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Endocrinology Vol. 139, No. 3 1038-1045
Copyright © 1998 by The Endocrine Society

ARTICLES

The Regulation of Gonadotropin-Releasing Hormone-Induced Calcium Signals in Male Rat Gonadotrophs by Testosterone Is Mediated by Dihydrotestosterone1

V. A. Tobin and B. J. Canny

Department of Physiology, Monash University, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Dr. B. J. Canny, Department of Physiology, Monash University, Clayton, Victoria 3168, Australia. E-mail: ben.canny{at}med.monash.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The biological effects of testosterone (T) may be mediated directly by T or indirectly by its metabolites, dihydrotestosterone (DHT) and estradiol. The present study examined whether the metabolism of T is involved in the regulation of GnRH-induced Ca2+ signaling at the pituitary. In gonadotrophs from castrated rats, a significantly greater percentage of gonadotrophs demonstrated oscillatory Ca2+ responses to 100 nM GnRH than cells from intact rats (72% vs. 24%; P < 0.05). This increase was prevented by the administration of T propionate (0.1 mg/kg·day), DHT benzoate (2 mg/kg·day,), estradiol benzoate (EB; 5 µg/kg·day), or the combination of the above doses of DHT benzoate and EB. In all cases the proportion of gonadotrophs from the steroid-treated rats having oscillatory Ca2+ responses to 100 nM GnRH was between 21–25% (P > 0.05, compared with intact rats). To assess the importance of T metabolism, intact male rats were treated with the aromatase inhibitor letrozole (1 mg/kg·day), the 5{alpha}-reductase inhibitor finasteride (50 mg/kg·day), or their respective vehicles for 7 days. Letrozole had no effect on GnRH-induced Ca2+ signals, serum LH concentrations, or ventral prostate or testes weight. Finasteride treatment, however, mimicked the effects of castration, with significantly more gonadotrophs exhibiting Ca2+ oscillations in response to 100 nM GnRH than gonadotrophs from the vehicle-treated group (71% vs. 20% respectively; P < 0.05). Finasteride also caused a significant (P < 0.05) decrease in prostatic weight and DHT concentration, but had no significant effect on either prostatic T or serum LH concentrations. These findings suggest that in the intact male rat, the effects of T on GnRH-induced Ca2+ signaling are preferentially mediated via DHT. The results of this study also show that in the absence of androgens, estradiol may regulate GnRH-induced Ca2+ signaling in the male rat pituitary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE MALE, gonadal steroids exert an inhibitory effect on gonadotroph function both via the hypothalamus, by regulating the secretion of GnRH into the hypophysial portal circulation (1), and directly at the pituitary gland (2, 3, 4). The mechanisms responsible for the inhibitory action of testosterone (T) at the pituitary may include regulation of the expression of GnRH receptors (5, 6, 7), the efficacy of second messenger signaling pathways (8, 9, 10, 11), and regulation of transcription and/or translation of gonadotropin subunit messenger RNAs (11, 12, 13, 14, 15, 16).

GnRH increases the concentration of free intracellular calcium ions ([Ca2+]i) in gonadotrophs, which has been shown to be crucial for mediating many of the actions of GnRH (17, 18, 19, 20). GnRH generates this increase in [Ca2+]i via both the inositol trisphosphate-stimulated release of Ca2+ from intracellular Ca2+ stores, and Ca2+ influx via plasma membrane calcium channels (21, 22). In both male (23) and female (24, 25, 26, 27) gonadotrophs, there is a complex concentration-dependent relationship between GnRH and the resulting Ca2+ signals. Low concentrations of GnRH induce oscillations in [Ca2+]i in the majority of gonadotrophs. With increasing concentrations of GnRH, the oscillation frequency increases, but fewer cells produce the oscillatory response. The remainder of gonadotrophs show a biphasic or spike-plateau response. We have previously shown that this concentration dependence of GnRH-induced calcium signals is lost after castration, but this loss is prevented if the castrated rats are treated with T from the time of castration (23). Furthermore, we demonstrated that this action of T is mediated directly at the pituitary, rather than by a modulation of hypothalamic GnRH secretion (28).

