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Endocrinology Vol. 140, No. 4 1767-1774
Copyright © 1999 by The Endocrine Society

ARTICLES

Androgen Modulation of Luteinizing Hormone Secretion by Female Rat Gonadotropes1

Judith L. Turgeon and Dennis W. Waring

Department of Human Physiology, University of California School of Medicine, Davis, California 95616

Address all correspondence and requests for reprints to: Dr. Judith L. Turgeon, Department of Human Physiology, University of California School of Medicine, Davis, California 95616. E-mail: jlturgeon{at}ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the female, androgens can have negative and positive actions in the regulation of LH, but it is not clear how they may function during the reproductive cycle. Toward resolving these potentially conflicting roles for androgen, we used an in vitro model of preovulatory gonadotropes to examine the effect of proestrous levels of testosterone (1.7 nM) or dihydrotestosterone (DHT; 0.7 nM) on LH secretion in response to pulsatile GnRH (1 nM) or elevated extracellular K+ (54 mM). For female rat pituitary cells cultured in 17ß-estradiol (E2)-containing medium, androgen treatment for 16 h, but not for 4 h, inhibited the LH secretory response to a pulse of either GnRH or K+ by about 60% and suppressed the acute augmentation action of 20 nM progesterone on GnRH- or K+-induced LH secretion. In the absence of E2, DHT also decreased LH secretion induced by a pulse of GnRH. DHT’s suppressive effect on progesterone could be partially overcome with increased progesterone (200 nM) or by removal of DHT during progesterone exposure. For pituitary cells transfected with a reporter plasmid containing three progesterone response elements, DHT only partially suppressed progesterone-stimulated transcriptional activity. The positive action of androgen (16 h) on LH secretion was elicited by multiple GnRH pulses with a latency of about 2 h after the first pulse; this facilitatory action of androgen did not require an E2 background and, therefore, is distinct from GnRH self priming. In summary, these data demonstrate both facilitatory and inhibitory actions of androgen on LH secretion function in female gonadotropes in vitro in the absence or presence of E2; these actions occur with a time course suggestive of a role for androgen in shaping the preovulatory LH surge. Androgen also markedly suppresses progesterone augmentation of stimulated LH secretion, which could be due in part to interference with the trans-activation function of the progesterone receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THAT ANDROGENS have the potential to suppress LH secretion in females in vitro as well as in vivo is well established (1, 2, 3, 4, 5, 6), but the physiological role, if any, for this androgen action in the female reproductive cycle has not been determined. Adding to the complexity, androgens also have the potential for a positive effect on LH secretion. It recently was reported that androgen is required to facilitate GnRH stimulation of LHß messenger RNA (mRNA) in female rats in vivo and in vitro (7, 8). For both negative and positive actions, either testosterone (T) or the nonaromatizable androgen, 5{alpha}-dihydrotestosterone (DHT), generally is found to be effective.

The mechanisms involved in the direct inhibitory action of androgens on GnRH-stimulated LH secretion from female gonadotropes have not been established, although T has been reported to inhibit LH release induced by phorbol ester-activated protein kinase C (9) and to either suppress (10) or potentiate (11) release induced by cAMP. There also is a report that DHT can decrease GnRH-binding sites in female pituitary cells (12). Most of the early studies that uncovered the suppressive effect of androgen on LH secretion were carried out with prolonged, nonpulsatile exposure of pituitary cells to GnRH, thus making it difficult to dissect out the complex actions. Given that androgens can have both negative and positive actions on the female gonadotrope and that the sites of action could involve steps in the GnRH signaling pathway, LH gene expression and protein synthesis, or the exocytotic machinery, it is not surprising that kinetic aspects of the stimulus and response as well as end points other than the aggregate LH secretory response must be considered.

Much of the recent work on androgen action in the gonadotrope has focused on cells from males and within the framework of its primary physiological role as a negative feedback regulator of LH secretion in the male (13, 14, 15). For the female, the physiological context is less obvious. During the rat estrous cycle, serum T and DHT levels are lowest during estrus and highest on proestrus after the onset of the preovulatory gonadotropin surges, and these fluctuations are independent of contributions from the adrenal glands (16, 17). The proestrous increase in serum T concentration appears to precede the onset of the LH surge by a few hours (16). Thus, there is a context in which androgens reasonably could be considered as playing a physiological role. Timing is paramount in the reproductive cycle. Most studies of androgen action on the gonadotrope have used extended exposure times (24 h or more), and the minimum exposure requirements or, in fact, whether there are different temporal requirements for the negative and positive actions of androgens have not been determined.

