, allowing an FSH rise that is crucial for ovarian follicular recruitment and maturation. E can have both positive and negative feedback effects on GnRH release (4, 10, 11, 12) and is also required for P receptor expression (13).
The normal cyclical changes in steroid levels and GnRH release do not occur in some hyperandrogenemic fertility disorders, most notably polycystic ovary syndrome (PCOS). Although the presentation of this disorder is variable, in most women it is characterized by disrupted reproductive cycles, elevated androgen levels, and high frequency LH pulses (14). In addition, animal models of this disorder in sheep, primates, and mice exhibit similar concomitant increases in LH and androgens (15, 16, 17). Studies in these animal models as well as in women with PCOS suggest that androgens may both interfere with the negative feedback actions of P and independently activate LH release (16, 18, 19). Of note, E positive and negative feedback also appear disrupted in these models (20, 21).
Previous studies in adult mice in which the steroid milieu was manipulated to reflect normal and hyperandrogenic states suggested that changes in -aminobutyric acid (GABA)-ergic postsynaptic currents in green fluorescent protein (GFP)-identified GnRH neurons may be one neurobiological mechanism underlying steroid feedback (22). Specifically, P inhibited GABA transmission, which can be excitatory in GnRH neurons (23, 24), dihydrotestosterone (DHT) increased GABA transmission relative to controls, and addition of DHT to P-treated females restored GABA transmission to control levels. The latter suggests that, as in humans, androgens (in this case DHT) and P may counteract one another in mice. These results indicate that GABAergic neurons participate in integrating and conveying steroid feedback to GnRH neurons. The recording conditions in that study, however, precluded simultaneous assessment of GnRH neuron firing patterns because of the high chloride pipette solution used to enhance the detection of GABAA receptor-mediated currents.
In the present study we used the same animal models to determine whether the GnRH neuron firing rate is altered by the steroid milieu. We used targeted extracellular recordings, which allow long-term monitoring of the activity of GnRH neurons while maintaining their native intracellular milieu (25), including chloride, and thus retaining the natural response to changes in GABAergic transmission. These experiments are critical for showing the overall effect of the steroid milieu on the activity of GnRH neurons. Our hypotheses were: 1) the combination of P and E has greater negative feedback than E alone; 2) androgens interfere with the ability of P to reduce the firing activity of GnRH neurons; and 3) androgens increase GnRH neuron firing activity.
Materials and Methods
Animals
GnRH neurons were recorded from transgenic female mice in which GFP is genetically targeted to GnRH neurons (26). Mice were on a 14 h light, 10 h dark cycle, with lights off at 1630 h, and were maintained on Harlan 2916 rodent chow (Harlan, Bartonsville, IL) and water ad libitum. All procedures were approved by the animal care and use committee of University of Virginia and were conducted within the guidelines of the National Research Council’s Guide for the Care and Use of Laboratory Animals.
We examined the effect of androgens and P on GnRH neuron firing pattern. Adult female mice were ovariectomized (OVX) under isoflurane (Abbott Laboratories, North Chicago, IL) anesthesia to remove ovarian steroid feedback and received steroid implants as described previously (22). Briefly, all mice received a SILASTIC brand capsule (Dow Corning, Midland, MI) containing 0.625 μg E in sesame oil. This was used as the control condition, because E is required for P receptor expression (13). In addition to E (OVX + E; eight cells from eight animals), groups of mice received a 2.5-mg P time-release pellet (Innovative Research of America, Sarasota, FL; OVX + E + P; eight cells from seven animals), a SILASTIC brand capsule containing 400 μg of the nonaromatizable androgen DHT in sesame oil (OVX + E + DHT; eight cells from eight animals), or both P and DHT (OVX + E + P + DHT; eight cells from five animals). All hormones were administered in vivo and were not present in any recording solutions; no more than two cells from a single animal were recorded. Postoperative analgesia was provided by a long-acting local anesthetic (0.25% bupivicaine; 7.5 μl/site; Abbott Laboratories). Recordings were made between 8 and 12 d after surgery and steroid replacement. No difference was noted in any parameter as a function of time after surgery, an observation that corresponds well with our previous experience with these models examining GABA transmission (22). This treatment duration was chosen because it is similar to the duration of the P rise during pseudopregnancy in rodents (27) 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. In the previous study, LH values in E alone, E + P and E + P + DHT groups were at the level of detection of the assay, whereas LH was significantly elevated in mice treated with E + DHT (22).
Brain slice preparation
Brain slices were prepared with a slight modification (28) of methods previously described (29). All solutions were bubbled with a 95% O2/5% CO2 mixture throughout the experiments and for at least 15 min before exposure to the tissue. Mice were decapitated, and the brain was rapidly removed and placed in ice-cold, high-sucrose saline solution containing 250 mM sucrose, 3.5 mM KCl, 26 mM NaHCO3, 10 mM glucose, 1.25 mM NaHPO4, 1.2 mM MgSO4, and 2.5 mM MgCl2. Coronal 200-μm brain 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 a solution of 50% high-sucrose saline and 50% normal saline (NS) containing (in mM) 135 mM NaCl, 3.5 mM KCl, 10 mM glucose, 1.3 mM NaHPO4, 1.2 mM MgSO4, and 2.5 mM CaCl2 and then were transferred to a solution of 100% NS at room temperature and kept at least 30 min and no more than 8 h before recording.