It is well established that in a variety of tissues, the actions of T may be mediated via the interaction of T with androgen receptors or the interaction of its metabolites dihydrotestosterone (DHT) or estradiol (E2) with androgen or estrogen receptors, respectively. The apparent necessity of aromatization and 5{alpha}-reduction in the control of gonadotroph function in the male varies between experimental paradigms and species. Investigators initially demonstrated the ability of both DHT and E2 to regulate gonadotroph function by in vivo and in vitro studies that involved testing the actions of either metabolite in the absence of T, either by DHT (29, 30, 31) or E2 (30, 31) treatment after castration or by inclusion of either steroid in tissue culture media (4, 32, 33). Under these conditions, both DHT and E2 could regulate gonadotroph function at least as well as T. However, with the development of specific aromatase and 5{alpha}-reductase inhibitors, investigators reexamined the importance of T metabolism for androgen-regulated feedback control of gonadotrophs. Studies with aromatase and 5{alpha}-reductase inhibitors have demonstrated that neither the aromatization of T to E2 (34) nor the reduction of T to DHT (35, 36) is essential for the regulation of LH secretion from gonadotrophs in male rats. The latter observation, in particular, presents a conundrum, as it has been shown that gonadotrophs in the male rat contain considerable 5{alpha}-reductase activity (37). The importance of aromatization or 5{alpha}-reduction to other aspects of gonadotroph function, such as intracellular signaling, has not been examined. This study has, therefore, examined the roles of DHT and E2 in mediating the actions of T on Ca2+ signaling in gonadotrophs from intact and castrated male rats. This was achieved by treating castrated male rats with T propionate (TP), DHT benzoate (DHTB), or E2 benzoate (EB) or by treating intact male rats with the specific 5{alpha}-reductase inhibitor finasteride, the aromatase inhibitor, letrozole or their respective vehicles. The resulting changes in GnRH-induced Ca2+ signaling were assessed using fura-2 microspectrofluorometry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague-Dawley rats (250–350 g; Central Animal Services, Monash University, Clayton, Australia) were housed under constant temperature conditions (22 C), and a 12-h light, 12-h dark cycle (lights on at 0700 h). Rats had free access to water and Purina rat chow (Barastoc, St. Arnaud, Australia). For anesthesia, a mixture of 3 parts xylazine (xylazine hydrochloride; 20 mg/ml; Parnell Laboratories, Alexandria, Australia) to 4 parts Ketamav (ketamine hydrochloride; 100 mg/ml; Mavlab, Slacks Creek, Australia) was given at a dose of 0.08 ml/100 g BW, im.

Experimental design
Exp 1: castration and steroid replacement. Rats were sham castrated or castrated under anesthesia, and treatments commenced immediately and continued for 7 days before death by decapitation and removal of the pituitary gland. Sham-castrated rats were anesthetized and underwent sham castration, where the scrotum was incised then closed without removal of the testes; these animals were then treated with the oil vehicle (0.2 ml sesame oil/day, sc; n = 5). Castrated male rats were treated with one of the following: vehicle (0.2 ml sesame oil/day, sc; n = 5), TP (100 µg/100 g BW in 0.2 ml sesame oil/day, sc; n = 5), EB (10 µg/100 g BW in 0.2 ml sesame oil/day, sc; n = 5), one of two concentrations of DHTB [DHTB1, 0.2 mg/100 g BW in 0.2 ml sesame oil/day, sc (n = 5); or DHTB2, 2 mg/100 g BW in 0.2 ml sesame oil/day, sc (n = 5)], or a combination of EB and DHTB2 (EB, 10 µg/100 g BW; DHTB, 2 mg/100 g BW in 0.2 ml sesame oil/day, sc; n = 5).

Exp 2: removal of DHT in vivo. To determine whether T must first be reduced to DHT via 5{alpha}-reductase to modulate GnRH-induced calcium signals in vivo, intact male rats were treated with the specific 5{alpha}-reductase inhibitor finasteride. Rats were treated daily with either finasteride (50 mg/kg in 0.2 ml 80% triolene-20% ethanol for 7 days; n = 10) or vehicle (0.2 ml of 80% triolene-ethanol 20% for 7 days; n = 10). This dose of finasteride was that recommended by Merck, Sharpe, and Dohme (Rahway, NJ), and lower doses have been demonstrated to be effective in inhibiting prostatic growth in adult male rats (38).

