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Testosterone Acts Directly at the Pituitary to Regulate Gonadotropin-Releasing Hormone-Induced Calcium Signals in Male Rat Gonadotropes¹

Department of Physiology, Monash University, Clayton, Victoria 3168, Australia; Research Unit for Molecular Reproductive Endocrinology, Department of Chemical Pathology, University of Cape Town, South Africa

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

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We have recently shown that castration alters GnRH-induced calcium (Ca²⁺) signaling in the gonadotropes of male rats. Instead of generating spike-plateau Ca²⁺ responses to high concentrations of GnRH (100 nM), the majority of gonadotropes from castrated rats have oscillatory Ca²⁺ responses, which are generally only seen with low concentrations of GnRH in the gonadotropes of intact rats. This change in the nature of GnRH-induced Ca²⁺ responses is prevented by in vivo testosterone treatment. The aims of the present study were, therefore, to determine if testosterone acts directly at the pituitary or via the regulation of hypothalamic GnRH secretion. Accordingly, castrated male rats were treated with a GnRH antagonist to ablate the effects of increased GnRH secretion at the pituitary gland. GnRH antagonist treatment (10 µg/100 g BW, twice daily for 7 days from the time of castration) decreased the concentration of LH in the serum of castrated rats (0.4 ± 0.1 ng/ml vs. 11.2 ± 0.4 ng/ml in untreated castrated rats, mean ± SEM) but had no effect on the proportion of gonadotropes having oscillatory Ca²⁺ responses to 100 nM GnRH when compared with untreated castrated rats (63% in antagonist-treated castrated rats vs. 70% in untreated castrated rats). The GnRH antagonist treatment did not, however, interfere with the ability of in vivo testosterone treatment (100 µg/100 g body weight/day) to decrease the proportion of gonadotropes having oscillatory Ca²⁺ responses to 100 nM GnRH (26% in testosterone-treated rats vs. 25% in testosterone and antagonist-treated rats). These findings indicate that testosterone acts directly at the pituitary, and not by altered GnRH secretion, to modulate GnRH-induced Ca²⁺ signals. To confirm this suggestion, cultured gonadotropes of castrated male rats were treated in vitro with 10 nM testosterone. Testosterone treatment for twelve, but not 4 h, restored the proportion of gonadotropes having oscillatory Ca²⁺ responses to that seen in gonadotropes from intact rats. The in vitro effects of testosterone over 12 h were prevented by concomitant treatment with the protein synthesis inhibitor cycloheximide (10 µM), which, when given alone, had no effect on GnRH-induced Ca²⁺ signals in cells from castrate male rats. Taken together, these findings suggest that testosterone has a direct genomic action at the pituitary to regulate GnRH-induced Ca²⁺ signals, via a process that involves new protein synthesis.

	Introduction

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GnRH REGULATES the synthesis and release of the gonadotropins, LH, and FSH, from gonadotrope cells of the anterior pituitary, which, in turn, regulate gametogenesis and hormone production by the gonads. In the male, the gonadal hormones testosterone and inhibin feedback at the hypothalamus and/or the pituitary gland to regulate the secretion of GnRH and the gonadotropins. While debate continues about the precise cellular mechanisms of action of GnRH, it is widely accepted that an increase in intracellular calcium ion concentration ([Ca²⁺]_i) is essential for the transduction of GnRH’s effects (1, 2, 3, 4). This increase in [Ca²⁺]_i occurs as a result of the mobilization of inositol-1,4,5 trisphosphate (InsP₃)-sensitive intracellular Ca²⁺ stores and the influx of extracellular Ca²⁺ via voltage-operated calcium channels (VOCCs) (5). The resulting patterns of changes in [Ca²⁺]_i following GnRH are complex and are dependent on the concentration of GnRH applied. Low concentrations of GnRH generate multiple oscillations in [Ca²⁺]_i in the majority of cells (the so-called oscillatory response), whereas high concentrations lead to a rapid increase in [Ca²⁺]_i, which is followed by a plateau phase above the preceding baseline (spike-plateau response) (1, 6, 7, 8, 9, 10).

