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0021-972X/00/$03.00/0 Vol. 85, No. 9
The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A.
Copyright © 2000 by The Endocrine Society
Aromatase Inhibition in the Human Male Reveals a
Hypothalamic Site of Estrogen Feedback*
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
ABSTRACT Blood samples were drawn daily between 0800 and 1000 h in the NL
The preponderance of evidence states that, in adult men, estradiol men and immediately before a GnRH bolus dose in the IHH men. In
(E2) inhibits LH secretion by decreasing pulse amplitude and respon- Exp 2, blood was drawn (every 10 min 12 h) from nine NL men at
siveness to GnRH consistent with a pituitary site of action. However, baseline and on day 7 of anastrozole. In a subset of five NL men, 5
this conclusion is based on studies that employed pharmacologic doses g/kg of the Nal-Glu GnRH antagonist was administered on comple-
of sex steroids, used nonselective aromatase inhibitors, and/or were tion of frequent blood sampling, then sampling continued every 20
performed in normal (NL) men, a model in which endogenous coun- min for a further 8 h.
terregulatory adaptations to physiologic perturbations confound in- Anastrozole suppressed E2 equivalently in the NL (136 10 to 52
terpretation of the results. In addition, studies in which estrogen 2 pmol/L, P 0.005) and IHH men (118 23 to 60 5 pmol/L, P
antagonists were administered to NL men demonstrated an increase 0.005). Testosterone levels rose significantly (P 0.005), with a mean
in LH pulse frequency, suggesting a potential additional hypotha- increase of 53 6% in NL vs. 56 7% in IHH men. Despite these
lamic site of E2 feedback. similar changes in sex steroids, the increase in gonadotropins was
To reconcile these conflicting data, we used a selective aromatase greater in NL than in IHH men (100 9 vs. 58 6% for LH, P 0.07;
inhibitor, anastrozole, to examine the impact of E2 suppression on the and 85 6 vs. 41 4% for FSH, P 0.002). Frequent sampling studies
hypothalamic-pituitary axis in the male. Parallel studies of NL men in the NL men demonstrated that this rise in mean LH levels, after
and men with idiopathic hypogonadotropic hypogonadism (IHH), aromatase blockade, reflected an increase in both LH pulse frequency
whose pituitary-gonadal axis had been normalized with long-term (10.2 0.9 to 14.0 1.0 pulses/24 h, P 0.05) and pulse amplitude
GnRH therapy, were performed to permit precise localization of the (5.7 0.7 to 8.4 0.7 IU/L, P 0.001). Percent LH inhibition after
site of E2 feedback. In this so-called tandem model, a hypothalamic acute GnRH receptor blockade was similar at baseline and after E2
site of action of sex steroids can thus be inferred whenever there is a suppression (69.2 2.4 vs. 70 1.9%), suggesting that there was no
difference in the gonadotropin responses of NL and IHH men to change in the quantity of endogenous GnRH secreted.
alterations in their sex steroid milieu. A selective GnRH antagonist From these data, we conclude that in the human male, estrogen has
was also used to provide a semiquantitative estimate of endogenous dual sites of negative feedback, acting at the hypothalamus to de-
GnRH secretion before and after E2 suppression. crease GnRH pulse frequency and at the pituitary to decrease re-
Fourteen NL men and seven IHH men were studied. In Exp 1, nine sponsiveness to GnRH. (J Clin Endocrinol Metab 85: 3027–3035,
NL and seven IHH men received anastrozole (10 mg/day po 7 days). 2000)
S TUDIES ON the site(s) of estrogen feedback on the hy-
pothalamic-pituitary (HP) axis in the human male are
conflicting. On the one hand, estradiol (E2) has been shown
tionships) 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
to inhibit LH secretion by decreasing LH pulse amplitude hormones. In addition, the aromatase inhibitor used, testo-
and LH responsiveness to GnRH consistent with a pituitary lactone, has been shown to have antiandrogenic properties
site of action (1– 8). On the other hand, administration of because of its ability to bind to the androgen receptor (15).
antiestrogens to NL men results in an increase in LH pulse This controversy in the literature reflects the difficulty in
frequency, suggesting a hypothalamic site of E2 feedback (6, interpreting studies on sex steroid feedback in the intact male
9 –12). Similarly, estrogen administration to castrated sheep because of the fact that gonadotropin secretion represents the
lowers mean LH levels by decreasing LH pulse frequency integrated response of both the hypothalamus and pituitary.