Exp 3: removal of E2 in vivo. To determine whether the modulation of GnRH-induced Ca2+ signals by T depended on the aromatization of T to E2, intact male rats were treated with the specific aromatase inhibitor letrozole. Intact male rats were treated daily by oral gavage with either letrozole (1 mg/kg in 0.2 ml 0.5% carboxymethylcellulose for 7 days; n = 4) or vehicle (0.2 ml 0.5% carboxymethylcellulose for 7 days; n = 4). This dose of letrozole was recommended by Ciba Geigy (Summit, NJ) and has been previously demonstrated to lead to a significant decrease in the concentration of E2 and an increase in the concentration of LH in the serum of female rats (39).

Ca2+ measurements
On a given experimental day, two animals from different groups within the same experiment were killed, and a separate single cell suspension was made of each pituitary gland. To achieve this, each pituitary gland was cut into approximately 1-mm3 pieces and dispersed using trypsin (0.2%, 60 min, 37 C) as previously described (23). Cells were resuspended in a standard buffer containing 117 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.8 mM CaCl2, 0.5 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, 10 mM glucose, and 0.1% BSA, pH 7.4, and plated onto poly-L-lysine (0.01%)-coated coverslips that formed the bases of temperature-controlled baths. The cells were incubated with fura-2/AM (1 µM, 20 min, 37 C) and then washed in the standard buffer only (20 min, 37 C). Intracellular Ca2+ measurements were made by illuminating the cells in a bath mounted on the stage of an inverted microscope (TMD, Nikon, Tokyo, Japan) that was attached to a SPEX Fluorolog system (SPEX Industries, Edison NJ) configured for Ca2+ measurement using fura-2. Single cells, chosen on the basis of a characteristic gonadotroph morphology (~25 µm in diameter and asymmetrical in appearance), were optically isolated, and changes in [Ca2+]i were recorded as changes in the fluorescent emission (510 nm) of fura-2 in response to 340- and 380-nm wavelength excitation light. All Ca2+ measurements were conducted between 4–10 h after obtaining the pituitary gland.

The experiments were conducted at 37 C in the standard buffer. The basal [Ca2+]i was recorded for 30 sec, after which time GnRH (100 nM, in standard buffer; Auspep, Melbourne, Australia) was added to the bath. This concentration of GnRH was used, as we have shown in a previous study (23) that the effect of castration on GnRH-induced Ca2+ signals in male rat gonadotrophs is most readily detected using high concentrations of GnRH. GnRH was added via a 26-gauge syringe needle positioned, using a micromanipulator, next to the cell to be studied. Approximately 100 µl GnRH-containing medium were added to a bath volume of 400 µl. The identity of the chosen cell was confirmed as a gonadotroph by an increase in [Ca2+]i in response to GnRH. In a previous study, approximately 95% of the cells that showed GnRH-induced increases in [Ca2+]i contained LHß, as detected by immunocytochemistry (23).

Verification of treatments
In Exp 1, 2, and 3, the ventral prostate and the spleen were removed and weighed to assess the effectiveness of the treatments. In Exp 2 and 3, the testes were also removed and weighed.

Serum hormone assays
In all three experiments, at the time of decapitation trunk blood was collected and allowed to clot at 4 C for 24 h. The blood was centrifuged, and the serum was removed and stored at -20 C until assayed for LH or E2.

Serum LH was measured using a previously described RIA protocol (40), with NIDDK rat LH RP-3 as standard. All samples were measured in two assays with an intraassay coefficient of variation (CV) of 13% and an interassay CV of 15%.

Serum E2 was measured in the rats from Exp 3 using a previously described RIA procedure (41) modified for rat serum. All samples were measured in one assay with an intraassay CV of 11%.

Prostatic DHT and T assay
As the pituitaries were required for the Ca2+ experiments, to demonstrate the effect of finasteride treatment on the relative levels of DHT and T, the concentrations of both were measured in the prostates collected from the rats at the time of decapitation. Finasteride has been shown to significantly decrease both the considerable 5{alpha}-reductase activity of the ventral prostate and the 5{alpha}-reductase activity of the pituitary of adult male rats (35).