A number of recent studies have suggested that, in addition to their well described actions on GnRH synthesis and secretion, gonadal hormones may regulate GnRH-induced changes in [Ca²⁺]_i in gonadotropes. These studies, using both in vivo and in vitro experimental paradigms, have shown effects on both the Ca²⁺ mobilization and Ca²⁺ influx phases of GnRH-induced Ca²⁺ responses. In the in vitro experiments, estradiol regulated the GnRH-induced mobilization of intracellular Ca²⁺ stores in both rat and ovine gonadotropes, and this action may be further modulated by progesterone or inhibin (7, 8). Estradiol has also been shown to modulate the influx of extracellular Ca²⁺, most probably via the regulation of VOCCs(11). In addition, GnRH-induced Ca²⁺ signals vary across the estrous cycle of the rat, and appear to change after ovariectomy (12). Previously, we have reported that following castration of male rats, there is a change in the nature of GnRH-induced Ca²⁺ responses in gonadotropes, and these changes are prevented by in vivo testosterone treatment (10). In gonadotropes from castrated rats there was no relationship between the concentration of GnRH applied and the Ca²⁺ signal generated. Over 70% of gonadotropes exhibited oscillatory Ca²⁺ responses to all GnRH concentrations tested (up to 1 µM), whereas only 15–25% of the gonadotropes from intact rats showed oscillatory responses to high concentrations of GnRH. While the in vitro studies discussed above suggest that the gonadal hormones may act directly at the pituitary to modulate GnRH-induced Ca²⁺ signaling, it was not clear from our in vivo studies whether the action of testosterone is directly at the pituitary, or via the regulation of GnRH secretion from the hypothalamus. A number of postcastration changes in gonadotropes have been linked to heightened GnRH secretion resulting in changes in GnRH-induced gene expression (13, 14, 15, 16, 17). Accordingly, the focus of the present study was to use both in vivo and in vitro models to determine the site of action of testosterone in regulating GnRH-induced Ca²⁺ signaling.

To examine the action of testosterone in modulating GnRH-induced Ca²⁺ signals, we treated castrate male rats with a GnRH antagonist with and without testosterone, from the time of castration, and recorded GnRH-induced Ca²⁺ signals in acutely dispersed gonadotropes. We also treated cultured anterior pituitary cells from castrate male rats with testosterone with and without the protein synthesis inhibitor cycloheximide in vitro and examined GnRH-induced Ca²⁺ signals.

	Materials and Methods

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Animals
Adult male Sprague-Dawley rats (150–200 g, Central Animal Services, Monash University) were housed under constant temperature conditions (22 C), and a 12-h, 12-h light-dark cycle (lights on 0700 h). Rats had free access to water and Purina Rat Chow (Barastoc, St. Arnaud, Victoria). The rats were anesthetized with a mixture of three parts Xylazine (Xylazine hydrochloride; 20 mg/ml; Parnell Laboratories, NSW, Australia) to four parts Ketamav (Ketamine hydrochloride; 100 mg/ml; Mavlab Pty. Ltd., Queensland, Australia), given at a dose of 0.08 ml/100 g BW im.

Exp 1: in vivo treatments
Seven days before experimentation rats were castrated under anesthesia. The rats immediately commenced treatment for the week with one of the following: 1) GnRH antagonist 27 ([Ac-d-Nal(2)¹, d--Me-pCl-Phe², d-Trp³, N--Ipr-Lys⁵, d-Tyr⁶, d-Ala¹⁰]GnRH; (GnRHA) (18); 10 µg/100 g BW in 0.2 ml 2.5% DMSO in saline, twice a day, sc; n = 6) and oil vehicle; 2) testosterone propionate (TP; 100 µg/100 g BW in 0.2 ml sesame oil/day sc; n = 6) and saline vehicle; 3) both GnRHA (10 µg/100 g BW in 0.2 ml 2.5% DMSO in saline, twice a day, sc) and TP (100 µg/100 g BW in 0.2 ml sesame oil/day sc; n = 6); or 4) both oil and saline vehicles (n = 6).