(13, 14). However, interpretation of these studies is con- In attempting to dissect the level of sex steroid negative
founded by their use of pharmacologic doses of sex steroids feedback, many investigators have made the assumptions
(precluding conclusions about physiologic feedback rela- that: 1) LH pulse frequency is determined solely by the fre-
quency of GnRH release from the hypothalamus; and 2) LH
Received December 7, 1999. Revision received May 11, 2000. Accepted pulse amplitude reflects pituitary sensitivity to GnRH. How-
June 4, 2000.
Address correspondence and requests for reprints to: Frances Hayes, ever, a linear relationship exists between the bolus dose of
MB, MRCPI, Reproductive Endocrine Unit and National Center for GnRH and the amplitude of the pituitary LH response (16,
Infertility Research, Massachusetts General Hospital, Fruit Street, Bos- 17). Therefore, it follows that any change in LH pulse am-
ton, Massachusetts 02114. E-mail: hayes.frances@mgh.harvard.edu. plitude could, in fact, reflect a pituitary and/or a hypotha-
* 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
lamic effect. In the intact human, it is not possible to distin-
in part at the 80th Annual Meeting of The Endocrine Society, New guish between these two effects by measuring GnRH in the
Orleans, Louisiana, 1998. peripheral blood because of its confinement to the hypo-
3027
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3028 HAYES ET AL. JCE & M • 2000
Vol. 85 • No. 9
physeal-portal circulation and short half-life. Therefore, pre- association with inappropriately low gonadotropin levels; 3) absence of
cise localization of the site of E2 feedback in the human endogenous gonadotropin pulsations during a 12- to 24-h period of
blood sampling; 4) otherwise normal reserve testing of anterior pituitary
requires a complimentary model in which both the dose and function; and 5) normal magnetic resonance imaging of the HP region.
frequency of GnRH administration can be experimentally At the time of participation in the study, all had normal serum concen-
controlled. Men with idiopathic hypogonadotropic hypogo- trations of T, LH, and FSH for at least 3 months, as a result of treatment
nadism (IHH), who lack endogenous hypothalamic GnRH with pulsatile sc GnRH therapy delivered at 2-h intervals (18).
secretion and whose pituitary-gonadal axis can be normal- The study was approved by the Human Research Committee at the
Massachusetts General Hospital, and all subjects provided written in-
ized with long-term pulsatile GnRH replacement (18), pro- formed consent.
vide such a model. Because the dose and frequency of ex-
ogenous GnRH administration can be experimentally Study protocol
controlled in this setting, this model, in effect, represents a
hypothalamic clamp. Therefore, any effects of altering go- Exp 1. Nine NL men and seven IHH men were treated with the aro-
matase inhibitor, anastrozole, 10 mg/day for 7 days. T, E2, LH, and FSH
nadal steroid levels on gonadotropin secretion in IHH men
were measured daily. Samples were drawn between 0800 and 1000 h in
can only reflect a pituitary site of action. In contrast, in NL the (NL) men and before a bolus dose of exogenous GnRH in the IHH
men with an intact HPG axis, gonadal steroids can modulate men.
gonadotropin secretion by direct inhibition at the level of the
Exp 2. Nine NL men, four of whom completed Exp 1, participated in a
pituitary, and/or by inhibiting GnRH secretion from the more intensive analysis of the gonadotropin response to E2 suppression.
hypothalamus. Thus, by the tandem study of these two hu- Subjects were admitted to the General Clinical Research Center of Mas-
man models, a hypothalamic site of action of sex steroids can sachusetts General Hospital and had an iv catheter inserted into a
be inferred whenever there is a difference in the gonadotro- forearm vein. Four of the nine NL men were admitted at 0700 h and had
pin responses of NL and IHH men to alterations in their sex 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
steroid milieu. extended protocol, which included administration of a GnRH antago-
Although it is not feasible to measure GnRH in peripheral nist. These men were admitted at 2300 h and had blood sampling from
blood in the human, we have previously validated use of a 2400 to 1200 h, after which they received a single sc injection of the
GnRH antagonist to provide a semiquantitative estimate of Nal-Glu GnRH antagonist (5 g/kg) to block the GnRH receptor. After
administration of the GnRH antagonist, blood samples were drawn
endogenous GnRH secretion (19, 20). This novel physiologic every 20 min, for a further 8 h, and subjects were then discharged. On
tool thus allows one to determine whether any given increase the morning after discharge, all nine NL men commenced taking anas-
in LH pulse amplitude reflects a hypothalamic (increase in trozole (10 mg/day for 7 days). On day 7 of anastrozole therapy, all
the GnRH bolus dose) or a pituitary (enhanced sensitivity to subjects were readmitted to the General Clinical Research Center for a
GnRH) effect. The basic premise of this approach is that the second 12-h frequent blood sampling study GnRH antagonist ad-
ministration, to examine the impact of E2 suppression on gonadotropin
response of a marker of GnRH action such as LH can be used secretion.