Electrophysiology and recordings
Targeted extracellular recordings (also known as loose-patch) were used for this study (25). Because low resistance seals (<50 M) do not influence the cell membrane and because the normal intracellular milieu, including native chloride concentrations, is maintained, this approach is a minimally invasive method for monitoring the endogenous electrical activity of a single cell. Although these events are not action potentials per se, they accurately reflect changes in the action potential firing rate. For simplicity, we have used the phrases firing rate, firing pattern, and/or firing activity to refer to these events. All recordings were conducted between 0900 and 1300 h; no effect of time of day was seen within this period.
Individual brain slices were placed in a recording chamber continuously superfused with oxygenated NS solution and kept at 29–31 C. Cells were visualized with an Olympus BX50WI upright fluorescent microscope with infrared differential interference contrast (Opelco, Dulles, VA). GnRH neurons were identified by brief illumination (15–45 sec) at 470 nm to visualize the GFP signal. Patch borosilicate pipettes (World Precision Instruments, Inc., Los Angeles, CA), which ranged from 1.5–2.5 M, were filled with normal HEPES-buffered solution containing 150 mM NaCl, 10 mM HEPES, 10 mM glucose, 2.5 mM CaCl2, 1.3 mM MgCl2, and 3.5 mM KCl. Pipettes were placed in contact with the GnRH neurons using an MP-285 micromanipulator (Sutter Instruments, Novato, CA). Seal resistances ranged from 5-19 M and either remained stable or increased during recording up to as high as 36 M. Minimal amounts of pipette drift were manually compensated to maintain contact with the cell. The location of each GnRH neuron was mapped on sketches of coronal sections obtained from a mouse brain atlas (30); results from the present study did not vary with cell location. The duration of recordings ranged from 44–220 min and was not different between groups (P > 0.10). If no activity was observed for 30 min, 15 mM KCl was added to the bath to check cell viability and recording integrity. If the cell did not respond to KCl, the dataset was truncated at the time of the last firing. If it fired in response to KCl, the dataset was truncated for analysis at the time of adding KCl.
Data collection
Current traces were obtained using an EPC-8 amplifier (HEKA, Mahone Bay, Nova Scotia, Canada) with the PulseControl XOP (Instrutech, Port Washington, NY) running in Igor Pro (Wavemetrics, Lake Oswego, OR) on the G4 Macintosh computer (Apple Computer, Cupertino, CA) to acquire data. A voltage-clamp mode with a pipette holding potential of 0 mV, filtering at 10 kHz, digitized with an ITC-18 acquisition interface (Instrutech) was used for the recordings. Pulse Control Event Tracker software was used to detect the cell membrane currents associated with the action potential firing (29), which were termed events.
Data analysis
Using custom programs (29) written for Igor Pro (Wavemetrics), events were counted and binned at 1-min intervals to identify changes in firing properties and at 5-min intervals for Cluster analysis to avoid oversampling errors in pulse detection. Binned event data were analyzed using Microsoft Excel (Microsoft Corp., Redmond, WA) for the following parameters: percentage of time in quiescence (1 event/min), maximum duration of quiescence, mean firing rate, instantaneous frequency, area under the curve of frequency, and peak amplitude. The mean firing rate was determined by dividing the total number of events detected by the duration of the recording. Instantaneous frequency is the interval between firing events converted to frequency. The area under the curve of firing frequency vs. time was determined using Igor Pro. Area was examined as an indicator of overall activity that is not subject to errors of pulse detection algorithms; this parameter was included because high activity in some cells made distinct patterns difficult to evaluate. The Cluster8 algorithm was used to identify changes in the firing pattern (31). Using peak and nadir clusters of one and two points, respectively, Cluster8 identified peaks and nadirs by pooled t testing and calculated peak amplitude and interpeak intervals. For group comparison, data were log-transformed as needed to normalize SD values, and parameters were compared by one-way ANOVA, followed by post hoc analysis with Student-Newman-Keuls test. Significance was set at P < 0.05.
Results
Steroid milieu alters GnRH neuron firing activity
In this study our goal was to determine the effects of steroid milieu, specifically P and DHT, on the firing patterns of individual GnRH neurons and also examine interactions between these steroids. Representative graphs of firing activity (firing events per 5 min) over time in hours are presented in Fig. 1A; these data are presented on the same scale to facilitate comparison. Figure 1B shows the pattern of action currents from three times during the recording shown in the lower left panel of Fig. 1A. There are two main points to be made from examining the raw data in Fig. 1A. First, inclusion of P with either E alone or E + DHT reduced activity (compare right to left in Fig. 1A). This was most evident as a reduction in baseline firing in cells from mice treated with E + P vs. those given E alone and a reduction in amplitude in cells from mice treated with E + P + DHT vs. E + DHT. Second, inclusion of DHT with either E or E + P (compare top to bottom in Fig. 1A) resulted in a marked increase in activity compared with absence of the androgen. This was noted as an overall increase in the level of firing as well as in the frequency of Cluster-identified firing peaks.
