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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 9 3027-3035
Copyright © 2000 by The Endocrine Society

From the Clinical Research Centers

Aromatase Inhibition in the Human Male Reveals a Hypothalamic Site of Estrogen Feedback1

Frances J. Hayes, Stephanie B. Seminara, Suzzunne DeCruz, Paul A. Boepple and William F. Crowley, Jr.

Reproductive Endocrine Unit of the Department of Medicine and National Center for Infertility Research, Massachusetts General Hospital, Boston, Massachusetts 02114

Address correspondence and requests for reprints to: Frances Hayes, MB, MRCPI, Reproductive Endocrine Unit and National Center for Infertility Research, Massachusetts General Hospital, Fruit Street, Boston, Massachusetts 02114. E-mail: hayes.frances{at}mgh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The preponderance of evidence states that, in adult men, estradiol (E2) inhibits LH secretion by decreasing pulse amplitude and responsiveness to GnRH consistent with a pituitary site of action. However, this conclusion is based on studies that employed pharmacologic doses of sex steroids, used nonselective aromatase inhibitors, and/or were performed in normal (NL) men, a model in which endogenous counterregulatory adaptations to physiologic perturbations confound interpretation of the results. In addition, studies in which estrogen antagonists were administered to NL men demonstrated an increase in LH pulse frequency, suggesting a potential additional hypothalamic site of E2 feedback.

To reconcile these conflicting data, we used a selective aromatase inhibitor, anastrozole, to examine the impact of E2 suppression on the hypothalamic-pituitary axis in the male. Parallel studies of NL men and men with idiopathic hypogonadotropic hypogonadism (IHH), whose pituitary-gonadal axis had been normalized with long-term GnRH therapy, were performed to permit precise localization of the site of E2 feedback. In this so-called tandem model, a hypothalamic site of action of sex steroids can thus be inferred whenever there is a difference in the gonadotropin responses of NL and IHH men to alterations in their sex steroid milieu. A selective GnRH antagonist was also used to provide a semiquantitative estimate of endogenous GnRH secretion before and after E2 suppression.

Fourteen NL men and seven IHH men were studied. In Exp 1, nine NL and seven IHH men received anastrozole (10 mg/day po x 7 days). Blood samples were drawn daily between 0800 and 1000 h in the NL men and immediately before a GnRH bolus dose in the IHH men. In Exp 2, blood was drawn (every 10 min x 12 h) from nine NL men at baseline and on day 7 of anastrozole. In a subset of five NL men, 5 µg/kg of the Nal-Glu GnRH antagonist was administered on completion of frequent blood sampling, then sampling continued every 20 min for a further 8 h.

Anastrozole suppressed E2 equivalently in the NL (136 ± 10 to 52 ± 2 pmol/L, P < 0.005) and IHH men (118 ± 23 to 60 ± 5 pmol/L, P < 0.005). Testosterone levels rose significantly (P < 0.005), with a mean increase of 53 ± 6% in NL vs. 56 ± 7% in IHH men. Despite these similar changes in sex steroids, the increase in gonadotropins was greater in NL than in IHH men (100 ± 9 vs. 58 ± 6% for LH, P = 0.07; and 85 ± 6 vs. 41 ± 4% for FSH, P < 0.002). Frequent sampling studies in the NL men demonstrated that this rise in mean LH levels, after aromatase blockade, reflected an increase in both LH pulse frequency (10.2 ± 0.9 to 14.0 ± 1.0 pulses/24 h, P < 0.05) and pulse amplitude (5.7 ± 0.7 to 8.4 ± 0.7 IU/L, P < 0.001). Percent LH inhibition after acute GnRH receptor blockade was similar at baseline and after E2 suppression (69.2 ± 2.4 vs. 70 ± 1.9%), suggesting that there was no change in the quantity of endogenous GnRH secreted.