to assess GnRH secretion in the presence of submaximal In the 12-h frequent blood sampling study, all samples were assayed
GnRH receptor blockade, such that the amount of GnRH for LH, whereas FSH was measured in hourly samples. T and E2 were
secreted is inversely proportional to the degree of LH inhi- determined at baseline and in serum pools composed of equal aliquots
of each sample obtained at 6-h intervals. After administration of the
bition (19, 20). Therefore, if removal of E2 negative feedback GnRH antagonist, LH was measured in all samples, whereas T was
were to increase endogenous GnRH secretion, one would measured in hourly pools.
expect that LH secretion would be less susceptible to GnRH
receptor blockade in the E2-deplete vs. E2-replete state. Evaluation of sex steroid and gonadotropin secretion
The aim of the present study was to examine the impact
of E2 suppression on the HPG axis in the human male. In an Frequent blood sampling study. Mean LH and FSH levels were calculated
for both frequent blood sampling studies. Pulsatile LH secretion was
effort to circumvent some of the limitations of previous stud- analyzed using the modified Santen and Bardin method, as recently
ies, we used: 1) the potent, highly selective aromatase in- validated by the investigators (23, 24). The mean LH pulse amplitude
hibitor, anastrozole (21), to deplete endogenous estrogen; 2) (defined as the difference between the peak and the preceding nadir)
a complimentary approach involving the tandem study of was calculated at baseline and on day 7 of anastrozole therapy.
NL and IHH men to permit precise localization of the site of LH inhibition after GnRH antagonist administration. The maximum degree
E2 feedback; and 3) the Nal-Glu GnRH antagonist to provide of gonadotropin suppression after administration of the Nal-Glu GnRH
a semiquantitative estimate of GnRH secretion, as previously antagonist was determined by calculating the percent inhibition from
described (19, 20). the preantagonist period [(mean PRE nadir)/mean PRE] 100, as
previously described (19). Nadir LH levels were calculated using a
3-point moving average, which is equivalent to 1 h of sampling.
Subjects and Methods
Subjects Hormone assays
NL men. Fourteen NL men (age, 22–50 yr) participated in the study. All Serum LH and FSH concentrations were determined by microparticle
study subjects met the following criteria: 1) normal pubertal develop- enzyme immunoassay using the automated Abbott AxSYM system (Ab-
ment, sexual function, and general health; 2) normal physical exami- bott Laboratories, Chicago, IL). The Second International Reference
nation, including a testicular volume 20 mL; 3) normal serum levels Preparation was used as the reference standard. The assay sensitivity for
of testosterone (T), E2, LH, FSH, TSH, and PRL; and 4) normal semen both LH and FSH was 1.6 IU/L. The intraassay coefficient of variation
analysis, according to World Health Organization criteria (22). (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%.
IHH men. Seven men (age, 30 – 46 yr) with isolated GnRH deficiency were Serum T concentrations were measured using the DPC Coat-A-Count
selected on the basis of the following criteria: 1) failure to undergo RIA kit (Diagnostic Products Corp., Los Angeles, CA), which had an
spontaneous puberty by the age of 18 yr; 2) serum T 3.5 nmol/L, in intra- and interassay CV less than 10%. E2 was measured by the Abbott
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HYPOTHALAMIC SITE OF ACTION OF E2 IN MEN 3029
AxSYM system, which had an analytical sensitivity of 36 pmol/L and Exp 2
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 Confirming the results of the single time point data ob-
using a commercially available (Serotec, Oxford, UK) double-antibody tained in Exp 1, frequent sampling studies in NL men
enzyme-linked immunosorbent assay, as previously described (25). In showed that anastrozole resulted in marked suppression of
our use, the clinical detection limit of this assay is 50 pg/mL, with a CV E2, accompanied by a significant increase in T, LH, and FSH
of 4 – 6% within plate and 15–18% between plates.