Prompted by the intriguing observations suggesting that androgens have a positive as well as a negative role in the regulation of LH, we asked whether androgens could modulate the LH secretory events in an in vitro model of preovulatory gonadotropes. Specifically, we examined the effect of androgens on GnRH self priming and on acute progesterone augmentation of stimulated LH secretion. Part of this study has been presented in preliminary form (18).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Adult female Sprague-Dawley rats (Simonsen Laboratories, Inc., Gilroy, CA) were maintained in controlled light conditions (14 h of light, 10 h of darkness). Media and sera for cell culture were described previously (19). For secretion experiments, cells were plated on Lux Thermanox coverslips (Nunc, Naperville, IL) coated with Matrigel (Collaborative Research, Bedford, MA). GnRH, 17ß-estradiol (E2), and androgens were obtained from Sigma Chemical Co. (St. Louis, MO); progesterone was obtained from Calbiochem (San Diego, CA). RU486 was a gift from Roussel-UCLAF (Romainville, France). Progesterone, androgens, E2, and RU486 were prepared as stock solutions in ethanol. The reporter plasmid TAT3LUC, which was provided by Keith Yamamoto (University of California, San Francisco, CA), contains three tandem progesterone/glucocorticoid-responsive elements (referred to as PREs in this report) derived from the tyrosine aminotransferase (TAT) gene located upstream of the minimal alcohol dehydrogenase promoter linked to the luciferase (LUC) gene (20). Luciferase activity was determined in cell extracts using the kit from Analytical Luminescence Laboratory (San Diego, CA). General chemicals were purchased from either Sigma Chemical Co. or Fisher Scientific International, Inc. (Pittsburgh, PA).

Pituitary cell culture
Rats were ovariectomized and maintained for 2 weeks, after which pituitaries were removed following CO2 narcosis and decapitation. Anterior pituitary cells, obtained by trypsin dispersion (day 0), were cultured in Eagle’s MEM containing D-valine (MEM), 0.2 mM kanamycin sulfate, and 10% charcoal-treated FBS (FBS-CT) with or without proestrous levels of E2 (0.2 nM) (21) in a humidified atmosphere of 5% CO2 in air as previously described (19). Residual steroid concentrations in FBS-CT were 10 pM for progesterone and less than 1 pM for E2 as determined by RIA. Short term ovariectomized rats were used to reduce the variability in steroid background for donor pituitaries and to allow the cells to be synchronized in vitro by the presence or absence of E2 in the culture medium, thus providing for an in vitro model of preovulatory gonadotropes. For secretion experiments, cells were plated at 3 x 105 on Matrigel-coated coverslips inserted into 22-mm wells. For transfection experiments, cells were plated at 6 x 105 in 35-mm dishes.

Secretion studies
The medium was changed on day 2; at that time, exposure to either T (1.7 nM) or DHT (0.7 nM) was initiated for some groups. Androgen concentrations were based on levels occurring during the preovulatory LH surge (16, 17) and correspond to the levels reported to result in facilitation of GnRH-stimulated LHß in female rats (7). Experiments were begun 16–19 h after the initiation of androgen treatment unless noted otherwise and were carried out in medium containing 1 mg/ml BSA without serum. For all experiments, successive 15-min incubations were collected before, during, and after the pulses to monitor LH secretion. Samples were stored at -70 C until assayed by RIA as described previously (19).

Single GnRH pulse protocol. Starting at time zero, cells were incubated in control medium with or without 20 or 200 nM progesterone and challenged with a single 1-nM GnRH pulse of 15-min duration, initiated at 90 min. In certain experiments, DHT exposure was either terminated or initiated at 30 min before progesterone stimulation was initiated.

Triple GnRH pulse protocol. Cells were challenged with three 15-min pulses of 1 nM GnRH at 60-min intervals.

Multiple K+ pulses. Cells were challenged with multiple pulses of 54 mM K+ of 15-min duration at 60-min intervals. In some groups, 20 nM progesterone was included in the medium beginning 90 min before the first K+ pulse. For the K+ pulse experiments, MEM/BSA medium was replaced with medium of similar composition, pH 7.4, that contained 1 mg BSA/ml, 15 mg phenol red/liter, 110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.6 mM MgSO4, 24 mM NaHCO3, 0.8 mM Na2HPO4, and 25 mM glucose; 54 mM K+ medium was prepared by equimolar replacement of NaCl with KCl.