From these data, we conclude that in the human male, estrogen has dual sites of negative feedback, acting at the hypothalamus to decrease GnRH pulse frequency and at the pituitary to decrease responsiveness to GnRH.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
STUDIES ON the site(s) of estrogen feedback on the hypothalamic-pituitary (HP) axis in the human male are conflicting. On the one hand, estradiol (E2) has been shown to inhibit LH secretion by decreasing LH pulse amplitude and LH responsiveness to GnRH consistent with a pituitary site of action (1, 2, 3, 4, 5, 6, 7, 8). On the other hand, administration of antiestrogens to NL men results in an increase in LH pulse frequency, suggesting a hypothalamic site of E2 feedback (6, 9, 10, 11, 12). Similarly, estrogen administration to castrated sheep lowers mean LH levels by decreasing LH pulse frequency (13, 14). However, interpretation of these studies is confounded by their use of pharmacologic doses of sex steroids (precluding conclusions about physiologic feedback relationships) and a general failure to account for the impact of such large steroid doses on sex hormone binding globulin and thus the ratio of free-to-bound endogenous gonadal hormones. In addition, the aromatase inhibitor used, testolactone, has been shown to have antiandrogenic properties because of its ability to bind to the androgen receptor (15).

This controversy in the literature reflects the difficulty in interpreting studies on sex steroid feedback in the intact male because of the fact that gonadotropin secretion represents the integrated response of both the hypothalamus and pituitary. In attempting to dissect the level of sex steroid negative feedback, many investigators have made the assumptions that: 1) LH pulse frequency is determined solely by the frequency of GnRH release from the hypothalamus; and 2) LH pulse amplitude reflects pituitary sensitivity to GnRH. However, a linear relationship exists between the bolus dose of GnRH and the amplitude of the pituitary LH response (16, 17). Therefore, it follows that any change in LH pulse amplitude could, in fact, reflect a pituitary and/or a hypothalamic effect. In the intact human, it is not possible to distinguish between these two effects by measuring GnRH in the peripheral blood because of its confinement to the hypophyseal-portal circulation and short half-life. Therefore, precise localization of the site of E2 feedback in the human requires a complimentary model in which both the dose and frequency of GnRH administration can be experimentally controlled. Men with idiopathic hypogonadotropic hypogonadism (IHH), who lack endogenous hypothalamic GnRH secretion and whose pituitary-gonadal axis can be normalized with long-term pulsatile GnRH replacement (18), provide such a model. Because the dose and frequency of exogenous GnRH administration can be experimentally controlled in this setting, this model, in effect, represents a hypothalamic clamp. Therefore, any effects of altering gonadal steroid levels on gonadotropin secretion in IHH men can only reflect a pituitary site of action. In contrast, in NL men with an intact HPG axis, gonadal steroids can modulate gonadotropin secretion by direct inhibition at the level of the pituitary, and/or by inhibiting GnRH secretion from the hypothalamus. Thus, by the tandem study of these two human models, a hypothalamic site of action of sex steroids can be inferred whenever there is a difference in the gonadotropin responses of NL and IHH men to alterations in their sex steroid milieu.

Although it is not feasible to measure GnRH in peripheral blood in the human, we have previously validated use of a GnRH antagonist to provide a semiquantitative estimate of endogenous GnRH secretion (19, 20). This novel physiologic tool thus allows one to determine whether any given increase in LH pulse amplitude reflects a hypothalamic (increase in the GnRH bolus dose) or a pituitary (enhanced sensitivity to GnRH) effect. The basic premise of this approach is that the response of a marker of GnRH action such as LH can be used to assess GnRH secretion in the presence of submaximal GnRH receptor blockade, such that the amount of GnRH secreted is inversely proportional to the degree of LH inhibition (19, 20). Therefore, if removal of E2 negative feedback were to increase endogenous GnRH secretion, one would expect that LH secretion would be less susceptible to GnRH receptor blockade in the E2-deplete vs. E2-replete state.