levels (Table 2). This increase in mean LH levels reflected an
increase in both LH pulse frequency (10.2 0.9 to 14.0 1.0
Statistical methods pulses/24 h, P 0.05) and LH pulse amplitude (5.7 0.7 to
Mean daily hormone levels in the NL and IHH men, over the 7 days 8.4 0.7 IU/L, P 0.05). Frequent blood sampling data from
of anastrozole therapy, were analyzed using ANOVA for repeated mea- a representative NL subject are indicated in Fig. 3. In this
sures, followed by post hoc Newman-Keuls testing for individual dif- individual, anastrozole resulted in a 60% increase in the
ferences. To compare the responses of the NL and IHH men, the data
were expressed as percent change from baseline, and the mean levels of
number of LH pulses despite a marked increase in T, which
the two groups were compared using ANOVA. For the frequent blood normally serves to restrain the hypothalamic GnRH pulse
sampling studies performed in the NL men at baseline and on day 7 of generator. Of the nine NL men studied, LH pulse frequency
anastrozole therapy, mean hormone levels, LH pulse frequency, and LH increased in seven, decreased in one, and was unchanged in
pulse amplitude were compared using a two-tailed paired t test. The another (Fig. 4). LH pulse amplitude increased in all study
maximum percent LH inhibition after GnRH antagonist administration
at baseline and on day 7 of anastrozole were compared using a paired subjects (Fig. 4).
t test. A P value less than 0.05 was taken to be statistically significant. Acute GnRH receptor blockade resulted in marked sup-
pression of LH, with mean levels falling from 9.7 1.5 to a
Results 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
Baseline
0.005). When the data were expressed as percent baseline, to
Baseline characteristics of the normal (NL) and IHH men allow comparison between studies performed in an E2-
are summarized in Table 1. The two groups were of similar replete vs. E2-deplete state, maximum percent LH inhibition
age, but the IHH men were slightly heavier (P 0.05). Mean was identical (69.2 2.4 vs. 70.0 1.9%) (Fig. 5).
T, E2, and LH levels were similar in both groups. FSH levels
tended to be higher in the IHH men, although this difference Discussion
did not achieve statistical significance. In keeping with their
smaller testicular size and higher FSH levels, IHH men had This parallel study of the response of NL and GnRH-
lower mean inhibin B levels than did NL men (P 0.05). deficient men to estrogen suppression clearly demonstrates
that, in the human male, E2 has both a pituitary and a hy-
pothalamic site of action. The increase in gonadotropin se-
Exp 1 cretion after aromatase inhibition, in IHH men on a fixed,
Treatment with anastrozole resulted in marked suppres- experimentally controlled GnRH regimen, confirms previ-
sion of E2 in both NL (P 0.005) and IHH men (P 0.005) ous studies indicating that E2 has a pituitary site of action
(Fig. 1). Mean absolute levels and percent suppression of E2 (1– 8) that, in this study, accounts for approximately half of
were similar in both groups for the duration of anastrozole the gonadotropin elevation observed in NL men. However,
therapy (52 2 pmol/L in the NL vs. 60 5 pmol/L in the the demonstration that estrogen suppression leads to a
IHH men; P, not significant). T levels rose significantly (P greater increase in gonadotropin levels in NL than in GnRH-
0.005) in both groups, with a mean increase of 53 6% in the deficient men also documents an additional hypothalamic
NL vs. 56 7% in the IHH men (Fig. 1). Despite these similar effect of E2.
changes in sex steroids, the increase in gonadotropin levels E2 could potentially alter GnRH secretion by increasing the
was greater in NL than in IHH men (100 9 vs. 58 6% for frequency and/or the amplitude of GnRH pulses from the
LH, P 0.07; and 85 6 vs. 41 4% for FSH, P 0.002) hypothalamus. Analysis of pulsatile LH secretion after E2
(Fig. 2). 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
TABLE 1. Baseline characteristics of normal men and men with several previous studies showing that a change in LH pulse
IHH
frequency can be detected when antiestrogens are adminis-
Parameter Normal men (n 14) IHH men (n 7) tered to NL men (6, 9 –12). Despite variations in the choice of
Age (yr) 31.0 2.1 36.7 2.6
antiestrogen (clomiphene vs. tamoxifen), duration of therapy
Body mass index (kg/m2) 26.0 0.7 30.3 1.9a (from 1– 6 weeks) and sampling paradigm used (10-min vs.