As most antibodies used in T RIAs have a high cross-reactivity with other androgens, in particular DHT, the androgens were separated by HPLC before determination of T and DHT concentrations by RIA. The protocol employed was a modification of that developed by O’Donnell et al. for the measurement of androgen concentrations in testes (42). The adapted protocol is as follows. After weighing, the prostates were frozen in liquid nitrogen and stored at -70 C until assayed. Prostates were thawed and homogenized individually in 60% acetonitrile, 0.1% trifluoroacetic acid (TFA), and approximately 5000 cpm each of radiolabeled [1,2,6,7-N-3H]T (DuPont-New England Nuclear, Boston, MA), [1{alpha},2{alpha}-N-3H]DHT (Amersham Life Sciences, Castle Hill, Australia) and [9,11-N-3H]androstenediol (DuPont-New England Nuclear, Melbourne, Australia). The homogenates were centrifuged (20 min, 10,000 rpm, 4 C), and the supernatant decanted and stored on ice. The pellet was resuspended in the homogenizing buffer and recentrifuged. The supernatants from both centrifugation steps from the same homogenates were combined and diluted 1:3 in 1% TFA in HPLC grade deionized H2O (MilliQ system, Millipore, Milford, MA), then loaded onto Sep-Pak C18 cartridges (Millipore, Waters). The androgens were eluted with 0.1% TFA in 60% acetonitrile, lyophylized, and stored at -20 C until further extracted. The samples were later resuspended in 200 µl HPLC buffer (0.1% TFA in 40% acetonitrile) and centrifuged (30 min, 4000 rpm, 4 C), and the supernatants were transferred to microfuge tubes.

A Waters µBondapak C18 column (30 x 0.39 cm; Millipore) and guard column were allowed to equilibrate in the HPLC buffer (30 min, 1 ml/min) before each sample was loaded. After the samples were loaded, the column was perfused with the HPLC buffer at 1 ml/min, and 0.5-ml fractions were collected. Once the retention times (17.5 min for T, 28.5 min for androstenediol, and 35.5 min for DHT) and the complete separation of the androgens was established using homogenates of prostates collected from other intact male rats, only radiolabeled [3H]T and [3H]DHT were included in the original homogenizing buffer. To determine the recoveries of each androgen, 2 ml scintillation fluid (Ecoscint, National Diagnostics, Atlanta, GA) were added to 50-µl samples of each fraction, and these were counted for 10 min/vial (Beckman ß-counter, Palo Alto, CA). The recoveries of radioactive standards were 42 ± 9% for T and 44 ± 3% for DHT (mean ± SEM; n = 28 runs). The appropriate fractions were then pooled and dried down overnight (Speed-Vac concentrator, Savant, Farmingdale, NY) and stored until assayed for T and DHT (-20 C).

The intraprostatic T and DHT concentrations were determined using the androgen RIA also developed by O’Donnell et al. (42). The primary antibody COX 0457 (Sirosera, Sydney, Australia) was diluted 1:50,000 in 1:800 normal sheep serum. Otherwise the assay protocol was unchanged. All samples were assayed in the one run, and the within-assay CV was 13%. The values shown in Results have been corrected for the removal of a 50-µl sample from each fraction to determine the recoveries of T and DHT standards, and the values were also corrected by the recovery percentages to compensate for the inconsistent losses of samples over the extraction procedures to give the prostatic T or DHT levels in nanograms per g prostate.

Statistical analysis
GnRH-induced Ca2+ signals consisted of either oscillatory signals, which were defined as having two or more spikes of greater than 2 fluorescence ratio units in amplitude or spike-plateau signals where the response was a single spike followed by a gradual decline to a plateau level greater than the prestimulatory baseline. As we have previously shown that little day to day variation exists in the response of male rat gonadotrophs to 100 nM GnRH (23, 28), the results from each experimental animal were pooled to give an overall proportion of gonadotrophs from each experimental group demonstrating a GnRH-induced oscillatory Ca2+ response to 100 nM GnRH. Within each experiment, these proportions were compared using {chi}2 analysis, with the Bonferroni correction applied to the analysis of Exp 1.