Transfection studies
Cells were incubated in MEM/FBS-CT medium plus E2. On day 2 of culture, cells were transfected with 2 µg TAT3LUC plasmid by the CaPO4 precipitation method and were subjected to glycerol shock 4 h later as previously described (22). After glycerol shock, incubation was continued in fresh medium with or without 0.7 nM DHT. Beginning 16 h later, the transfected cells were rinsed in MEM/BSA and challenged for 6 h in MEM/BSA with medium only (control), progesterone (20 or 200 nM), DHT (continued), DHT (continued) plus progesterone, or acute DHT plus progesterone.

Extract preparation
Cells were rinsed in cold PBS, incubated in lysis buffer at 4 C for 15 min, scraped and lysed in a final volume of 180 µl, and stored at -70 C until assayed at 50 µl in duplicate in a luminometer.

Data analysis
Data are presented as the mean ± SEM. Each experiment represents a separate pool of dispersed pituitary cells; n refers to the number of times an experiment was repeated. For LH secretion studies in response to pulsatile secretagogue administration, the integrated secretory response was calculated as the total amount of LH secreted during the 15-min exposure to a secretagogue plus that secreted in the subsequent 15 min. In some cases, the t distribution was used to test the hypothesis that the response was significantly different from 100%. All other statistical analyses were performed using SigmaStat (version 2.0, SPSS, Inc., Chicago, IL). For multiple comparisons, differences between groups were determined by ANOVA followed by the Tukey test; where differences are indicated as significant, P < 0.05. Where appropriate, differences between two groups were determined using Student’s t test, with the level of significance noted in the report of the results. In simple linear regression we tested the hypothesis that the slope of the regression line was significantly different from zero.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect on response to single GnRH pulse
To confirm the previously observed inhibitory action of an androgen background on the LH secretory response to GnRH, we exposed pituitary cells, cultured in the presence of E2, to either T or DHT overnight (16 h) and then challenged the cells with a pulse of GnRH. As shown in Fig. 1Go, the LH secretory response is significantly suppressed in the presence of either androgen to about 40% of the control value (compared with control, P < 0.01 for either T or DHT). Also shown in Fig. 1Go is the acute augmentation by progesterone of GnRH-stimulated LH secretion; exposure to 20 nM progesterone for 90 min before a pulse of GnRH led to a doubling of the LH secretory response. When the experiment was repeated for cells incubated in the presence of T or DHT for 16 h, the progesterone-induced augmentation was completely suppressed; no significant difference was found regardless of whether progesterone was present in either androgen-treated group.



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  		Figure 1. Integrated LH secretory response to a GnRH pulse. Female pituitary cells cultured in 0.2 nM E2-containing medium for 3 days were challenged with a 15-min pulse of 1 nM GnRH. For androgen-treated groups, cells were exposed to either 1.7 nM T or 0.7 nM DHT beginning 16 h before the GnRH pulse and continuing. For progesterone-treated groups, cells were exposed to 20 nM progesterone beginning 90 min before the GnRH challenge. For this and all subsequent secretion experiments, successive 15-min incubations were collected before, during, and after a secretagogue pulse to monitor LH secretion. The integrated secretory response is calculated as the total amount of LH secreted during the 15-min exposure to GnRH plus that secreted in the subsequent 15 min. Results are expressed as the mean ± SEM from three to eight independent experiments. Bars not sharing the same letter are significantly different from each other (P < 0.01).

 
The latency of onset of this pronounced suppressive effect of androgens on GnRH-stimulated LH secretion was examined in experiments with shorter preexposure times. When DHT was added to the incubation medium 4 h before stimulation with a pulse of GnRH, the LH secretory response was not significantly different from the control value (91 ± 4% of control; n = 3); when the DHT exposure time was extended to 8 h before stimulation of LH secretion, the response was slightly, but significantly, reduced to 81 ± 1% of the control value (n = 3; P < 0.01).