Testicular volume (mL) 23.0 0.7 12.0 1.2b 20-min intervals), an increase in GnRH pulse frequency was
Testosterone (nmol/L) 19 1 15 1 observed in the vast majority of individuals studied (6, 9 –12).
E2 (pmol/L) 136 11 118 22 In the single study where clomiphene administration was
LH (IU/L) 11.1 1.1 12.9 1.8
FSH (IU/L) 7.1 0.8 15.8 4.0 reported to have no impact on LH pulse frequency, the sam-
Inhibin B (pg/mL) 192 24 99 19a ple size of two precludes any conclusions (28). Confirmatory
Results are expressed as mean SEM. evidence for a role of estrogen in modulating GnRH pulse
a
P 0.05 compared with normal men. frequency is provided by studies in sheep that indicate that
b
P 0.005 compared with normal men. estrogen administration to long-term castrated rams lowers
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3030 HAYES ET AL. JCE & M • 2000
Vol. 85 • No. 9
FIG. 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.
mean LH levels, mainly by decreasing LH pulse frequency as an estrogen agonist at the pituitary, causing decreased
(13, 14). A hypothalamic action of E2 is also evident from responsiveness to exogenous GnRH (11).
studies in the primate demonstrating that direct administra- In this study, we adopted a different approach, to dissect
tion of E2 into the hypothalamus (29) or third ventricle (30) the basis for the increase in LH pulse amplitude after E2
suppresses LH secretion. suppression, using a GnRH antagonist to provide a semi-
The increase in LH pulse amplitude, observed after aro- quantitative estimate of endogenous GnRH secretion. The
matase inhibition, could potentially reflect an increase in the basic premise of this approach is that the response of a
amplitude of GnRH pulses stimulating the pituitary, and/or marker of GnRH action, such as LH, can be used to assess
enhanced pituitary sensitivity to the same amount of endog- GnRH secretion in the presence of submaximal GnRH re-
enous GnRH. Previous studies have attempted to distinguish ceptor blockade, such that the amount of GnRH secreted will
between these two mechanisms by examining pituitary be inversely proportional to the degree of LH inhibition (19,
responsiveness to pharmacological doses of exogenous 20). Such an approach is only possible because GnRH is the
GnRH before and during antiestrogen therapy (11, 31, 32). only known secretagogue for LH, GnRH and its antagonist
These studies paradoxically demonstrated that clomiphene bind to a single receptor type, and there is no evidence of any
blunted pituitary responsiveness to exogenous GnRH de- change in GnRH receptor affinity over a wide range of both
spite increasing both mean LH levels and the amplitude of physiologic and pharmacologic conditions (19). In the
spontaneous LH pulses (11, 31, 32). The mechanism pro- present study, the degree of LH inhibition, after acute GnRH
posed for this divergence between spontaneous pulse height receptor blockade, was unaltered by E2 suppression. We feel
and acute pituitary responsiveness to exogenous GnRH was that it is unlikely that use of a lower GnRH antagonist dose
that clomiphene was having tissue-specific mixed agonist/ would have identified differences in GnRH secretion after
antagonist effects. The authors concluded that clomiphene estrogen suppression, for the following reasons. First, the
was acting as an estrogen antagonist at the hypothalamus, degree of LH suppression achieved was submaximal, with
resulting in an increase in endogenous GnRH secretion, but nadir levels above the limit of detection of the LH assay.
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HYPOTHALAMIC SITE OF ACTION OF E2 IN MEN 3031
FIG. 2. Changes in gonadotropin concentrations in NL men (left) and IHH men (right), in response to administration of anastrozole, as in Fig.
1. 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.