The group mean ± SEM are provided for quiescence measures in Fig. 2, for measures of firing activity in Fig. 3, and for Cluster analysis parameters in Fig. 4. Data are presented below to address the questions. 1) Does P provide additional negative feedback over E alone 2) Does DHT increase the activity of GnRH neurons 3) Does DHT interfere with the ability of P to provide negative feedback
P negative feedback reduces GnRH neuron activity
In our previous work with this animal model, E + P treatment reduced GABA transmission to GnRH neurons compared with the effect of E alone, but this was not reflected in LH levels, because E alone reduced LH to the limits of detection of the assay (22). We first asked whether P provides additional negative feedback compared with E alone at the level of the GnRH neuron (compare middle two bars in Figs. 2–4). E + P treatment vs. E treatment increased the percentage of time that GnRH neurons were quiescent (P < 0.05; Fig. 2A) without affecting the duration of quiescence (Fig. 2B). Addition of P also reduced the instantaneous frequency (P < 0.05; Fig. 3B) and the area under the curve of the firing rate (P < 0.05; Fig. 3C), but did not alter the mean firing rate compared with that in cells from mice treated with only E (Fig. 3A). Cluster analysis revealed no change in either firing rate peaks per hour or peak amplitude between cells from E + P- and E only-treated mice (Fig. 4). Some, but not all, measures of GnRH neuron activity thus suggest that P provides additional negative feedback over E alone.
GnRH neuron firing activity is increased in the presence of DHT
In women with PCOS and hyperandrogenemic animal models, there is evidence of increased activity of the hypothalamo-pituitary axis coincident with elevated androgen levels. In a previous study using these same animal models (22), LH was elevated in mice treated with E + DHT. We examined the effect of DHT, a nonaromatizable androgen, on GnRH neuron activity (compare first two bars in Figs. 2–4). For all parameters measured, except amplitude of Cluster8-detected peaks (Fig. 4B), DHT markedly increased measures of GnRH neuron activity (P < 0.05; Figs. 3 and 4), concomitantly reducing measures of GnRH neuron quiescence (P < 0.05; Fig. 2). It should be noted that Cluster analysis of data from GnRH neurons from E + DHT-treated animals was particularly problematic, because these neurons were nearly constantly active (Fig. 1, lower left) at very high levels. These data must thus be interpreted with caution. Nonetheless, it is clear that mild elevations in DHT produce a marked activation of the GnRH neuronal system.
DHT reduces efficacy of P negative feedback
In women with PCOS and animal models of this disorder, androgens appear to interfere with E and P feedback effects on LH release. Consistent with this, addition of P was unable to counteract the DHT-induced reduction in GnRH neuron quiescence (Fig. 2). Likewise, addition of P was unable to counteract the DHT-induced activation of GnRH neurons with regard to instantaneous frequency (Fig. 3B), area under the curve (Fig. 3C), or approximate doubling of both Cluster peak frequency and amplitude induced by DHT (Fig. 4). In contrast, the mean firing rate was lower (P < 0.05) in cells from mice treated with E + P + DHT than in those from mice treated with E + DHT, but was not different compared with E only-treated controls (Fig. 3A). Together, these data suggest that the activating effects of DHT predominate, and largely obviate, the inhibitory actions of P on GnRH neuron firing activity. An alternative way to state this is that P is largely ineffective in reversing the stimulatory effects of DHT on GnRH neuron firing activity.
Discussion
The feedback regulation of GnRH neurons by steroid hormones is critical to maintain normal reproductive function. In women with hyperandrogenemic infertility, such as PCOS, the high androgen levels may perturb the function of the hypothalamo-pituitary-gonadal axis. Through direct examination of GnRH neurons from mice treated with various steroid regimens, we show that not only do increased levels of androgens decrease the efficacy of P negative feedback effects on GnRH neuron activity, they separately have strong stimulatory effects on these cells. This may contribute to the high frequency pulsatile LH release and concomitant suppression of FSH seen in these disorders and the subsequent impairment of follicular development leading to infertility.
This work employed mouse models that were previously established to mimic distinct reproductive states (22). The levels of E and P are physiological. The DHT dose is a mild elevation to about twice the normal circulating testosterone level in females. This is intended to mimic the hyperandrogenemia in women with PCOS, in whom androgen levels are elevated, but still below normal male levels (14). In this regard, in gonadectomized males, the same DHT treatment used in this study fails to return seminal vesicle weight to that of intact males (Pielecka, J., and S. M. Moenter, unpublished observations). The use of physiological levels of steroids may account for some of the differences between the results of the present study and those of other studies in mice that used high levels of steroid replacement. Those studies indicated inhibitory actions of DHT on LH levels in males; the high dose was consistent with the goal of those studies to establish if steroid feedback existed at all in knockout mice (32).
Another difference in the present approach is the direct examination of GnRH neuron firing activity. This allows independent effects at the central level to be evaluated. This is important, because LH levels reflect feedback action at both the hypothalamus and the pituitary. In our earlier studies, we found that P reduced GABA transmission to GnRH neurons compared with that in E alone controls, but failed to find any difference in LH levels due to limitations of assay sensitivity. The present data indicate that P indeed inhibits several parameters of GnRH neuron firing over and above the effect of E. This is consistent with previous work showing that P inhibits the frequency of LH and GnRH release (33, 34). Of note, the present studies were conducted largely during the morning, when E negative feedback is strongest (35), perhaps precluding measuring a difference in LH levels. Together, these data indicate that even under strong E inhibition, P has an important role in quieting the reproductive neuroendocrine system.
Data from adult women (18, 36) and from models of prenatal androgenization in primates, sheep, and rodents (15, 16, 17, 20) suggest that in females, excess androgens may disrupt communication of feedback signals from P and E. The independent effects of androgens are important to consider in addition to their ability to interfere with feedback via other steroids. This is particularly true when one considers hyperandrogenemic infertility, because the infrequency of ovulation in these women means that there is rarely a corpus luteum to produce P. Our results showed that when DHT is present in addition to E + P or E alone, there is a marked increase in the activity of GnRH neurons. Thus, in androgenized animals, the inhibitory effects of E and P were largely counteracted. In the presence of DHT, the ability of P to inhibit GnRH neurons was reflected only in a single parameter, the mean firing rate, which was reduced to control values (E alone). For all other measures of general GnRH neuron activity examined, DHT overcame E and P negative feedback, producing high levels of GnRH neuron firing. DHT also altered the patterning of this activity. Specifically, DHT roughly doubled both the frequency of peaks in firing rate as well as the amplitude of these peaks. Together, these data suggest a strong activating effect of androgens on GnRH neurons.