Effect on response to multiple GnRH pulses
To address whether E2 is required for the suppressive action of androgen on GnRH-stimulated LH secretion, pituitary cells cultured in the absence of E2 were subjected to hourly GnRH pulses, which led to repetitive, small amplitude secretory responses. As shown in Fig. 2AGo, exposure to DHT for 16–19 h in the absence of E2 significantly suppressed the response to the first GnRH pulse (P < 0.001) just as was found in the presence of E2 (Fig. 1Go). However, although the response for the control cells remained constant over the three GnRH pulses, the response for the androgen-treated cells increased with each pulse, such that by the third pulse, LH secretion was doubled compared with the response to the first (P < 0.002). The integrated secretory data are presented in Table 1Go.



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  		Figure 2. Time course of the LH secretory response to multiple GnRH pulses. Female pituitary cells were incubated in the absence of E2 (A) or in the presence of 0.2 nM E2 (B) for 3 days. All groups were challenged with three 15-min GnRH pulses (1 nM) at 1-h intervals. DHT (0.7 nM) was present in the indicated groups for 16–19 h before and continuing through the GnRH pulses. Results are expressed as the mean ± SEM from four to seven independent experiments. Note the difference in scale for the ordinates in A and B.

 

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  		Table 1. Suppressive and facilitatory actions of androgen on LH secretion in response to multiple GnRH pulses

 
In contrast to the secretory response in the absence of E2, GnRH self priming was elicited in the control cells with an E2 background (Fig. 2BGo); the response to the third GnRH pulse was 145 ± 2% of that to the first pulse (significantly greater at P < 0.01). When E2-treated cells were exposed to DHT or T (16–19 h), the initial secretory response was significantly suppressed, as demonstrated for the DHT-treated group in Fig. 1Go, but the responses to subsequent GnRH pulses increased (P < 0.002, response to third pulse vs. response to first pulse) similar to that shown in the absence of E2 (Fig. 2BGo and Table 1Go). Although this is reminiscent of a self-priming response, the result showing that estrogen is not required for the apparent facilitatory effect of androgen on GnRH-stimulated LH secretion (Fig. 2AGo) suggested that the androgen action was distinct from GnRH self priming, which is absolutely dependent on a background of E2 for its expression. To further distinguish between the positive action of androgen in the presence of multiple GnRH pulses and GnRH self priming, we repeated the experiments in the presence of RU486, which we have shown to eliminate GnRH self priming even in the absence of progesterone (23). As indicated in Table 1Go, the LH secretory response in DHT-treated cells was unaffected by RU486, whereas the same treatment in the absence of androgen abolished self priming (responses not significantly different from each other within the control plus RU486 group). This result provides further evidence that the facilitatory action of androgen on LH secretion in response to multiple GnRH pulses is unrelated to the self-priming action of GnRH.

Effect on response to pulses of elevated extracellular K+
In the next series, the GnRH receptor was bypassed and LH secretion was elicited with hourly pulses of 54 mM K+ resulting in depolarization and repetitive secretory episodes (Fig. 3Go). The aim was to test whether GnRH receptor activation is required for either the inhibitory or facilitatory actions of androgen on LH secretion. As shown in Fig. 3AGo, exposure to DHT for 16–19 h resulted in a significant and unambiguous suppression of depolarization-stimulated LH secretion to less than 40% of the control value for the first pulse of K+ (P < 0.001). Although the responses in the DHT-treated cells continued to be suppressed compared with those in control cells over the next four pulses of K+, unlike the responses in the controls there was a gradual increase in the LH secretory response for the DHT-treated cells. The rise was significant, as shown by the significantly positive slope of the regression line drawn through the peak secretory responses to the five K+ pulses (P < 0.005). However, the increase seen after multiple depolarization pulses over 5 h was not as dramatic as the 2- to 3-fold increases seen during stimulation with multiple GnRH pulses over 3 h (Fig. 2Go and Table 1Go).



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  		Figure 3. Time course of LH secretory response to multiple K+ pulses. A, Female pituitary cells cultured in 0.2 nM E2-containing medium for 3 days were challenged with 15-min pulses of 54 mM K+ at 1-h intervals. For the androgen-treated group, cells were exposed to 0.7 nM DHT beginning 16–19 h before and continuing through the pulses of K+. B, The protocol was identical to that in A, except that 20-nM progesterone treatment was initiated 90 min before the first pulse of elevated K+. Results are expressed as the mean ± SEM from three or four independent experiments. Note the difference in scale for the ordinates in A and B.