The aim of the present study was to examine the impact of E2 suppression on the HPG axis in the human male. In an effort to circumvent some of the limitations of previous studies, we used: 1) the potent, highly selective aromatase inhibitor, anastrozole (21), to deplete endogenous estrogen; 2) a complimentary approach involving the tandem study of NL and IHH men to permit precise localization of the site of E2 feedback; and 3) the Nal-Glu GnRH antagonist to provide a semiquantitative estimate of GnRH secretion, as previously described (19, 20).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

NL men. Fourteen NL men (age, 22–50 yr) participated in the study. All study subjects met the following criteria: 1) normal pubertal development, sexual function, and general health; 2) normal physical examination, including a testicular volume >= 20 mL; 3) normal serum levels of testosterone (T), E2, LH, FSH, TSH, and PRL; and 4) normal semen analysis, according to World Health Organization criteria (22).

IHH men. Seven men (age, 30–46 yr) with isolated GnRH deficiency were selected on the basis of the following criteria: 1) failure to undergo spontaneous puberty by the age of 18 yr; 2) serum T < 3.5 nmol/L, in association with inappropriately low gonadotropin levels; 3) absence of endogenous gonadotropin pulsations during a 12- to 24-h period of blood sampling; 4) otherwise normal reserve testing of anterior pituitary function; and 5) normal magnetic resonance imaging of the HP region. At the time of participation in the study, all had normal serum concentrations of T, LH, and FSH for at least 3 months, as a result of treatment with pulsatile sc GnRH therapy delivered at 2-h intervals (18).

The study was approved by the Human Research Committee at the Massachusetts General Hospital, and all subjects provided written informed consent.

Study protocol

Exp 1. Nine NL men and seven IHH men were treated with the aromatase inhibitor, anastrozole, 10 mg/day for 7 days. T, E2, LH, and FSH were measured daily. Samples were drawn between 0800 and 1000 h in the (NL) men and before a bolus dose of exogenous GnRH in the IHH men.

Exp 2. Nine NL men, four of whom completed Exp 1, participated in a more intensive analysis of the gonadotropin response to E2 suppression. Subjects were admitted to the General Clinical Research Center of Massachusetts General Hospital and had an iv catheter inserted into a forearm vein. Four of the nine NL men were admitted at 0700 h and had blood sampling every 10 min for 12 h, from 0800 to 2000 h, after which they were discharged home. The other five subjects participated in an extended protocol, which included administration of a GnRH antagonist. These men were admitted at 2300 h and had blood sampling from 2400 to 1200 h, after which they received a single sc injection of the Nal-Glu GnRH antagonist (5 µg/kg) to block the GnRH receptor. After administration of the GnRH antagonist, blood samples were drawn every 20 min, for a further 8 h, and subjects were then discharged. On the morning after discharge, all nine NL men commenced taking anastrozole (10 mg/day for 7 days). On day 7 of anastrozole therapy, all subjects were readmitted to the General Clinical Research Center for a second 12-h frequent blood sampling study ± GnRH antagonist administration, to examine the impact of E2 suppression on gonadotropin secretion.

In the 12-h frequent blood sampling study, all samples were assayed for LH, whereas FSH was measured in hourly samples. T and E2 were determined at baseline and in serum pools composed of equal aliquots of each sample obtained at 6-h intervals. After administration of the GnRH antagonist, LH was measured in all samples, whereas T was measured in hourly pools.

Evaluation of sex steroid and gonadotropin secretion

Frequent blood sampling study. Mean LH and FSH levels were calculated for both frequent blood sampling studies. Pulsatile LH secretion was analyzed using the modified Santen and Bardin method, as recently validated by the investigators (23, 24). The mean LH pulse amplitude (defined as the difference between the peak and the preceding nadir) was calculated at baseline and on day 7 of anastrozole therapy.