Ca²⁺ measurements
Pituitary glands were dispersed into single cells using trypsin (0.2%) as previously described (10). Cells were suspended in buffer A containing (in mM) NaCl (117), KCl (5), MgCl₂ (2), CaCl₂ (1.8), KH₂PO₄ (0.5), NaHCO₃ (5), N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES) (10), glucose (10), and BSA (0.1%), pH 7.4) and plated onto poly-L-lysine (0.01%) coated coverslips that formed the bases of temperature-controlled baths. The cells in each bath were loaded with fura-2/AM (1 µM, 20 min, 37 C) and then underwent a wash period with buffer A (20 min, 37 C). The GnRH-induced [Ca²⁺]_i signals in single gonadotropes were measured using standard microfluorimetry techniques for the calcium-sensitive probe fura-2/AM (10). Briefly, each potential gonadotrope was chosen on the basis of a characteristic morphology, with the cells chosen being larger than most other anterior pituitary cells, and while being essentially spherical, had a number of small membrane protrusions. Changes in [Ca²⁺]_i were monitored by recording the fluorescent emission (510 nm) of fura-2 in response to 340 and 380 nm wavelength excitation light. The ratio of these emissions was calculated off-line and used as an index of a change in [Ca²⁺]_i. Gonadotropes were subsequently defined as those cells showing an increase in [Ca²⁺]_i in response to GnRH. We have previously demonstrated (10) that of the cells selected using these criteria, approximately 95% are immunopositive for LHß and have Ca²⁺ responses to GnRH. When choosing cells from castrated rats, care was taken to choose cells that had roughly the same characteristics as gonadotropes from intact rats, avoiding cells that were very large or had gross distortions from a spherical shape. There was no apparent difference in the proportion of gonadotropes having a Ca²⁺ response to GnRH between any of the experimental groups, including cells from the GnRH antagonist-treated groups that had none of the castration-induced changes in gonadotrope morphology.

Ca²⁺ measurements were conducted at 37 C in buffer A. The basal [Ca²⁺]_i was recorded for 30 sec, after which time GnRH (100 nM, Auspep, Melbourne, Australia) was added to the bath. GnRH was added via a 26G syringe needle positioned, using a micromanipulator, next to the cell to be studied. Approximately 100 µl of 100 nM GnRH-containing buffer A was added to a fixed bath volume of 400 µl. A one-hundred nanomolar concentration GnRH was used in all experiments because this concentration reveals the greatest differences between gonadotropes (10).

Exp 2
As an initial test of a direct action of testosterone in regulating GnRH-induced Ca²⁺ signals, cells from castrated male rats were obtained and plated as above, and treated with either testosterone (10 nM in 0.2% EtOH in buffer A) or 0.2% EtOH in buffer A alone for 200 min before, and during, the loading with fura-2/AM (1 µM, 20 min) and a subsequent wash period of 20 min. As pituitary cells treated for 4 h with testosterone failed to demonstrate any significant effects on GnRH-induced Ca²⁺ signaling, longer periods of testosterone treatment were investigated, using sterile culture techniques and supplemented medium.

Cells from the anterior pituitaries of either intact male rats or rats castrated for 7 days were harvested as above, but under sterile conditions. Cells were suspended in medium 199 (Life Technologies, Inc., Grand Island, NY) containing 20 mM HEPES, 10% FCS (which had been charcoal-stripped to remove endogenous steroids) and antibiotics (final concentrations, 100 U/ml penicillin, 100 pg/ml streptomycin, and 250 ng/ml amphotericin B; Life Technologies). The residual concentration of testosterone in the charcoal-stripped FCS was less that 0.08 nM (minimum detectable concentration), where the concentration in unstripped serum was 1.2 nM. Cells were then plated onto coverslips that were placed into a humidified incubator (37 C, 80% humidity, 5% CO₂) for at least 12 h before measuring [Ca²⁺]_i.

During the 12-h incubation period, cells were exposed to vehicle (0.2% EtOH in medium), testosterone (10 nM) or cycloheximide (CHX; 10 µM dissolved in medium) alone, or a combination of testosterone (10 nM) and CHX (10 µM). Forty minutes before measuring [Ca²⁺]_i, the cells were loaded with fura-2/AM (1 µM) as above. Both the fura-2 loading and the wash period were in the absence of steroid or cycloheximide treatments. GnRH(100 nM)-induced Ca²⁺ signals were measured as in Exp 1. Each treatment was repeated in at least three separate cultures.