The effects of the treatments in Exp 1 on serum LH concentrations and organ weights (corrected for the body weight of each animal) were compared using one-way ANOVA. Differences between the treatment groups and the castrate and oil group were determined using Dunnett’s test. The effects of treatments in Exp 2 and 3 on serum LH and organ weights (corrected for the body weight of each animal) and in Exp 3 prostatic DHT and T and serum E2 were determined using Student’s t test for unpaired samples. In all cases, P < 0.05 was considered statistically significant.

Materials
GnRH was obtained from Auspep (Australia), fura-2/AM was purchased from Molecular Probes (Eugene, OR), Xylaze was obtained from Parnell Laboratories, and Ketamav from Mavlab. Letrozole was generously provided by Ciba-Geigy, and finasteride was donated by Merck, Sharpe, and Dohme (Rahway, NJ). The HPLC grade acetonitrile was obtained from Malinckrodt (St. Louis, MO). The T tracer used in the androgen assay was [125I]histamine-T, obtained from the Department of Clinical Biochemistry Monash Medical Center (Clayton, Australia). TP, DHTB, and EB were purchased from Sigma Chemical Co. (St. Louis, MO) as were all other reagents unless otherwise stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: castration and steroid replacement
As expected, there was a significant increase in the serum concentration of LH after castration and removal of the negative feedback provided by T (Table 1Go). There was also a significant decrease in the average weight of the androgen-dependent organ, the ventral prostate. The decrease in the weight of the ventral prostate was prevented by the TP treatment, the higher dose of DHTB, and the combination of EB and the higher dose of DHTB (Table 1Go). The postcastration increase in serum LH, however, was prevented by all of the steroid treatments, except the lower dose of DHTB. The bioactivity of the preparation of DHTB used in the present study was assessed by administering a dose of 32/mg day for 7 days to chronically castrated male rams. This treatment, as previously established (43), abolished LH pulses over a 3-h sampling period and significantly reduced the mean concentrations of LH in the serum compared with the serum LH concentration measured over a 3-h sampling period in the same rams before DHTB treatment (data not shown).


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  		Table 1. Effect of castration and steroid replacement on the ventral prostate and the serum LH concentration in male rats

 
As we have previously shown (23), after castration there was a significant increase in the percentage of gonadotrophs that responded to 100 nM GnRH with the oscillatory Ca2+ signal compared with the percentage of gonadotrophs from the sham castrate plus oil-treated rats (Fig. 1Go). Treatment of castrated male rats with TP from the time of castration prevented the castration-induced increase in the proportion of gonadotrophs with oscillatory Ca2+ signals to 100 nM GnRH. Indeed, the castration-induced change in the response to GnRH was prevented by the administration of EB or the higher dose of DHTB (DHTB2), whether given alone or in combination (Fig. 1Go). The lower dose of DHTB was, however, ineffective in changing GnRH-induced Ca2+ signals in gonadotrophs of castrated rats. A typical example of GnRH-induced Ca2+ signals from each of the treatment groups is shown in Fig. 1Go.



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  		Figure 1. Effect of castration and steroid replacement on the number and proportion of cells exhibiting different GnRH (100 nM)-induced [Ca2+]i signals in gonadotrophs from male rats. Rats were castrated and treated daily for 7 days from the day of castration with sesame oil (1 ml/kg) or one of the following steroid preparations: TP (100 µg/100 g BW), EB (5 µg/100 g BW), DHTB1 (0.2 mg/100 g BW), DHTB2 (2 mg/100 g BW), or EB (5 µg/100 g BW) and DHTB2 (2 mg/100 g BW). Sham-castrated rats were treated for 7 days with sesame oil alone as an additional control. Five rats were used in each group. A, Typical [Ca2+]i signals from single gonadotrophs in response to 100 nM GnRH from each of the seven groups. There was no effect of any treatment on the amplitude of the Ca2+ responses. B, The percentage of gonadotrophs from each group showing oscillatory Ca2+ signals to 100 nM GnRH is indicated, and the number in parentheses shows the number of gonadotroph cells recorded to give these results. The asterisk denotes a significant (P < 0.05) difference in the percentage compared with that found in the control (sham-castrated+vehicle) group.