LH inhibition after GnRH antagonist administration. The maximum degree of gonadotropin suppression after administration of the Nal-Glu GnRH antagonist was determined by calculating the percent inhibition from the preantagonist period [(mean PRE - nadir)/mean PRE] x 100, as previously described (19). Nadir LH levels were calculated using a 3-point moving average, which is equivalent to 1 h of sampling.

Hormone assays

Serum LH and FSH concentrations were determined by microparticle enzyme immunoassay using the automated Abbott AxSYM system (Abbott Laboratories, Chicago, IL). The Second International Reference Preparation was used as the reference standard. The assay sensitivity for both LH and FSH was 1.6 IU/L. The intraassay coefficient of variation (CV) values for LH and FSH were less than 7% and less than 6%, respectively, with interassay CVs for both hormones of less than 7.4%. Serum T concentrations were measured using the DPC Coat-A-Count RIA kit (Diagnostic Products Corp., Los Angeles, CA), which had an intra- and interassay CV less than 10%. E2 was measured by the Abbott AxSYM system, which had an analytical sensitivity of 36 pmol/L and a functional sensitivity of 73 pmol/L. The intraassay CV was less than 6.4%, with an interassay CV less than 10.6%. Inhibin B was measured using a commercially available (Serotec, Oxford, UK) double-antibody enzyme-linked immunosorbent assay, as previously described (25). In our use, the clinical detection limit of this assay is 50 pg/mL, with a CV of 4–6% within plate and 15–18% between plates.

Statistical methods

Mean daily hormone levels in the NL and IHH men, over the 7 days of anastrozole therapy, were analyzed using ANOVA for repeated measures, followed by post hoc Newman-Keuls testing for individual differences. To compare the responses of the NL and IHH men, the data were expressed as percent change from baseline, and the mean levels of the two groups were compared using ANOVA. For the frequent blood sampling studies performed in the NL men at baseline and on day 7 of anastrozole therapy, mean hormone levels, LH pulse frequency, and LH pulse amplitude were compared using a two-tailed paired t test. The maximum percent LH inhibition after GnRH antagonist administration at baseline and on day 7 of anastrozole were compared using a paired t test. A P value less than 0.05 was taken to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Baseline

Baseline characteristics of the normal (NL) and IHH men are summarized in Table 1Go. The two groups were of similar age, but the IHH men were slightly heavier (P < 0.05). Mean T, E2, and LH levels were similar in both groups. FSH levels tended to be higher in the IHH men, although this difference did not achieve statistical significance. In keeping with their smaller testicular size and higher FSH levels, IHH men had lower mean inhibin B levels than did NL men (P < 0.05).


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  		Table 1. Baseline characteristics of normal men and men with IHH

 
Exp 1

Treatment with anastrozole resulted in marked suppression of E2 in both NL (P < 0.005) and IHH men (P < 0.005) (Fig. 1Go). Mean absolute levels and percent suppression of E2 were similar in both groups for the duration of anastrozole therapy (52 ± 2 pmol/L in the NL vs. 60 ± 5 pmol/L in the IHH men; P, not significant). T levels rose significantly (P < 0.005) in both groups, with a mean increase of 53 ± 6% in the NL vs. 56 ± 7% in the IHH men (Fig. 1Go). Despite these similar changes in sex steroids, the increase in gonadotropin levels was greater in NL than in IHH men (100 ± 9 vs. 58 ± 6% for LH, P = 0.07; and 85 ± 6 vs. 41 ± 4% for FSH, P < 0.002) (Fig. 2Go).



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  		Figure 1. Changes in gonadal steroids in NL men (left) and IHH men (right), in response to administration of anastrozole (10 mg/day for 7 days). Data are expressed both as percent change (in the bar graph) and absolute values (in the line graph). Asterisks, Significant change from baseline (BL): *, P < 0.05; **, P < 0.005.

 


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  		Figure 2. Changes in gonadotropin concentrations in NL men (left) and IHH men (right), in response to administration of anastrozole, as in Fig. 1Go. Data are expressed as percent change in the bar graph and absolute values in the line graph. Asterisks, Significant change from BL: *, P < 0.05; **, P < 0.005.