Verification of treatments
Treatments were verified by determining the concentration of LH in the serum of trunk blood collected following decapitation. In all cases seminal vesicles and ventral prostate were removed and weighed to assess the effect of the treatments on androgen-dependent organs.

Serum LH assay.
LH in serum was measured using a previously described RIA protocol, using NIADDK rLH-RP-3 as standard. All samples were measured in a single assay with an intraassay coefficient of variation (C.V.) of 5% (n = 4) and had a minimum detectable concentration of 0.27 ng/ml.

Testosterone assay.
To verify the efficacy of the charcoal stripping process to remove endogenous steroids from the FCS used in Exp 2, samples of stripped and unstripped FCS were assayed for testosterone. Samples of both FCS and media were stored at -20 C until assayed. Testosterone was measured using a previously described assay (19) following ethylacetate:hexane extraction. All samples were measured in one assay, with an intraassay C.V. of 5%.

Statistical analysis
The Ca²⁺ signals of individual cells were analyzed and allocated to the categories of either oscillatory or spike-plateau, as these were the only categories of Ca²⁺ signal observed. Responses of two or more spikes where the fluorescence ratio increased to a value that was 3 or greater above the baseline (generally 1.5–2.5) were termed oscillatory. All other responses consisted of a sharp increase in the fluorescence ratio with a gradual decline to a plateau above prestimulatory baseline, and were classified as spike-plateau. As there was no day-to-day difference in the behavior of single cell preparations within each treatment goup, the results from each replicate of each treatment goup (n = 6 for Exp 1, n = 3 or greater for Exp 2) have been pooled. The results are therefore presented as the proportion of cells showing specific responses in each treatment group. These data were analyzed using ² analysis for independence. When a significant effect was demonstrated by this ² analysis, differences between individual groups were analyzed using the Bonferroni correction of the ² statistic.

The effect of treatments on serum LH concentrations, and organ weights (corrected for the body weight of each animal) were compared using one-way ANOVA, Differences between treatment groups were analyzed post hoc with Fisher’s least significant difference test.

Materials
Unless otherwise stated reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

	Results

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Exp 1
Serum LH concentrations were significantly (P < 0.05) decreased in rats that received testosterone propionate (TP) or GnRH antagonist (GnRHA) treatment, when compared with vehicle-treated castrated controls (Table 1). However, rats receiving both GnRHA and TP had serum LH concentrations that were not lower than when either treatment was given alone. Both groups of rats receiving TP treatment (i.e. castrate + saline + TP and castrate + GnRHA + TP) had androgen-dependent organ weights that were not significantly (P > 0.05) different from each other, but greater (P < 0.05, Table 1) than the two groups that did not receive TP (castrate + saline + oil and castrate + GnRHA + oil).

View larger version (19K): [in this window] [in a new window]   Figure 1. The effect of 1) castration; 2) castration and TP treatment; 3) castration and GnRHA treatment; or 4) castration and GnRHA and TP treatment in male rats on the proportion of gonadatropes exhibiting GnRH (100 nM)-induced oscillatory Ca2+ responses. The resulting [Ca2+]i responses were classified as oscillatory or spike-plateau. A, Typical GnRH (100 nM) Ca2+ signals from each treatment group. B, The percentage of gonadotropes having Ca2+ oscillations to 100 nM GnRH, from each of the treatment goups (with the number (n) of gonadotropes tested from six rats in each goup). Different superscripts denote significantly different proportions of gonadotropes (P < 0.01) showing oscillatory Ca2+ signals.