TABLE 2. Impact of the aromatase inhibitor, anastrozole (10 mg/ LH to GnRH. This conclusion is at variance with the studies
day 7 days) on gonadal steroid and gonadotropin levels in reporting that clomiphene diminishes pituitary responsive-
normal men (n 9)
ness to exogenous GnRH administration (11, 31, 32). How-
Baseline Day 7 of anastrozole ever, the major limitations to using GnRH tests to assess
E2 (pmol/L) 162 19 95 7a pituitary sensitivity in an intact system are: 1) the fact that the
Testosterone (nmol/L) 18 2 33 3b pituitary LH response to an exogenous GnRH bolus varies
Mean LH (IU/L) 9.5 0.9 16.8 1.4b significantly with the previous LH interpulse interval (34);
LH pulses/24 h 10.2 0.9 14.0 1.0a and 2) the pharmacological nature of the doses used. Ac-
LH pulse amplitude (IU/L) 5.7 0.7 8.4 0.7b
Mean FSH (IU/L) 6.0 0.6 11.2 1.3b
cordingly, there is marked variability in the LH response to
Inhibin B (pg/mL) 171 16 207 15a a single bolus dose of GnRH in NL men (35). Therefore, the
Results are mean SEM and are based on blood samples drawn
results of single-dose GnRH testing are difficult to interpret
every 10 min 12 h. unless endogenous gonadotropin secretion is blocked so that
a
P 0.05 compared with baseline. the confounder of variable endogenous interpulse intervals
b
P 0.001 compared with baseline. is eliminated.
Having excluded an increase in GnRH pulse amplitude,
Second, the same antagonist dose used in this study (5 g/kg other potential mechanisms for the increased LH pulse am-
Nal-Glu) has previously been shown to be capable of de- plitude after E2 suppression include an increase in pituitary
tecting differences in GnRH secretion in different physiologic responsiveness to GnRH as a result of an increase in: 1) the
and pathophysiologic states in both men (33) and women number of gonadotropes; 2) the number of GnRH receptors;
(19). Therefore, we conclude that the hypothalamic effect of and 3) the affinity of GnRH for its receptor. It seems unlikely
E2 suppression is to increase the frequency, rather than the that 7 days of estrogen suppression would alter gonadotrope
bolus dose, of GnRH. This finding, in turn, implies that the number in these adult men. In cultured pituitary cells from
increase in LH pulse amplitude observed after aromatase ovariectomized ewes, estrogen administration has been
inhibition is attributable to enhanced pituitary sensitivity of shown to increase GnRH receptor number and to have no
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3032 HAYES ET AL. JCE & M • 2000
Vol. 85 • No. 9
FIG. 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 12 h. Triangles represent LH pulses detected using the
modified Santen and Bardin pulse-detection algorithm (23, 24).
effect on binding affinity (36). Indeed, there has been no ies in both prepubertal boys (42, 43) and adult males (1–3,
demonstration of changes in GnRH receptor affinity over a 7, 8, 41, 44), indicating that, on a molar basis, the steroid
wide range of physiologic and pharmacologic conditions dose required to suppress gonadotropin secretion is ap-
(37). To our knowledge, no data are available on the effect of proximately 200-fold less for E2 than for T.
estrogen withdrawal on GnRH receptor number in the male. A number of other approaches can be taken to study sex
Therefore, the mechanism(s) underlying the increase in LH steroid regulation of gonadotropins in the human male, all
pulse amplitude remains speculative. of which have inherent limitations. A series of experiments
In the present study, E2 suppression resulted in a sig- of nature comprising patients with E2 receptor mutations and
nificant increase in LH pulse frequency. This change in congenital aromatase deficiency provide models that permit
pulse frequency was all the more impressive given that it the impact of selective estrogen ablation to be examined in
occurred despite a concomitant rise in T levels, which the human. However, the major limitation of this genetic
normally has a restraining influence on the GnRH pulse approach is the small number of patients available for study.
generator (2, 4, 38 – 41). Therefore, the net effect of remov- To date, only one estrogen receptor (ER) mutation (45) and
ing E2 negative feedback, while allowing T levels to rise to two cases of congenital aromatase deficiency (46, 47) have
the supraphysiologic range, is an increase in gonadotropin been described in adult males. However, consistent with the
secretion. These data therefore speak to the importance of data we obtained using an aromatase inhibitor, congenital E2
E2 in the negative feedback control of gonadotropin se- deficiency was associated with a 2- to 3-fold increase in FSH
cretion in the male. This concept that E2 is a more potent in all three patients, and in LH in two individuals, despite
suppressor of LH secretion than is T is supported by stud- normal-to-elevated T levels (45– 47). In addition, estrogen
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HYPOTHALAMIC SITE OF ACTION OF E2 IN MEN 3033
FIG. 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.