 
Exp 2: removal of DHT in vivo
Exp 1 demonstrated that DHT replacement, like T, can modulate GnRH-induced calcium signals in gonadotrophs from castrated male rats that have no endogenous androgens. To study the relative importance of the reduction of T to DHT, intact male rats were treated with the 5{alpha}-reductase inhibitor, finasteride. This treatment selectively reduced the concentrations of DHT, allowing measurement of the actions of T and its metabolite E2 in the absence of DHT on the hypothalamo-pituitary axis. As has been previously reported (35), serum LH levels were not different in the finasteride-treated animals compared with those in the vehicle-treated control rats (Table 2Go).


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  		Table 2. Effect of finasteride on the androgen-dependent organ weights and serum LH concentration in intact male rats

 
The finasteride treatment was successful, however, in blocking the conversion of T to DHT, as prostate weights were significantly decreased in animals treated with finasteride (P < 0.05; Table 2Go) compared with those in vehicle-treated animals. The 30% decrease in ventral prostate weight observed is similar to that reported by other workers (44). In keeping with this finding there was also a significant reduction in the prostatic DHT concentrations in the finasteride-treated animals (P < 0.05; Fig. 2Go). The mean prostatic T concentration of finasteride-treated rats was approximately double that of the respective controls, although this difference did not attain statistical significance (P < 0.1; Fig. 2Go). There was no change in the weight of the testes (Table 2Go).



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  		Figure 2. Effect of finasteride treatment of intact rats on the number and proportion of gonadotrophs exhibiting different GnRH (100 nM)-induced [Ca2+]i signals and prostatic T metabolism. Intact male rats were treated with finasteride (50 mg/kg·day in 80% triolene-20% ethanol; n = 10) or vehicle (0.2 ml/day 80% triolene-20% ethanol; n = 10). A, Typical Ca2+ signals in response to 100 nM GnRH in single gonadotrophs from the two treatment groups. There was no effect of finasteride treatment on the amplitude of the Ca2+ responses. B, The percentage of gonadotrophs showing oscillatory Ca2+ signals in response to 100 nM GnRH is indicated, and the number in parentheses shows the number of gonadotroph cells recorded to give these results. C, The prostatic DHT concentrations from each treatment group described above, expressed as nanograms per g prostate. D, The prostatic T concentrations from each treatment group described above, expressed as nanograms per g prostate. The asterisk denotes a significant (P < 0.05) difference between the values obtained for the two groups.

 
Finasteride treatment of intact male rats did, however, mimic the effect of castration on GnRH-induced Ca2+ signals. A significantly greater proportion of gonadotrophs from finasteride-treated intact male rats showed an oscillatory [Ca2+]i signal in response to 100 nM GnRH compared with gonadotrophs from intact male rats treated with vehicle (P < 0.05; Fig. 2Go). It would appear, therefore, that the GnRH-induced Ca2+ signals are preferentially regulated by DHT, rather than T.

Exp 3: removal of E2 in vivo
Exp 1 demonstrated that E2, like T, can modulate GnRH-induced calcium signals in castrated male rats. We, therefore, used the nonsteroidal aromatase inhibitor letrozole in intact male rats to determine whether T modulates GnRH-induced calcium signals after its aromatization to E2.

Letrozole treatment had no effect on the average weights of either the testes or the ventral prostates or on the serum LH levels (Table 3Go) of the male rats. In addition, treating male rats with letrozole did not affect GnRH-induced Ca2+ signals (Fig. 3Go). We were also unable to demonstrate that letrozole caused a significant reduction in serum E2 concentrations in male rats (data not shown), as has been previously reported for a related aromatase inhibitor in male rats (34). This lack of effect may be a reflection of the normally low concentrations of E2 in male rat serum.