These data support and extend our previous examination of GABA transmission to GnRH neurons in these same animal models. E + P reduced both the frequency and the amplitude of GABA postsynaptic currents (PSCs) in GnRH neurons compared with E alone, whereas DHT increased both PSC frequency and amplitude. Although the consequence of GABAA receptor activation is controversial in GnRH neurons (23, 24, 37), previous work from our laboratory has shown that treatments that suppress GnRH release, such as P or fasting, reduce the frequency and amplitude of GABAergic PSCs, whereas prenatal androgenization, which increases LH (and presumably GnRH) release, increases the frequency and amplitude of these currents (16, 22, 38). In addition, activation of metabotropic glutamate receptors reduces both GABA transmission to GnRH neurons and GnRH neuron firing activity, whereas antagonism has the opposite effect (28). The present data also support the hypothesis that GABAA receptor activation can be excitatory in GnRH neurons by demonstrating suppressed GnRH neuron activity in the same animal model in which GABA transmission was reduced (E + P) and increased GnRH neuron activity in an animal model (E + DHT) in which GABA transmission was increased.
In the previous study of GABAergic transmission, the inhibitory action of P and the enhancing action of DHT counteracted each other, so that GABA transmission to GnRH neurons in animals treated with P and DHT was similar to control levels. In the present work, the activating effect of DHT was stronger than the inhibitory action of P at the level of GnRH neuron activity. It is important to note that only two parameters (GABA transmission and firing rate) that might be affected by steroid milieu have been examined in GnRH neurons to date. If we consider the firing rate to be a measure of the output of GnRH neurons, and GABA transmission to be one of the several variables that contribute to this output, it is reasonable to postulate that other variables are differentially altered to produce a weighting toward increased output from GnRH neurons. These might include other synaptic inputs, changes in glial interactions, or changes in intrinsic conductances.
In this regard, steroid hormones can induce synaptic plasticity (39). In females, E increases spine density in the hippocampus and ventromedial hypothalamus, whereas P decreased spine density after 6 h of exposure in the former (40). Interestingly, androgens increase hippocampal spine density in both males and females (41, 42). In monkey GnRH neurons, spine density is reduced, and glial apposition is increased by ovariectomy, but the active steroid has not been identified (43). Spines are thought to be the termination point for excitatory synapses; thus, this morphological change may reflect altered connectivity. Steroids can also alter intrinsic conductances. For example, E alters potassium currents in GnRH neurons (44). Likewise, firing properties in oxytocin neurons are altered in pregnancy and lactation, possibly as a result of changes in steroid milieu (45). These observations in combination with the present data suggest multiple neurobiological mechanisms by which P and DHT may act to alter the ultimate output of GnRH neurons.
In summary, the present data indicate that P and androgens differentially regulate GnRH neuron activity. This is probably due in part to the previously reported alterations in GABA transmission to these cells. The strong activation of GnRH neurons that occurs in response to mild elevations in androgen levels may have implications for understanding the causes of infertility due to hyperandrogenemic disorders in women. Additional studies will explore additional intrinsic and synaptic mechanisms that may account for this activational effect of androgens.
Acknowledgments
We thank Xu-Zhi Xu for expert technical assistance, and Catherine Christian, Zhiguo Chu, Debra Fisher, Alison Roland, Talent Shevchenko, and Pei-San Tsai for editorial comments.
Footnotes
This work was supported by the National Institute of Child Health and Human Development, National Institutes of Health (Cooperative Agreement U54-HD-28934) and the U54 Ligand and Assay Core.
J.P., S.D.Q. and S.M.M. have nothing to declare.
First Published Online December 8, 2005
Abbreviations: DHT, Dihydrotestosterone; E, estradiol; GFP, green fluorescent protein; GABA, -aminobutyric acid; NS, normal saline; OVX, ovariectomized; P, progesterone; PCOS, polycystic ovary syndrome; PSC, postsynaptic current.
Accepted for publication November 23, 2005.
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Teruyama R, Armstrong WE 2002 Changes in the active membrane properties of rat supraoptic neurons during pregnancy and lactation. Neuroendocrinology 14:933–944(Justyna Pielecka, Samuel D. Quaynor and )
Abstract
GnRH neurons are the central regulators of fertility, and their activity is modulated by steroid feedback. In women with hyperandrogenemic infertility and in animal models of these disorders, elevated androgen levels interfere with progesterone (P) negative feedback. Our previous work showed that steroids altered the frequency and amplitude of -aminobutyric acid (GABA) transmission to GnRH neurons. Specifically, P inhibited GABA transmission, which can excite GnRH neurons, whereas dihydrotestosterone (DHT) increased GABA transmission. In this study the GnRH neuron firing rate was examined in the same animal models. Adult (>2 months) female mice were ovariectomized and treated for 8–12 d with implants containing estradiol (E), E and P, E and DHT, or E, P, and DHT. Targeted extracellular recordings were used to examine the long-term firing activity of green fluorescent protein-identified GnRH neurons in brain slices from these mice. In comparing E alone to E plus P animals, P increased the percentage of time that GnRH neurons were quiescent and reduced the area under the curve of the firing rate and the instantaneous firing frequency, suggesting that P provides additional negative feedback over E alone. The addition of DHT markedly increased GnRH neuron activity in both the presence and absence of P. DHT also altered the firing pattern of GnRH neurons, such that peaks in the firing rate detected by the Cluster8 algorithm were approximately doubled in frequency and amplitude. These data support and extend our previous findings and are consistent with the hypothesis that the changes in GABAergic transmission observed in these animal models impact upon the activity of GnRH neurons, and central androgen action probably stimulates GnRH release.