 
Acute progesterone treatment augments K+-stimulated LH secretion (24, 25); therefore, we next asked whether this progesterone effect was susceptible to DHT suppression. Figure 3BGo shows that incubation in the presence of 20 nM progesterone led to an augmentation of the response to depolarizing K+ pulses (note differences in scale for the ordinate in B compared with that in A). This augmentation by progesterone was unambiguously inhibited by preexposure to androgen. As an example for direct comparison, the integrated secretory data for the second K+ pulse for all groups are shown in Fig. 4Go; by 150 min of exposure to progesterone, the depolarization-induced LH secretory response was doubled, but this augmentation was significantly suppressed for cells incubated in DHT before exposure to progesterone.



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  		Figure 4. Integrated LH secretory response to a K+ pulse. The responses to the second pulse of 54 mM K+ for all groups in Fig. 3Go (A and B) are shown as the integrated responses, calculated as the total amount of LH secreted during the 15-min exposure to the second pulse of K+ plus that secreted during the subsequent 15 min. For the progesterone groups, treatment began 150 min before the K+ challenge. Bars not sharing the same letter are significantly different from each other (P < 0.05).

 
Effect on progesterone-enhanced transcription
Because the acute stimulatory action of progesterone could be suppressed by androgen whether the secretagogue was GnRH or depolarization, we questioned whether DHT interfered with progesterone receptor-related transcriptional activation. For these studies, rat pituitary cells were transfected with a plasmid containing three tandem PREs linked to a minimal promoter and a luciferase reporter; although the plasmid would be expected to be taken up by multiple pituitary cell types, in the rat only gonadotropes express the progesterone receptor (26 26A ). As shown in Fig. 5Go, exposure to 20 nM progesterone for 6 h resulted in a 3-fold increase in luciferase activity. When transfected cells were pretreated with DHT for 16 h before and during stimulation with progesterone, the progesterone-stimulated luciferase response was significantly dampened by about 30% (Fig. 5Go). DHT by itself had no effect on luciferase expression. When the progesterone concentration was increased to 200 nM, or exposure to DHT was limited to the acute period during which the cells were exposed to progesterone, there was still a tendency for the progesterone-stimulated luciferase response to be dampened by DHT, but the differences were not significant (data not shown).



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  		Figure 5. Changes in luciferase activity. Female pituitary cells cultured in 0.2 nM E2-containing medium were transfected with TAT3LUC, a plasmid containing three PREs. The day after transfection, cells were challenged for 6 h with medium only (control) or 20 nM progesterone and/or 0.7 nM DHT; cell extracts were assayed for luciferase reporter activity. For the androgen-treated groups, DHT was present for 16 h before and during the 6-h test period. Luciferase activity is expressed as arbitrary light units (ALU), representing the mean ± SEM from four independent experiments. Bars not sharing the same letter are significantly different from each other (P < 0.05).

 
The possibility of a trans-repressor action of DHT at the progesterone receptor was further examined in LH secretion studies in which DHT was either acutely added or removed from the medium 2 h before a GnRH pulse. As shown in Table 2Go for control pituitary cells that have a chronic (16-h) background of DHT exposure, the LH secretory response to a GnRH pulse was suppressed by about 40% regardless of whether the DHT was continued or removed before the GnRH pulse; however, DHT was without significant effect when control cells were exposed to the steroid for only 2 h. For cells responding to the acute augmentation action of progesterone, the temporal pattern of DHT suppression differed, in that 1) removal of DHT 30 min before initiation of the acute 20-nM progesterone treatment resulted in a slight, but significant, abatement in the suppression compared with the response when DHT was continuously present (P < 0.02); and 2) acute addition of DHT just before progesterone exposure is initiated results in a significant decrease in the response to GnRH (75% of that found when DHT is not present; P < 0.001; Table 2Go). Although the maximal effective concentration of progesterone in these cells was in the range of 10-7 M based on augmentation of GnRH-stimulated LH secretion (19), we tested whether increasing the concentration of progesterone would diminish the ability of DHT to interfere with progesterone. As shown in Table 2Go, although there is a slight, but significant, lessening of the chronic DHT-induced suppression in the 200-nM progesterone group (P < 0.005 for 200-nM compared with 20-nM progesterone groups), the GnRH response remained suppressed to about 40%; for acute DHT treatment there were no differences between the two concentrations of progesterone. In conjunction with the transfection studies these results suggest that in addition to the posttranscriptional consequences of DHT’s action, another component of the suppressive effect of DHT may involve interference with progesterone’s action at the transcriptional level.