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  		Table 3. Effect of letrozole on androgen-dependent organ weights and serum LH concentration in intact male rats

 


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  		Figure 3. Effect of letrozole treatment on the number and proportion of cells exhibiting different GnRH (100 nM)-induced [Ca2+]i signals in gonadotrophs from intact male rats. Intact rats were treated with letrozole (1 mg/kg·day in carboxymethylcellulose; n = 4) or vehicle (0.2 ml/day carboxymethylcellulose; n = 4). A, Typical Ca2+ signals in response to 100 nM GnRH in single gonadotrophs from the two treatment groups. B, The percentage of gonadotrophs showing oscillatory Ca2+ signals in response to 100 nM GnRH is indicated, and the number in parentheses shows the number of gonadotroph cells recorded to give these results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has shown that in intact male rats, the regulation of GnRH-induced Ca2+ signals by T is preferentially mediated by DHT. In addition, E2 can modulate GnRH-induced Ca2+ signals in the absence of T, but it is not clear whether E2 is involved in the action of endogenous T. We were unable to demonstrate that conversion of T to E2 was inhibited in the letrozole-treated rats, and therefore, we must rely on previously published reports that the dose of letrozole administered is effective in inhibiting aromatase activity (34, 39). If this is so, it seems unlikely that aromatization of T is critical in regulating GnRH-induced Ca2+ signals in intact male rats. We have also confirmed our previous observations that castration alters the pattern of Ca2+ signals seen in gonadotrophs in response to GnRH, and that in vivo TP treatment from the time of castration prevents this change (23). As the inhibition of the metabolism of T to DHT had no effect on serum LH concentrations, these findings suggest that the androgen feedback control of GnRH-induced Ca2+ signals and that of LH secretion occur via separate mechanisms.

The 5{alpha}-reduction of T appears to be a critical step in the regulation of GnRH-induced Ca2+ signals. The first experiment in the present study established the efficacy of DHT in suppressing the castration-induced increase in serum LH concentrations and the change in Ca2+ signaling. Although the dose of DHT required in the present study to modulate serum LH concentrations and prostatic weights was greater than that reported by other workers (45) and prompted the test for biological activity of the DHTB used in this study, it is important to note that GnRH-induced Ca2+ signaling was only affected by a dose of DHT that was also successful in preventing the effects of castration on serum LH concentrations and prostatic weights. The anterior pituitary and gonadotrophs in particular have very high levels of 5{alpha}-reductase activity (37), suggesting that the metabolism of T plays a role in gonadotroph function. To investigate this possibility, intact male rats were treated with finasteride to prevent the local production of DHT. The effectiveness of finasteride treatment was established by the resulting decrease in the weight of the ventral prostate and in prostatic DHT concentrations, and we assume that a similar inhibition of 5{alpha}-reductase activity would be observed in the pituitary, as has been previously reported (35). Removing DHT did not alter serum LH concentrations, as has been previously reported (35, 36), suggesting that T plays a more important role than DHT in regulating LH levels in male rats. It is important to note that treatment of males, both human and rat, with 5{alpha}-reductase inhibitors has been previously demonstrated to either have a minimal effect (38, 46, 47), as in the present study, or to increase T levels (48, 49, 50). Finasteride treatment did significantly decrease prostatic DHT and produced a castration-like change in GnRH-induced Ca2+ signals, despite levels of T not significantly different from those in untreated male rats. This suggests that DHT plays a more important role than T in modulating GnRH-induced Ca2+ signals in male rat gonadotrophs. In a variety of tissues, both T and DHT act via the androgen receptor, with reported differences in potency thought to be due to different stabilities of the steroid-receptor complex when bound to T or DHT (51). Recently, however, it has been shown that prostatic epithelial cell function and apoptosis of prostate cells are differentially regulated by T and DHT (52), a finding which implies that individual genes may have different sensitivities to the activated androgen receptor complex, depending on the specific androgen bound. Although any actions of T at the hypothalamus in regulating LH secretion cannot be excluded, the present studies also suggest differential regulation of cellular function by T and DHT in gonadotrophs, with DHT preferentially regulating Ca2+ signaling, whereas T regulates LH secretion.