When gonadotropes from intact male rats were cultured for 12 h in steroid-free medium, only 24% of the cells showed the oscillatory Ca²⁺ signal in response to 100 nM GnRH (Fig. 2). In contrast, 80% of gonadotropes from castrated male rats, cultured under similar conditions had oscillatory Ca²⁺ responses (Fig. 2). These findings indicate that the differences between intact and castrated male rats in GnRH-induced Ca²⁺ responses that were previously observed using acutely dispersed cells (10) persist following 12 h of culture. Gonadotropes from castrated rats that were incubated in 10 nM testosterone for 12 h showed a reversal of the effect of castration, with significantly fewer cells (19%) showing the oscillatory response to 100 nM GnRH (P < 0.05 vs. vehicle-treated cells, Fig. 2). When the cells were treated with both testosterone (10 nM) and the protein synthesis inhibitor cycloheximide (10 µM) for 12 h, the effect of testosterone was not apparent, with the majority of gonadotropes (91%, Fig. 2) still having oscillatory Ca²⁺ responses when stimulated with GnRH (100 nM). Treatment of cells from castrate male rats with CHX (10 µM) alone did not affect the responses of gonadotropes, with 89% of gonadotropes showing Ca²⁺ oscillations (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of testosterone treatment in vitro on the proportion of gonadotropes from male rats exhibiting GnRH (100 nM)-induced oscillatory Ca2+ responses. Cells were taken from intact male rats and treated in vitro with vehicle (0.2% EtOH, 12 h) or cells were taken from male rats castrated for 7 days and treated in vitro for 12 h with one of the following: vehicle (0.2% EtOH); 10 nM testosterone (T); 10 µM cycloheximide (CHX); or a combination of 10 nM T and 10 µM CHX. The Ca2+ responses were classified as oscillatory or spike-plateau. A,Typical GnRH(100 nM) Ca2+ signals from each treatment goup. There was no difference between the different treatment groups in the frequency of the Ca2+ oscillations. B, The percentage of gonadotropes with oscillatory responses to 100 nM GnRH, from each of the treatment groups (with the number (n) of gonadotropes tested in at least three separate cultures in each experimental group). Different superscripts denote significantly different proportion of gonadotropes (P < 0.01) showing oscillatory Ca2+ signals.

	Discussion

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Following castration, there are a number of changes in the function of gonadotropes of the anterior pituitary gland. In the male rat, numerous studies have established that these changes in gonadotrope function are mediated by both the hypothalamus and a direct action of gonadal factors at the pituitary (20, 21, 22, 23). After castration, there is an increase in gonadotrope size and number (24, 25), GnRH receptor messenger RNA expression and GnRH receptor number (26, 27, 28), and both gonadotropin subunit expression and gonadotropin secretion (reviewed in 13 . These changes may reflect increased GnRH expression in, and release from, the hypothalamus following castration (16, 17, 21), though not all studies have demonstrated an increase in these parameters (29, 30, 31). The majority of the gonadectomy-induced changes are restored by treatment with gonadal steroids, though inhibin plays an important role in regulating FSH synthesis and secretion (32, 33, 34).

Many of the studies that have demonstrated a pituitary effect of steroids on the parameters that change following castration have been conducted in vitro (23, 26, 35, 36, 37), and while they clearly establish the potential for steroids to act directly at the pituitary, they leave open the question of what happens in vivo. Accordingly, researchers have utilized models of surgical manipulation of the hypothalamus (38), or pharmacological and immunological interventions (25, 39, 40, 41, 42, 43), to inhibit GnRH secretion or action. Studies, similar to ours, which have employed the use of GnRH antagonists from the time of castration, have demonstrated that steroids act via the hypothalamus to regulate gonadotrope size and number, GnRH receptor expression, gonadotropin subunit expression, and LH secretion (40). Indeed, the present study shows that inhibiting the action of GnRH with the GnRH antagonist in the absence of testosterone, is sufficient to decrease LH concentrations in the serum and block the usual castration-induced change in gonadotrope morphology (data not shown). However, the GnRH antagonist treatment did not modulate GnRH-induced Ca²⁺ signaling in gonadotropes after castration, suggesting that the change in signal transduction is not due to an increased post castration GnRH drive, and results from a direct action of testosterone on the pituitary in vivo. The decrease in LH concentrations, without changes in the nature of GnRH-induced Ca²⁺ signals, also throws into question the relationship between specific Ca²⁺ signals and the secretory response of the gonadotropes. It has previously been suggested that the spike-plateau response triggers secretion, where the oscillatory response does not (6). The findings of the present, and other studies (44), makes it likely that there is a far more subtle and complex relationship between GnRH-induced Ca²⁺ signals and exocytosis.