most likely reflects the fact that ERKO mice are not totally
deficient in estrogen action, because they continue to express
the ER- 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 ERKO mice (53). Although gonadotropin lev-
els were not reported for the male ERKO mice, LH levels
in the female double knockouts were higher than those seen
in the ERKO mice, suggesting that some of estrogen’s feed-
back effects are mediated by the ER- 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
FIG. 5. Percent LH inhibition in five NL men after administration of of subjects were matched for gonadal steroids at baseline,
the Nal-Glu GnRH antagonist at baseline and on day 7 (D7) of anas- inhibin B levels were significantly higher in the NL than the
trozole therapy. The Nal-Glu GnRH antagonist was administered sc IHH men. However, given that inhibin B is an important
at a dose of 5 g/kg at time zero.
negative feedback regulator of FSH secretion (54 –59), one
treatment resulted in complete suppression of serum gonad- would expect that the lower inhibin B levels in the IHH men
otropin levels (47). Characterization of the HPG axis in these would have facilitated a greater FSH response to estrogen
cases was based on a single time point estimation. Whereas suppression than that seen in NL men. The fact that the rise
a single sample is adequate to obtain an accurate estimate of in FSH was 2-fold greater in the NL than in the IHH men is
FSH secretion, it does not accurately reflect mean LH levels, therefore all the more significant. In a previous study em-
given the pulsatile pattern of LH secretion. Therefore, it is ploying this same tandem model, we found that estrogen
possible that the normal LH concentration observed in one administration suppressed gonadotropin secretion to the
individual with congenital aromatase deficiency (47) repre- same degree in NL and GnRH-deficient men, suggesting that
sented the trough level of a pulse. the major site of E2 feedback was at the pituitary (7). It is
Though the phenotype of the ER mutation and congenital important to note that a significant change in LH pulse fre-
aromatase deficiency patients is similar, in terms of effect on quency was observed in the NL men in that study, and it is
the HP axis, the phenotype of the corresponding male mice possible that a difference in the responses of NL and GnRH-
knockouts, created by targeted disruption of the ER- gene deficient men would have been detected if physiologic (as
(ERKO mice) (48) and the aromatase CYP19 gene (ArKO opposed to pharmacologic) doses of sex steroids had been
mice) (49), respectively, is different. As in cases of congenital used.
aromatase deficiency in the human, adult male ArKO mice Until recently, the precise cellular mechanism by which E2
exhibit elevated levels of gonadotropins despite high circu- suppresses GnRH secretion was controversial. On the one
lating T concentrations (49). In contrast, adult ERKO males hand, estrogen response elements had been demonstrated in
exhibit normal levels of hypothalamic GnRH, pituitary FSH the promoter region of the primate GnRH gene (60). In ad-
messenger RNA (mRNA), and serum FSH, but elevated LH dition, there were reports of low levels of ER mRNA in two
levels and markedly diminished fertility (50). This difference different immortalized GnRH cell lines (60, 61). On the other
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3034 HAYES ET AL. JCE & M • 2000
Vol. 85 • No. 9
hand, immunocytochemical studies using ER double-label- anism for increased luteinizing hormone secretion. J Clin Endocrinol Metab.
48:315–319.
ing had failed to demonstrate ER expression on GnRH neu- 11. Winters SJ, Troen P. 1985 Evidence for a role of endogenous estrogen in the
rons in a variety of species, including the rat (62), guinea pig hypothalamic control of gonadotropin secretion in men. J Clin Endocrinol
(63), sheep (64, 65), and monkey (66, 67). The absence of ER Metab. 61:842– 845.
12. Veldhuis JD, Dufau ML. 1987 Estradiol modulates the pulsatile secretion of
immunoreactivity, combined with the demonstration that biologically active luteinizing hormone in man. J Clin Invest. 80:631– 638.
GnRH neurons did not concentrate E2 (68), suggested that 13. Schanbacher BD. 1984 Regulation of luteinizing hormone secretion in male
estrogen effects on GnRH were not occurring through a sheep by endogenous estrogen. Endocrinology. 115:944 –950.