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  		Table 2. Comparison of suppressive effect of chronic or acute DHT on progesterone augmentation of GnRH-stimulated LH secretion

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the female, androgens can have both negative and positive effects on the regulation of LH production and secretion (1, 2, 3, 4, 5, 6, 7, 8, 27). In this work we demonstrate that both the suppressive action and the stimulatory effect of T or DHT are evident in vitro when LH secretion is elicited from female pituitary cells with pulses of either GnRH or elevated K+.

Inhibitory androgen action
The temporal requirements for the suppression of secretagogue-induced LH secretion by androgens provide some clue to its possible role in the female reproductive cycle. In humans, ovarian androgen secretion varies throughout the cycle, with the peak occurring during the periovulatory period (28, 29, 30). In the rat, the serum T concentration appears to exhibit three phases. The lowest levels are found on estrus; by the first day of diestrus the levels have doubled and stay in that range through the second day of diestrus and the morning of proestrus; from there the levels increase 2- to 3-fold on the afternoon of proestrus beginning about 4 h before the onset of the preovulatory LH surge (16). Using an androgen concentration similar to that observed during the proestrous gonadotropin surge, we found that 8 h, but not 4 h, of exposure in vitro led to only a slight reduction in stimulus-induced LH secretion, but that 16 h of androgen resulted in a 60% decrease in the response. Given this latency, it is unlikely that the negative action of androgen is operable or at least has any physiological consequences during the approximately 4-h duration of the rat LH surge. However, it is not unreasonable to speculate that the timing of the onset of the inhibitory action of androgen would be consistent with a role for the steroid in the termination of the surge. Several candidate mechanisms have been suggested to participate in the termination of the surge (31, 32, 33). Indicative of the potential for redundancy in the system, which would be expected, is a report from 1974 suggesting that antiserum to T administered on the evening before the day of the preovulatory surge was without effect on the LH surge (34).

Facilitatory androgen action
A positive action of androgens on LH in female rats has been shown as a facilitation of GnRH pulse-induced gonadotropin subunit mRNA expression (7, 8). An up-regulation of LHß message was seen after 6 h of GnRH pulses in vivo or 24 h in vitro for pituitary glands or cells that had been exposed to T for 1 day; shorter exposure times to either the steroid or to the GnRH pulse regimens were not examined in these studies (7, 8). In our work we found that after the initial suppressed response in cells incubated in an androgen background, there was a gradual increase in the LH secretory response to GnRH pulses that reached significance by 2 h after the first pulse, and this occurred whether the cells had been cultured in the presence or absence of E2. Whether this correlates with an increase in LH available for secretion remains to be established, but it is interesting that once the androgen background is in place, the relatively short time course for a putative up-regulation of LHß in response to GnRH pulses is consistent with the rapid events occurring during the preovulatory LH surge in rats. The lowest androgen concentration required for the facilitatory effect was not determined in the in vivo work of Yasin et al. (7), but they do report that a pre-LH surge level of T was more effective than higher concentrations. The requirement for pulsatile GnRH delivery to observe a positive modulatory role for androgens could explain why many of the previous studies that used long duration GnRH exposures failed to detect positive androgen actions in female pituitary cells (2, 3, 4, 5, 27).

Androgen and depolarization-induced secretion
In the current study, depolarization of gonadotropes with hourly pulses of elevated extracellular K+ resulted in LH secretion episodes of constant dimensions. On a background of DHT that continued through the K+ challenges, the secretory episodes were reduced by more than half, but by the fourth hour of pulsing there was evidence for some abatement of the suppression. This increase in responsiveness to depolarization-induced calcium influx even in the presence of the inhibitory action of androgen on the secretion process correlates with the observation by Haisenleder et al. that in the presence of T, pulsatile calcium influx stimulated an increase in gonadotropin subunit mRNAs after 24 h of pulsing, the only time point examined (8). Whether the increase in secretion reported in our studies is a reflection of increased available LH remains to be established. It is of interest, however, that, for LH secretion stimulated by either depolarization or GnRH, androgen treatment led to an initial suppression that was gradually overcome with subsequent secretagogue pulses. This indicates that neither the suppressive effect nor the facilitatory action of androgen has an absolute requirement for a change in the GnRH receptor. However, the more rapid time course for the positive response to GnRH pulses compared with depolarization is probably due to activation of pathways in addition to calcium-related signals and perhaps to an increase in GnRH receptor as well. It would be important in future studies to test for a possible effect of androgen on GnRH receptor expression and binding.