Our in vitro studies confirm other recent studies that sex steroids can act directly at the pituitary to regulate GnRH-induced Ca²⁺ signaling. Ortmann et al. (7, 8) have shown that progesterone alters the nature of GnRH-induced Ca²⁺ signaling in both gonadotropes from ovariectomized female rats, and T3–1 cells. Using sheep cells, Ghosh et al. have demonstrated that both estradiol and inhibin modulate the number cells responding, and the nature of the Ca²⁺ response to GnRH (45). Estradiol treatment of cultured ovine pituitary cells has a time-dependent effect on the amplitude of basal Ca²⁺ currents and the nature of GnRH-stimulated changes in the cell membrane potential (11). It has also been demonstrated that estradiol and progesterone may alter GnRH-induced InsP₃ production in rat anterior pituitary cells and T3–1 cells. Estradiol treatment of T3–1 cells increases the EC₅₀ for GnRH-induced InsP₃ production (46), whereas long-term treatment of anterior pituitary cells and T3–1 cells by either estradiol or progesterone enhances GnRH-induced inositol phosphate production (47). Together with the observations presented in this report, it is clear that gonadal factors modulate components of Ca²⁺ homeostasis. This novel feedback mechanism may not be restricted to gonadotropes, as glucocorticoids appear able to regulate CRF-induced cAMP generation (48) and Ca²⁺ currents (49) in corticotropes. Glucocorticoids may also regulate CRF-induced Ca²⁺ responses (50), although this finding has not been observed in all studies (51).

In the present in vitro studies, we have demonstrated that changes in gonadotrope function acquired in vivo persist for at least 12 h in culture, suggesting that there has been a profound alteration in the cellular mechanisms controlling Ca²⁺ homeostasis in these cells. The gonadotropes, however, maintain considerable plasticity and are able to revert to normal GnRH-induced Ca²⁺ signaling when treated for sufficient time with testosterone. It is possible, however, that not all gonadotropes exhibit this plasticity. We have previously shown that in gonadotropes from intact rats approximately 25% of gonadotropes will have spike-plateau responses to low (10 pM) concentrations of GnRH, whereas not all (75%) gonadotropes have spike-plateau responses to 100 nM GnRH (10). This observation suggests that there may be subsets of gonadotropes that are programmed to respond to GnRH with either an oscillatory or spike-plateau response (irrespective of the concentration applied), but that there is majority (50%) that have concentration-dependent responses, which can be manipulated by their endocrine milieu. It is unclear if there are any functional differences between these putative subsets of gonadotropes.

The effects of testosterone also depend upon the synthesis of new protein(s), and, with the time-dependence of testosterone’s effect, these data suggest a genomic mechanism of action consistent with the involvement of a steroid receptor. It appears, therefore, that in the intact rat, testosterone maintains the production of a protein(s) required for the transition of oscillatory response to spike-plateau in the face of high concentrations of GnRH. The identity of this protein(s) is unclear, though a recently published biophysical model of the control of Ca²⁺ homeostasis in gonadotropes suggests several candidates (52). This model predicts, among other possibilities, that Ca²⁺ oscillations will be favored in gonadotropes if there is 1) reduced InsP₃ production in response to given concentration of GnRH; 2) reduced expression of the InsP₃ receptor on the endoplasmic reticulum; or 3) decreased activity of the Ca²⁺-ATPase pump on the endoplasmic reticulum. While data regarding the role of testosterone in regulating these aspects of gonadotrope function are lacking, it is of interest to note that both estradiol and progesterone have been implicated in regulated the activity of Ca²⁺ATPases in myometrium (53) and GnRH-induced InsP₃ production (46, 47). The possibility that these aspects of Ca²⁺ homeostasis in gonadotropes are regulated by testosterone is presently under investigation in this laboratory. Defining the specific actions of testosterone on Ca²⁺ homeostasis in gonadotropes will be of considerable importance to our understanding of the control of gonadotrope function.

	Acknowledgments

 
The authors would like to thank Dr. Paul Farnworth for his assistance with the LH assay and Drs. Richard Lang and Alan Tilbrook for helpful discussions.

	Footnotes

 
¹ This work was funded by a grant from the National Health and Medical Research Council of Australia (to B.J.C.). R.P.M. acknowledges support from the Medical Research Council of South Africa.

Received February 19, 1997.

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