14. Scott CJ, Kuehl DE, Ferreira SA, Jackson GL. 1997 Hypothalamic sites of
classic ER-mediated process. It was thus postulated that es- action for testosterone, dihydrotestosterone, and estrogen in the regulation of
trogen-receptive neurons were acting as intermediaries in luteinizing hormone secretion in male sheep. Endocrinology. 138:3686 –3694.
the nongenomic regulation of GnRH by estrogen (for review, 15. Vigersky RA, Mozingo D, Eil C, Purohit V, Bruton J. 1982 The antiandrogenic
effects of delta 1-testolactone (Teslac) in vivo in rats and in vitro in human
see Ref. 69). However, recent studies using the novel and cultured fibroblasts, rat mammary carcinoma cells, and rat prostate cytosol.
highly sensitive technique of single-cell multiplex RT-PCR Endocrinology. 110:214 –219.
demonstrated, for the first time, the presence of both ER and 16. Levine JE, Pau KYF, Ramirez VD, Jackson GL. 1982 Simultaneous measure-
ment of luteinizing hormone-releasing hormone and luteinizing hormone
ER messenger RNA in native GnRH neurons (70). In ad- release in unanesthetized, ovariectomized sheep. Endocrinology. 111:
dition, evidence has now been provided that estrogen can 1449 –1455.
17. Spratt DI, Finkelstein JS, Badger TM, Butler JP, Crowley WF. 1986 Bio- and
directly suppress GnRH gene expression in ER - and ER - immunoreactive luteinizing hormone responses to low doses of gonadotropin-
expressing GT1–7 GnRH neurons (71). releasing hormone (GnRH):dose response curves in GnRH deficient men.
From these clinical investigative studies on the impact of J Clin Endocrinol Metab. 63:143–150.
18. Hoffman AR, Crowley WF. 1982 Induction of puberty in men by long-term
aromatase inhibition in NL and GnRH-deficient men, em- pulsatile administration of low-dose gonadotropin-releasing hormone. N Engl
ploying frequent blood sampling combined with adminis- J Med. 307:1237–1241.
tration of a GnRH antagonist, we conclude that, in the human 19. Hall JE, Taylor AE, Martin KA, Rivier J, Schoenfeld DA, Crowley Jr WF. 1994
Decreased release of gonadotropin-releasing hormone during the preovula-
male, estrogen has dual sites of negative feedback, acting at tory midcycle luteinizing hormone surge in normal women. Proc Natl Acad
the hypothalamus to decrease GnRH pulse frequency and at Sci USA. 91:6894 – 6898.
the pituitary to decrease pituitary responsiveness to GnRH. 20. Hayes FJ, Taylor AE, Martin KM, Hall JE. 1998 Use of a GnRH antagonist as
a physiologic probe in polycystic ovary syndrome: assessment of neuroendo-
crine and androgen dynamics. J Clin Endocrinol Metab. 83:2343–2349.
Acknowledgments 21. Plourde PV, Dyroff M, Dukes M. 1994 Arimidex: a potent and selective
fourth-generation aromatase inhibitor. Breast Cancer Res Treat. 30:103–111.
We gratefully acknowledge the nurses of the General Clinical Re- 22. World Health Organization. 1992 WHO laboratory manual for the examina-
search Center for excellent clinical care and the technicians of the Re- tion of human semen and sperm-cervical mucus interaction. 3rd ed. Cam-
productive Endocrine Sciences Center Radioimmunoassay Core for su- bridge: Cambridge University Press.
23. Santen RJ, Bardin CW. 1973 Episodic luteinizing hormone secretion in man.
perb technical contributions to this study. The Nal-Glu GnRH antagonist
Pulse analysis, clinical interpretation, physiologic mechanisms. J Clin Invest.
was synthesized at the Salk Institute under contract with the NIH and 52:2617–2628.
made available by the Contraceptive Development Branch, Center for 24. Hayes FJ, McNicholl DJ, Schoenfeld D, Marsh EE, Hall JE. 1999 Free alpha-
Population Research, National Institute of Child Health and Human subunit is superior to luteinizing hormone as a marker of gonadotropin-
Development. We thank Jean Rivier, Ph.D., and Marvin Karten, Ph.D., releasing hormone despite desensitization at fast pulse frequencies. J Clin
for support in these studies. Endocrinol Metab. 84:1028 –1036.
25. Groome KP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimeric
inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab.
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