 
Exp 2

Confirming the results of the single time point data obtained in Exp 1, frequent sampling studies in NL men showed that anastrozole resulted in marked suppression of E2, accompanied by a significant increase in T, LH, and FSH levels (Table 2Go). This increase in mean LH levels reflected an increase in both LH pulse frequency (10.2 ± 0.9 to 14.0 ± 1.0 pulses/24 h, P < 0.05) and LH pulse amplitude (5.7 ± 0.7 to 8.4 ± 0.7 IU/L, P < 0.05). Frequent blood sampling data from a representative NL subject are indicated in Fig. 3Go. In this individual, anastrozole resulted in a 60% increase in the number of LH pulses despite a marked increase in T, which normally serves to restrain the hypothalamic GnRH pulse generator. Of the nine NL men studied, LH pulse frequency increased in seven, decreased in one, and was unchanged in another (Fig. 4Go). LH pulse amplitude increased in all study subjects (Fig. 4Go).


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  		Table 2. Impact of the aromatase inhibitor, anastrozole (10 mg/day x 7 days) on gonadal steroid and gonadotropin levels in normal men (n = 9)

 


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  		Figure 3. Pulsatile LH secretion, in a representative NL male, before and after 7 days of anastrozole (10 mg/day), demonstrating an increase in both LH pulse frequency and amplitude. Blood samples were drawn every 10 min x 12 h. Triangles represent LH pulses detected using the modified Santen and Bardin pulse-detection algorithm (23 24 ).

 


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  		Figure 4. Individual mean LH levels, LH pulse frequency, and LH pulse amplitude in nine NL men before and after 7 days of anastrozole-induced E2 suppression. The group mean ± SEM for each parameter is indicated to the side of each graph. E2 (+), Estrogen-replete state at baseline; E2 (-), estrogen-deplete state after aromatase inhibition; asterisks, significant change from baseline.

 
Acute GnRH receptor blockade resulted in marked suppression of LH, with mean levels falling from 9.7 ± 1.5 to a nadir of 2.8 ± 0.4 IU/L at baseline (P < 0.005) and from 19.0 ± 1.9 to a nadir of 5.6 ± 0.7 IU/L on day 7 of anastrozole (P < 0.005). When the data were expressed as percent baseline, to allow comparison between studies performed in an E2-replete vs. E2-deplete state, maximum percent LH inhibition was identical (69.2 ± 2.4 vs. 70.0 ± 1.9%) (Fig. 5Go).



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  		Figure 5. Percent LH inhibition in five NL men after administration of the Nal-Glu GnRH antagonist at baseline and on day 7 (D7) of anastrozole therapy. The Nal-Glu GnRH antagonist was administered sc at a dose of 5 µg/kg at time zero.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This parallel study of the response of NL and GnRH-deficient men to estrogen suppression clearly demonstrates that, in the human male, E2 has both a pituitary and a hypothalamic site of action. The increase in gonadotropin secretion after aromatase inhibition, in IHH men on a fixed, experimentally controlled GnRH regimen, confirms previous studies indicating that E2 has a pituitary site of action (1, 2, 3, 4, 5, 6, 7, 8) that, in this study, accounts for approximately half of the gonadotropin elevation observed in NL men. However, the demonstration that estrogen suppression leads to a greater increase in gonadotropin levels in NL than in GnRH-deficient men also documents an additional hypothalamic effect of E2.