Introduction
GnRH NEURONS FORM the final common pathway regulating reproductive function. The secretion of GnRH occurs in discrete bursts or pulses, with essentially no secretion between bursts of release (1, 2, 3), except during the preovulatory period in females when a GnRH surge occurs (4, 5, 6). In normal females, pulsatile and surge modes of GnRH secretion are regulated by the ovarian steroids estradiol (E) and progesterone (P). P, produced during the luteal phase of the cycle, provides negative feedback to lower GnRH/LH pulse frequency (7, 8, 9). Low frequency GnRH pulses continue into the early follicular phase, allowing an FSH rise that is crucial for ovarian follicular recruitment and maturation. E can have both positive and negative feedback effects on GnRH release (4, 10, 11, 12) and is also required for P receptor expression (13).
The normal cyclical changes in steroid levels and GnRH release do not occur in some hyperandrogenemic fertility disorders, most notably polycystic ovary syndrome (PCOS). Although the presentation of this disorder is variable, in most women it is characterized by disrupted reproductive cycles, elevated androgen levels, and high frequency LH pulses (14). In addition, animal models of this disorder in sheep, primates, and mice exhibit similar concomitant increases in LH and androgens (15, 16, 17). Studies in these animal models as well as in women with PCOS suggest that androgens may both interfere with the negative feedback actions of P and independently activate LH release (16, 18, 19). Of note, E positive and negative feedback also appear disrupted in these models (20, 21).
Previous studies in adult mice in which the steroid milieu was manipulated to reflect normal and hyperandrogenic states suggested that changes in -aminobutyric acid (GABA)-ergic postsynaptic currents in green fluorescent protein (GFP)-identified GnRH neurons may be one neurobiological mechanism underlying steroid feedback (22). Specifically, P inhibited GABA transmission, which can be excitatory in GnRH neurons (23, 24), dihydrotestosterone (DHT) increased GABA transmission relative to controls, and addition of DHT to P-treated females restored GABA transmission to control levels. The latter suggests that, as in humans, androgens (in this case DHT) and P may counteract one another in mice. These results indicate that GABAergic neurons participate in integrating and conveying steroid feedback to GnRH neurons. The recording conditions in that study, however, precluded simultaneous assessment of GnRH neuron firing patterns because of the high chloride pipette solution used to enhance the detection of GABAA receptor-mediated currents.
In the present study we used the same animal models to determine whether the GnRH neuron firing rate is altered by the steroid milieu. We used targeted extracellular recordings, which allow long-term monitoring of the activity of GnRH neurons while maintaining their native intracellular milieu (25), including chloride, and thus retaining the natural response to changes in GABAergic transmission. These experiments are critical for showing the overall effect of the steroid milieu on the activity of GnRH neurons. Our hypotheses were: 1) the combination of P and E has greater negative feedback than E alone; 2) androgens interfere with the ability of P to reduce the firing activity of GnRH neurons; and 3) androgens increase GnRH neuron firing activity.
Materials and Methods
Animals
GnRH neurons were recorded from transgenic female mice in which GFP is genetically targeted to GnRH neurons (26). Mice were on a 14 h light, 10 h dark cycle, with lights off at 1630 h, and were maintained on Harlan 2916 rodent chow (Harlan, Bartonsville, IL) and water ad libitum. All procedures were approved by the animal care and use committee of University of Virginia and were conducted within the guidelines of the National Research Council’s Guide for the Care and Use of Laboratory Animals.
We examined the effect of androgens and P on GnRH neuron firing pattern. Adult female mice were ovariectomized (OVX) under isoflurane (Abbott Laboratories, North Chicago, IL) anesthesia to remove ovarian steroid feedback and received steroid implants as described previously (22). Briefly, all mice received a SILASTIC brand capsule (Dow Corning, Midland, MI) containing 0.625 μg E in sesame oil. This was used as the control condition, because E is required for P receptor expression (13). In addition to E (OVX + E; eight cells from eight animals), groups of mice received a 2.5-mg P time-release pellet (Innovative Research of America, Sarasota, FL; OVX + E + P; eight cells from seven animals), a SILASTIC brand capsule containing 400 μg of the nonaromatizable androgen DHT in sesame oil (OVX + E + DHT; eight cells from eight animals), or both P and DHT (OVX + E + P + DHT; eight cells from five animals). All hormones were administered in vivo and were not present in any recording solutions; no more than two cells from a single animal were recorded. Postoperative analgesia was provided by a long-acting local anesthetic (0.25% bupivicaine; 7.5 μl/site; Abbott Laboratories). Recordings were made between 8 and 12 d after surgery and steroid replacement. No difference was noted in any parameter as a function of time after surgery, an observation that corresponds well with our previous experience with these models examining GABA transmission (22). This treatment duration was chosen because it is similar to the duration of the P rise during pseudopregnancy in rodents (27) 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. In the previous study, LH values in E alone, E + P and E + P + DHT groups were at the level of detection of the assay, whereas LH was significantly elevated in mice treated with E + DHT (22).