Androgen and the acute action of progesterone
Acute progesterone exposure (<6 h) of pituitary cells in culture results in a severalfold increase in the LH secretory response to GnRH (19, 35, 36, 37), and this acute action of progesterone shares common characteristics with the GnRH self-priming effect (22, 23). In the female reproductive cycle, the augmentation by progesterone of stimulated secretion probably plays a role in GnRH signal amplification during the preovulatory gonadotropin surge; therefore, our studies showing that an extended background of elevated androgen levels can interfere with the acute effect of progesterone may have some relevance to the actions of hyperandrogenism in the disruption of cyclicity.

The sites through which androgen achieves this dampening of the acute actions of progesterone on secretagogue-stimulated LH secretion are not known. Based on these studies, however, we can speculate that the primary transcriptional targets of androgen for both the suppression of LH secretion in general as well as the inhibition of progesterone’s ability to augment LH secretion are likely to overlap; these consequences of androgen’s genomic actions could be proteins involved in the proximal secretagogue signaling pathway and/or the LH exocytotic pathways. We suggest that an additional target specific to the acute action of progesterone is interference by androgen with the transcriptional activity of the progesterone receptor.

Androgen receptor
The androgen receptor has been shown to have both trans-activation and trans-repression modes of regulation of gene expression and to use a variety of strategies involving regions outside of its DNA-binding domain to achieve some of its multiple functions (38, 39, 40, 41, 42, 43, 44). In relation to our work showing DHT suppression of progesterone’s augmentation action on stimulated LH secretion, Yen et al. (41) demonstrated in cotransfection studies in CV-1 cells that wild-type androgen receptor can have striking dominant negative activity on glucocorticoid receptor- and progesterone receptor-mediated transcription. For rat pituitary cells, we found that DHT was able to only partially repress progesterone receptor-mediated transcription and that this could be overcome with increased progesterone concentration. The reporter plasmid used for our transfection studies contained PRE sequences similar to that used by Yen et al. (41), but differences in promoter context, e.g. the use of three tandem PREs in the construct we used, as well as cell-specific factors, agonist concentration, and receptor abundance could explain the lack of an effect of androgen alone on trans-activation as well as the relatively modest cross-talk between the androgen and progesterone receptors in our pituitary cell study (38, 45). However, as only one construct was tested, we cannot exclude the possibility of significant androgen receptor interference with the trans-activation function of the progesterone receptor at target genes in gonadotropes. In fact, our secretion results with acute androgen addition or removal in the presence of progesterone suggest that at least part of the DHT-induced abolition of the augmentation effect of progesterone on stimulated LH secretion could be due to a direct inhibitory action at the level of the progesterone receptor as well as to downstream targets, e.g. a short-lived protein specific to the progesterone augmentation pathway.

In summary, the facilitatory and inhibitory functions suggest that androgens have complex modulatory roles in the cyclic regulation of LH secretion that are context dependent, particularly with regard to temporal requirements, GnRH pulse patterns, and the presence of progesterone. Based on the results reported here, a possible scenario during the estrous cycle would include an androgen background-dependent facilitation of GnRH pulse-induced increase in gonadotropin subunit expression that becomes manifest during the preovulatory period as GnRH pulse frequency and/or amplitude increases. The latency of the inhibitory action attributable to the increase in androgen occurring before the onset of the gonadotropin surges is consistent with a role for androgen in the termination of the LH surge. These studies provide the framework for further investigation of the potential multiple targets and interplay of steroids within the gonadotrope for the complex androgen action.


   Acknowledgments
 
We are grateful to Dr. Keith Yamamoto for providing the TAT3LUC plasmid, and to Dr. Stanko Stojilkovic and his colleagues for providing us with a preprint of their work. We thank Coralie Munro for the RIA measurement of progesterone and E2.


   Footnotes
 
1 This work was supported by NIH Grant HD-12137. Back

Received September 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Beyer C, Jaffe RB, Gay VL 1972 Testosterone metabolism in target tissues: effects of testosterone and dihydrotestosterone injection and hypothalamic implantation on serum LH in ovariectomized rats. Endocrinology 91:1372–1375[Medline]
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