Despite the abundance of 5{alpha}-reductase activity in the male rat anterior pituitary, its function has to date been somewhat cryptic. As stated above, the inhibition of 5{alpha}-reduction does not lead to an elevation of LH levels in male rats (35, 36), and in vitro experiments also generally suggest the 5{alpha}-reduction is of little importance in the regulation of gonadotroph function (53). The present study, therefore, hints at a possible function of the 5{alpha}-reductase in the pituitary, viz. the regulation of GnRH-induced Ca2+ signaling. Although it is clear that increases in Ca2+ are obligatory for the expression of GnRH action at the gonadotroph (54), the precise cellular function of oscillatory vs. spike-plateau Ca2+ responses remains obscure. Spike-plateau Ca2+ responses are most readily seen in the gonadotrophs of intact rats when challenged with high (supraphysiological?) concentrations of GnRH, whereas more physiological concentrations generate predominantly oscillatory responses. In gonadotrophs of castrated rats, however, the oscillatory Ca2+ response persists at all concentrations of GnRH (23). It was initially hypothesized that the oscillation and spike-plateau responses were linked to the secretion of LH from gonadotrophs (24), although there is mounting evidence, from the present and a previous study (28), that the relationship between Ca2+ responses and LH secretion can be disrupted. In addition, exocytosis has been directly measured from gonadotrophs that have Ca2+ oscillations (55), and a recent study (56) suggested that the mobilization of Ca2+ from intracellular stores, which occurs with both Ca2+ oscillations and spikes-plateaux, is the critical step in the initiation of exocytosis. It, therefore, seems highly unlikely that the various Ca2+ signals in gonadotrophs are related in a simple, direct fashion to LH secretion. In other cell types, oscillatory or spike-plateau Ca2+ responses have been specifically linked to the activation of mitochondrial enzymes, gene expression, and cellular differentiation (reviewed in Ref.57). It is not clear if any of these or other functions of the gonadotroph are regulated by Ca2+ oscillations and/or DHT.

In contrast, the metabolism of T to E2 does not appear to be important in the regulation of GnRH-induced Ca2+ signaling. The castration-induced changes in both GnRH-induced Ca2+ signaling and serum LH concentrations were prevented by the administration of E2. This latter finding is in keeping with previous observations (6) and suggests that E2 may play a role in the regulation of gonadotroph function in males. It has been recently demonstrated that E2 treatment in vitro can modulate aspects of Ca2+ homeostasis in a variety of gonadotroph preparations, including female ovine cells in primary pituitary culture and clonal gonadotroph cell lines. E2 treatment has been shown to regulate inositol trisphosphate production by female rat gonadotrophs (58) and {alpha}T3-1 cells (58, 59). In addition, treatment with E2 in vitro can regulate the amplitude of Ca2+ conductance, in a time-dependent fashion, in ovine gonadotrophs (60). Numerous studies have demonstrated, however, that the relative importance of the aromatization of T to E2 in the modulation of gonadotroph function in the male varies among species. Although male sheep (61), humans (34), and nonhuman primates (62, 63) treated with aromatase inhibitors demonstrate significant increases in circulating gonadotropins, rats do not (34), and consequently, E2 is not thought to play an important role in providing negative feedback control in the male rat. The results of the present study show that there was no change in serum LH levels in intact male rats treated with letrozole, and we have extended the observations with this compound by showing that it also failed to affect GnRH-induced Ca2+ signals.

In summary, this study has demonstrated the importance of the conversion of T to DHT in the regulation of GnRH-induced Ca2+ signals in anterior pituitary gonadotrophs. As such, they hint at an important function of the abundant 5{alpha}-reductase activity found in the anterior pituitary gland. As the changes in GnRH-induced Ca2+ signaling observed after finasteride treatment appear separate from the regulation of LH secretion, the present findings leave open the precise role of the Ca2+ signals in gonadotroph function and suggest that the modulation of GnRH-induced Ca2+ signals by T is not an important element in its negative feedback control of LH secretion. The findings of the present study provide new avenues for investigation of the importance of 5{alpha}-reduction of T and Ca2+ signaling in the regulation of reproductive function in males.


   Acknowledgments
 
The authors thank Dr. Paul Farnworth for his assistance with the LH assay, Dr. Alan Tilbrook for his assistance in verifying the bioactivity of the DHTB and helpful discussions, Drs. Peter Stanton and Liza O’Donnell for assistance with the determination of prostatic T and DHT, Elise Coghill for performing the rat serum E2 assay, and Dr. Rick Lang for helpful discussions. We also thank both Ciba-Geigy and Merck, Sharpe, and Dohme for their generous gifts of letrozole and finasteride, respectively.


   Footnotes
 
1 A preliminary report of this work was presented at the 10th International Congress of Endocrinology, San Fransisco, CA, June 12–15th, 1996. Back

Received September 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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