Brain slice preparation
Brain slices were prepared with a slight modification (28) of methods previously described (29). All solutions were bubbled with a 95% O2/5% CO2 mixture throughout the experiments and for at least 15 min before exposure to the tissue. Mice were decapitated, and the brain was rapidly removed and placed in ice-cold, high-sucrose saline solution containing 250 mM sucrose, 3.5 mM KCl, 26 mM NaHCO3, 10 mM glucose, 1.25 mM NaHPO4, 1.2 mM MgSO4, and 2.5 mM MgCl2. Coronal 200-μm brain 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 a solution of 50% high-sucrose saline and 50% normal saline (NS) containing (in mM) 135 mM NaCl, 3.5 mM KCl, 10 mM glucose, 1.3 mM NaHPO4, 1.2 mM MgSO4, and 2.5 mM CaCl2 and then were transferred to a solution of 100% NS at room temperature and kept at least 30 min and no more than 8 h before recording.
Electrophysiology and recordings
Targeted extracellular recordings (also known as loose-patch) were used for this study (25). Because low resistance seals (<50 M) do not influence the cell membrane and because the normal intracellular milieu, including native chloride concentrations, is maintained, this approach is a minimally invasive method for monitoring the endogenous electrical activity of a single cell. Although these events are not action potentials per se, they accurately reflect changes in the action potential firing rate. For simplicity, we have used the phrases firing rate, firing pattern, and/or firing activity to refer to these events. All recordings were conducted between 0900 and 1300 h; no effect of time of day was seen within this period.
Individual brain slices were placed in a recording chamber continuously superfused with oxygenated NS solution and kept at 29–31 C. Cells were visualized with an Olympus BX50WI upright fluorescent microscope with infrared differential interference contrast (Opelco, Dulles, VA). GnRH neurons were identified by brief illumination (15–45 sec) at 470 nm to visualize the GFP signal. Patch borosilicate pipettes (World Precision Instruments, Inc., Los Angeles, CA), which ranged from 1.5–2.5 M, were filled with normal HEPES-buffered solution containing 150 mM NaCl, 10 mM HEPES, 10 mM glucose, 2.5 mM CaCl2, 1.3 mM MgCl2, and 3.5 mM KCl. Pipettes were placed in contact with the GnRH neurons using an MP-285 micromanipulator (Sutter Instruments, Novato, CA). Seal resistances ranged from 5-19 M and either remained stable or increased during recording up to as high as 36 M. Minimal amounts of pipette drift were manually compensated to maintain contact with the cell. The location of each GnRH neuron was mapped on sketches of coronal sections obtained from a mouse brain atlas (30); results from the present study did not vary with cell location. The duration of recordings ranged from 44–220 min and was not different between groups (P > 0.10). If no activity was observed for 30 min, 15 mM KCl was added to the bath to check cell viability and recording integrity. If the cell did not respond to KCl, the dataset was truncated at the time of the last firing. If it fired in response to KCl, the dataset was truncated for analysis at the time of adding KCl.
Data collection
Current traces were obtained using an EPC-8 amplifier (HEKA, Mahone Bay, Nova Scotia, Canada) with the PulseControl XOP (Instrutech, Port Washington, NY) running in Igor Pro (Wavemetrics, Lake Oswego, OR) on the G4 Macintosh computer (Apple Computer, Cupertino, CA) to acquire data. A voltage-clamp mode with a pipette holding potential of 0 mV, filtering at 10 kHz, digitized with an ITC-18 acquisition interface (Instrutech) was used for the recordings. Pulse Control Event Tracker software was used to detect the cell membrane currents associated with the action potential firing (29), which were termed events.
Data analysis
Using custom programs (29) written for Igor Pro (Wavemetrics), events were counted and binned at 1-min intervals to identify changes in firing properties and at 5-min intervals for Cluster analysis to avoid oversampling errors in pulse detection. Binned event data were analyzed using Microsoft Excel (Microsoft Corp., Redmond, WA) for the following parameters: percentage of time in quiescence (1 event/min), maximum duration of quiescence, mean firing rate, instantaneous frequency, area under the curve of frequency, and peak amplitude. The mean firing rate was determined by dividing the total number of events detected by the duration of the recording. Instantaneous frequency is the interval between firing events converted to frequency. The area under the curve of firing frequency vs. time was determined using Igor Pro. Area was examined as an indicator of overall activity that is not subject to errors of pulse detection algorithms; this parameter was included because high activity in some cells made distinct patterns difficult to evaluate. The Cluster8 algorithm was used to identify changes in the firing pattern (31). Using peak and nadir clusters of one and two points, respectively, Cluster8 identified peaks and nadirs by pooled t testing and calculated peak amplitude and interpeak intervals. For group comparison, data were log-transformed as needed to normalize SD values, and parameters were compared by one-way ANOVA, followed by post hoc analysis with Student-Newman-Keuls test. Significance was set at P < 0.05.
Results
Steroid milieu alters GnRH neuron firing activity
In this study our goal was to determine the effects of steroid milieu, specifically P and DHT, on the firing patterns of individual GnRH neurons and also examine interactions between these steroids. Representative graphs of firing activity (firing events per 5 min) over time in hours are presented in Fig. 1A; these data are presented on the same scale to facilitate comparison. Figure 1B shows the pattern of action currents from three times during the recording shown in the lower left panel of Fig. 1A. There are two main points to be made from examining the raw data in Fig. 1A. First, inclusion of P with either E alone or E + DHT reduced activity (compare right to left in Fig. 1A). This was most evident as a reduction in baseline firing in cells from mice treated with E + P vs. those given E alone and a reduction in amplitude in cells from mice treated with E + P + DHT vs. E + DHT. Second, inclusion of DHT with either E or E + P (compare top to bottom in Fig. 1A) resulted in a marked increase in activity compared with absence of the androgen. This was noted as an overall increase in the level of firing as well as in the frequency of Cluster-identified firing peaks.