E2 could potentially alter GnRH secretion by increasing the frequency and/or the amplitude of GnRH pulses from the hypothalamus. Analysis of pulsatile LH secretion after E2 suppression, in this study, demonstrates a clear-cut increase in LH (and, by inference, GnRH) pulse frequency (16, 26, 27). This estrogen effect on LH pulse frequency is consistent with several previous studies showing that a change in LH pulse frequency can be detected when antiestrogens are administered to NL men (6, 9, 10, 11, 12). Despite variations in the choice of antiestrogen (clomiphene vs. tamoxifen), duration of therapy (from 1–6 weeks) and sampling paradigm used (10-min vs. 20-min intervals), an increase in GnRH pulse frequency was observed in the vast majority of individuals studied (6, 9, 10, 11, 12). In the single study where clomiphene administration was reported to have no impact on LH pulse frequency, the sample size of two precludes any conclusions (28). Confirmatory evidence for a role of estrogen in modulating GnRH pulse frequency is provided by studies in sheep that indicate that estrogen administration to long-term castrated rams lowers mean LH levels, mainly by decreasing LH pulse frequency (13, 14). A hypothalamic action of E2 is also evident from studies in the primate demonstrating that direct administration of E2 into the hypothalamus (29) or third ventricle (30) suppresses LH secretion.

The increase in LH pulse amplitude, observed after aromatase inhibition, could potentially reflect an increase in the amplitude of GnRH pulses stimulating the pituitary, and/or enhanced pituitary sensitivity to the same amount of endogenous GnRH. Previous studies have attempted to distinguish between these two mechanisms by examining pituitary responsiveness to pharmacological doses of exogenous GnRH before and during antiestrogen therapy (11, 31, 32). These studies paradoxically demonstrated that clomiphene blunted pituitary responsiveness to exogenous GnRH despite increasing both mean LH levels and the amplitude of spontaneous LH pulses (11, 31, 32). The mechanism proposed for this divergence between spontaneous pulse height and acute pituitary responsiveness to exogenous GnRH was that clomiphene was having tissue-specific mixed agonist/antagonist effects. The authors concluded that clomiphene was acting as an estrogen antagonist at the hypothalamus, resulting in an increase in endogenous GnRH secretion, but as an estrogen agonist at the pituitary, causing decreased responsiveness to exogenous GnRH (11).

In this study, we adopted a different approach, to dissect the basis for the increase in LH pulse amplitude after E2 suppression, using a GnRH antagonist to provide a semiquantitative estimate of endogenous GnRH secretion. The basic premise of this approach is that the response of a marker of GnRH action, such as LH, can be used to assess GnRH secretion in the presence of submaximal GnRH receptor blockade, such that the amount of GnRH secreted will be inversely proportional to the degree of LH inhibition (19, 20). Such an approach is only possible because GnRH is the only known secretagogue for LH, GnRH and its antagonist bind to a single receptor type, and there is no evidence of any change in GnRH receptor affinity over a wide range of both physiologic and pharmacologic conditions (19). In the present study, the degree of LH inhibition, after acute GnRH receptor blockade, was unaltered by E2 suppression. We feel that it is unlikely that use of a lower GnRH antagonist dose would have identified differences in GnRH secretion after estrogen suppression, for the following reasons. First, the degree of LH suppression achieved was submaximal, with nadir levels above the limit of detection of the LH assay. Second, the same antagonist dose used in this study (5 µg/kg Nal-Glu) has previously been shown to be capable of detecting differences in GnRH secretion in different physiologic and pathophysiologic states in both men (33) and women (19). Therefore, we conclude that the hypothalamic effect of E2 suppression is to increase the frequency, rather than the bolus dose, of GnRH. This finding, in turn, implies that the increase in LH pulse amplitude observed after aromatase inhibition is attributable to enhanced pituitary sensitivity of LH to GnRH. This conclusion is at variance with the studies reporting that clomiphene diminishes pituitary responsiveness to exogenous GnRH administration (11, 31, 32). However, the major limitations to using GnRH tests to assess pituitary sensitivity in an intact system are: 1) the fact that the pituitary LH response to an exogenous GnRH bolus varies significantly with the previous LH interpulse interval (34); and 2) the pharmacological nature of the doses used. Accordingly, there is marked variability in the LH response to a single bolus dose of GnRH in NL men (35). Therefore, the results of single-dose GnRH testing are difficult to interpret unless endogenous gonadotropin secretion is blocked so that the confounder of variable endogenous interpulse intervals is eliminated.