The group mean ± SEM are provided for quiescence measures in Fig. 2, for measures of firing activity in Fig. 3, and for Cluster analysis parameters in Fig. 4. Data are presented below to address the questions. 1) Does P provide additional negative feedback over E alone 2) Does DHT increase the activity of GnRH neurons 3) Does DHT interfere with the ability of P to provide negative feedback
P negative feedback reduces GnRH neuron activity
In our previous work with this animal model, E + P treatment reduced GABA transmission to GnRH neurons compared with the effect of E alone, but this was not reflected in LH levels, because E alone reduced LH to the limits of detection of the assay (22). We first asked whether P provides additional negative feedback compared with E alone at the level of the GnRH neuron (compare middle two bars in Figs. 2–4). E + P treatment vs. E treatment increased the percentage of time that GnRH neurons were quiescent (P < 0.05; Fig. 2A) without affecting the duration of quiescence (Fig. 2B). Addition of P also reduced the instantaneous frequency (P < 0.05; Fig. 3B) and the area under the curve of the firing rate (P < 0.05; Fig. 3C), but did not alter the mean firing rate compared with that in cells from mice treated with only E (Fig. 3A). Cluster analysis revealed no change in either firing rate peaks per hour or peak amplitude between cells from E + P- and E only-treated mice (Fig. 4). Some, but not all, measures of GnRH neuron activity thus suggest that P provides additional negative feedback over E alone.
GnRH neuron firing activity is increased in the presence of DHT
In women with PCOS and hyperandrogenemic animal models, there is evidence of increased activity of the hypothalamo-pituitary axis coincident with elevated androgen levels. In a previous study using these same animal models (22), LH was elevated in mice treated with E + DHT. We examined the effect of DHT, a nonaromatizable androgen, on GnRH neuron activity (compare first two bars in Figs. 2–4). For all parameters measured, except amplitude of Cluster8-detected peaks (Fig. 4B), DHT markedly increased measures of GnRH neuron activity (P < 0.05; Figs. 3 and 4), concomitantly reducing measures of GnRH neuron quiescence (P < 0.05; Fig. 2). It should be noted that Cluster analysis of data from GnRH neurons from E + DHT-treated animals was particularly problematic, because these neurons were nearly constantly active (Fig. 1, lower left) at very high levels. These data must thus be interpreted with caution. Nonetheless, it is clear that mild elevations in DHT produce a marked activation of the GnRH neuronal system.
DHT reduces efficacy of P negative feedback
In women with PCOS and animal models of this disorder, androgens appear to interfere with E and P feedback effects on LH release. Consistent with this, addition of P was unable to counteract the DHT-induced reduction in GnRH neuron quiescence (Fig. 2). Likewise, addition of P was unable to counteract the DHT-induced activation of GnRH neurons with regard to instantaneous frequency (Fig. 3B), area under the curve (Fig. 3C), or approximate doubling of both Cluster peak frequency and amplitude induced by DHT (Fig. 4). In contrast, the mean firing rate was lower (P < 0.05) in cells from mice treated with E + P + DHT than in those from mice treated with E + DHT, but was not different compared with E only-treated controls (Fig. 3A). Together, these data suggest that the activating effects of DHT predominate, and largely obviate, the inhibitory actions of P on GnRH neuron firing activity. An alternative way to state this is that P is largely ineffective in reversing the stimulatory effects of DHT on GnRH neuron firing activity.
Discussion
The feedback regulation of GnRH neurons by steroid hormones is critical to maintain normal reproductive function. In women with hyperandrogenemic infertility, such as PCOS, the high androgen levels may perturb the function of the hypothalamo-pituitary-gonadal axis. Through direct examination of GnRH neurons from mice treated with various steroid regimens, we show that not only do increased levels of androgens decrease the efficacy of P negative feedback effects on GnRH neuron activity, they separately have strong stimulatory effects on these cells. This may contribute to the high frequency pulsatile LH release and concomitant suppression of FSH seen in these disorders and the subsequent impairment of follicular development leading to infertility.
This work employed mouse models that were previously established to mimic distinct reproductive states (22). The levels of E and P are physiological. The DHT dose is a mild elevation to about twice the normal circulating testosterone level in females. This is intended to mimic the hyperandrogenemia in women with PCOS, in whom androgen levels are elevated, but still below normal male levels (14). In this regard, in gonadectomized males, the same DHT treatment used in this study fails to return seminal vesicle weight to that of intact males (Pielecka, J., and S. M. Moenter, unpublished observations). The use of physiological levels of steroids may account for some of the differences between the results of the present study and those of other studies in mice that used high levels of steroid replacement. Those studies indicated inhibitory actions of DHT on LH levels in males; the high dose was consistent with the goal of those studies to establish if steroid feedback existed at all in knockout mice (32).
Another difference in the present approach is the direct examination of GnRH neuron firing activity. This allows independent effects at the central level to be evaluated. This is important, because LH levels reflect feedback action at both the hypothalamus and the pituitary. In our earlier studies, we found that P reduced GABA transmission to GnRH neurons compared with that in E alone controls, but failed to find any difference in LH levels due to limitations of assay sensitivity. The present data indicate that P indeed inhibits several parameters of GnRH neuron firing over and above the effect of E. This is consistent with previous work showing that P inhibits the frequency of LH and GnRH release (33, 34). Of note, the present studies were conducted largely during the morning, when E negative feedback is strongest (35), perhaps precluding measuring a difference in LH levels. Together, these data indicate that even under strong E inhibition, P has an important role in quieting the reproductive neuroendocrine system.
Data from adult women (18, 36) and from models of prenatal androgenization in primates, sheep, and rodents (15, 16, 17, 20) suggest that in females, excess androgens may disrupt communication of feedback signals from P and E. The independent effects of androgens are important to consider in addition to their ability to interfere with feedback via other steroids. This is particularly true when one considers hyperandrogenemic infertility, because the infrequency of ovulation in these women means that there is rarely a corpus luteum to produce P. Our results showed that when DHT is present in addition to E + P or E alone, there is a marked increase in the activity of GnRH neurons. Thus, in androgenized animals, the inhibitory effects of E and P were largely counteracted. In the presence of DHT, the ability of P to inhibit GnRH neurons was reflected only in a single parameter, the mean firing rate, which was reduced to control values (E alone). For all other measures of general GnRH neuron activity examined, DHT overcame E and P negative feedback, producing high levels of GnRH neuron firing. DHT also altered the patterning of this activity. Specifically, DHT roughly doubled both the frequency of peaks in firing rate as well as the amplitude of these peaks. Together, these data suggest a strong activating effect of androgens on GnRH neurons.
These data support and extend our previous examination of GABA transmission to GnRH neurons in these same animal models. E + P reduced both the frequency and the amplitude of GABA postsynaptic currents (PSCs) in GnRH neurons compared with E alone, whereas DHT increased both PSC frequency and amplitude. Although the consequence of GABAA receptor activation is controversial in GnRH neurons (23, 24, 37), previous work from our laboratory has shown that treatments that suppress GnRH release, such as P or fasting, reduce the frequency and amplitude of GABAergic PSCs, whereas prenatal androgenization, which increases LH (and presumably GnRH) release, increases the frequency and amplitude of these currents (16, 22, 38). In addition, activation of metabotropic glutamate receptors reduces both GABA transmission to GnRH neurons and GnRH neuron firing activity, whereas antagonism has the opposite effect (28). The present data also support the hypothesis that GABAA receptor activation can be excitatory in GnRH neurons by demonstrating suppressed GnRH neuron activity in the same animal model in which GABA transmission was reduced (E + P) and increased GnRH neuron activity in an animal model (E + DHT) in which GABA transmission was increased.
In the previous study of GABAergic transmission, the inhibitory action of P and the enhancing action of DHT counteracted each other, so that GABA transmission to GnRH neurons in animals treated with P and DHT was similar to control levels. In the present work, the activating effect of DHT was stronger than the inhibitory action of P at the level of GnRH neuron activity. It is important to note that only two parameters (GABA transmission and firing rate) that might be affected by steroid milieu have been examined in GnRH neurons to date. If we consider the firing rate to be a measure of the output of GnRH neurons, and GABA transmission to be one of the several variables that contribute to this output, it is reasonable to postulate that other variables are differentially altered to produce a weighting toward increased output from GnRH neurons. These might include other synaptic inputs, changes in glial interactions, or changes in intrinsic conductances.
In this regard, steroid hormones can induce synaptic plasticity (39). In females, E increases spine density in the hippocampus and ventromedial hypothalamus, whereas P decreased spine density after 6 h of exposure in the former (40). Interestingly, androgens increase hippocampal spine density in both males and females (41, 42). In monkey GnRH neurons, spine density is reduced, and glial apposition is increased by ovariectomy, but the active steroid has not been identified (43). Spines are thought to be the termination point for excitatory synapses; thus, this morphological change may reflect altered connectivity. Steroids can also alter intrinsic conductances. For example, E alters potassium currents in GnRH neurons (44). Likewise, firing properties in oxytocin neurons are altered in pregnancy and lactation, possibly as a result of changes in steroid milieu (45). These observations in combination with the present data suggest multiple neurobiological mechanisms by which P and DHT may act to alter the ultimate output of GnRH neurons.
In summary, the present data indicate that P and androgens differentially regulate GnRH neuron activity. This is probably due in part to the previously reported alterations in GABA transmission to these cells. The strong activation of GnRH neurons that occurs in response to mild elevations in androgen levels may have implications for understanding the causes of infertility due to hyperandrogenemic disorders in women. Additional studies will explore additional intrinsic and synaptic mechanisms that may account for this activational effect of androgens.
Acknowledgments
We thank Xu-Zhi Xu for expert technical assistance, and Catherine Christian, Zhiguo Chu, Debra Fisher, Alison Roland, Talent Shevchenko, and Pei-San Tsai for editorial comments.
Footnotes
This work was supported by the National Institute of Child Health and Human Development, National Institutes of Health (Cooperative Agreement U54-HD-28934) and the U54 Ligand and Assay Core.
J.P., S.D.Q. and S.M.M. have nothing to declare.
First Published Online December 8, 2005
Abbreviations: DHT, Dihydrotestosterone; E, estradiol; GFP, green fluorescent protein; GABA, -aminobutyric acid; NS, normal saline; OVX, ovariectomized; P, progesterone; PCOS, polycystic ovary syndrome; PSC, postsynaptic current.
Accepted for publication November 23, 2005.
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