Having excluded an increase in GnRH pulse amplitude, other potential mechanisms for the increased LH pulse amplitude after E2 suppression include an increase in pituitary responsiveness to GnRH as a result of an increase in: 1) the number of gonadotropes; 2) the number of GnRH receptors; and 3) the affinity of GnRH for its receptor. It seems unlikely that 7 days of estrogen suppression would alter gonadotrope number in these adult men. In cultured pituitary cells from ovariectomized ewes, estrogen administration has been shown to increase GnRH receptor number and to have no effect on binding affinity (36). Indeed, there has been no demonstration of changes in GnRH receptor affinity over a wide range of physiologic and pharmacologic conditions (37). To our knowledge, no data are available on the effect of estrogen withdrawal on GnRH receptor number in the male. Therefore, the mechanism(s) underlying the increase in LH pulse amplitude remains speculative.

In the present study, E2 suppression resulted in a significant increase in LH pulse frequency. This change in pulse frequency was all the more impressive given that it occurred despite a concomitant rise in T levels, which normally has a restraining influence on the GnRH pulse generator (2, 4, 38, 39, 40, 41). Therefore, the net effect of removing E2 negative feedback, while allowing T levels to rise to the supraphysiologic range, is an increase in gonadotropin secretion. These data therefore speak to the importance of E2 in the negative feedback control of gonadotropin secretion in the male. This concept that E2 is a more potent suppressor of LH secretion than is T is supported by studies in both prepubertal boys (42, 43) and adult males (1, 2, 3, 7, 8, 41, 44), indicating that, on a molar basis, the steroid dose required to suppress gonadotropin secretion is approximately 200-fold less for E2 than for T.

A number of other approaches can be taken to study sex steroid regulation of gonadotropins in the human male, all of which have inherent limitations. A series of experiments of nature comprising patients with E2 receptor mutations and congenital aromatase deficiency provide models that permit the impact of selective estrogen ablation to be examined in the human. However, the major limitation of this genetic approach is the small number of patients available for study. To date, only one estrogen receptor (ER) mutation (45) and two cases of congenital aromatase deficiency (46, 47) have been described in adult males. However, consistent with the data we obtained using an aromatase inhibitor, congenital E2 deficiency was associated with a 2- to 3-fold increase in FSH in all three patients, and in LH in two individuals, despite normal-to-elevated T levels (45, 46, 47). In addition, estrogen treatment resulted in complete suppression of serum gonadotropin levels (47). Characterization of the HPG axis in these cases was based on a single time point estimation. Whereas a single sample is adequate to obtain an accurate estimate of FSH secretion, it does not accurately reflect mean LH levels, given the pulsatile pattern of LH secretion. Therefore, it is possible that the normal LH concentration observed in one individual with congenital aromatase deficiency (47) represented the trough level of a pulse.

Though the phenotype of the ER mutation and congenital aromatase deficiency patients is similar, in terms of effect on the HP axis, the phenotype of the corresponding male mice knockouts, created by targeted disruption of the ER-{alpha} gene (ERKO mice) (48) and the aromatase CYP19 gene (ArKO mice) (49), respectively, is different. As in cases of congenital aromatase deficiency in the human, adult male ArKO mice exhibit elevated levels of gonadotropins despite high circulating T concentrations (49). In contrast, adult {alpha}ERKO males exhibit normal levels of hypothalamic GnRH, pituitary FSHß messenger RNA (mRNA), and serum FSH, but elevated LH levels and markedly diminished fertility (50). This difference most likely reflects the fact that {alpha}ERKO mice are not totally deficient in estrogen action, because they continue to express the ER-ß isoform predominantly in the hypothalamus and, to a limited extent, in the pituitary (51, 52). This hypothesis is supported by recently published data on the phenotype of the double {alpha}ßERKO mice (53). Although gonadotropin levels were not reported for the male {alpha}ßERKO mice, LH levels in the female double knockouts were higher than those seen in the {alpha}ERKO mice, suggesting that some of estrogen’s feedback effects are mediated by the ER-ß receptor (53).

Given the limitations of these genetic and animal models, we chose to use disease models and pharmacologic tools to create an E2-deplete milieu in the male. The human model that we chose to use, i.e. the tandem study of NL and GnRH-deficient men, also has limitations. Though the two groups of subjects were matched for gonadal steroids at baseline, inhibin B levels were significantly higher in the NL than the IHH men. However, given that inhibin B is an important negative feedback regulator of FSH secretion (54, 55, 56, 57, 58, 59), one would expect that the lower inhibin B levels in the IHH men would have facilitated a greater FSH response to estrogen suppression than that seen in NL men. The fact that the rise in FSH was 2-fold greater in the NL than in the IHH men is therefore all the more significant. In a previous study employing this same tandem model, we found that estrogen administration suppressed gonadotropin secretion to the same degree in NL and GnRH-deficient men, suggesting that the major site of E2 feedback was at the pituitary (7). It is important to note that a significant change in LH pulse frequency was observed in the NL men in that study, and it is possible that a difference in the responses of NL and GnRH-deficient men would have been detected if physiologic (as opposed to pharmacologic) doses of sex steroids had been used.

Until recently, the precise cellular mechanism by which E2 suppresses GnRH secretion was controversial. On the one hand, estrogen response elements had been demonstrated in the promoter region of the primate GnRH gene (60). In addition, there were reports of low levels of ER mRNA in two different immortalized GnRH cell lines (60, 61). On the other hand, immunocytochemical studies using ER{alpha} double-labeling had failed to demonstrate ER expression on GnRH neurons in a variety of species, including the rat (62), guinea pig (63), sheep (64, 65), and monkey (66, 67). The absence of ER immunoreactivity, combined with the demonstration that GnRH neurons did not concentrate E2 (68), suggested that estrogen effects on GnRH were not occurring through a classic ER-mediated process. It was thus postulated that estrogen-receptive neurons were acting as intermediaries in the nongenomic regulation of GnRH by estrogen (for review, see Ref. 69). However, recent studies using the novel and highly sensitive technique of single-cell multiplex RT-PCR demonstrated, for the first time, the presence of both ER{alpha} and ERß messenger RNA in native GnRH neurons (70). In addition, evidence has now been provided that estrogen can directly suppress GnRH gene expression in ER{alpha}- and ERß-expressing GT1–7 GnRH neurons (71).

From these clinical investigative studies on the impact of aromatase inhibition in NL and GnRH-deficient men, employing frequent blood sampling combined with administration of a GnRH antagonist, we conclude that, in the human male, estrogen has dual sites of negative feedback, acting at the hypothalamus to decrease GnRH pulse frequency and at the pituitary to decrease pituitary responsiveness to GnRH.


   Acknowledgments
 
We gratefully acknowledge the nurses of the General Clinical Research Center for excellent clinical care and the technicians of the Reproductive Endocrine Sciences Center Radioimmunoassay Core for superb technical contributions to this study. The Nal-Glu GnRH antagonist was synthesized at the Salk Institute under contract with the NIH and made available by the Contraceptive Development Branch, Center for Population Research, National Institute of Child Health and Human Development. We thank Jean Rivier, Ph.D., and Marvin Karten, Ph.D., for support in these studies.


   Footnotes
 
1 Supported in part by Grants R01-HD15788-15, DK-07028-24, M01-RR-01066, P30-HD-28138, and NIH Grant N01-HD-02906; and presented in part at the 80th Annual Meeting of The Endocrine Society, New Orleans, Louisiana, 1998. Back

Received December 7, 1999.

Revised May 11, 2000.

Accepted June 4, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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