0013-7227/04/$15.00/0
Printed in U.S.A.
Endocrinology 145(10):4558 – 4564
Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2004-0517
Alcohol Alters Luteinizing Hormone Secretion in
Immature Female Rhesus Monkeys by a
Hypothalamic Action
GREGORY A. DISSEN, ROBERT K. DEARTH, H. MORGAN SCOTT, SERGIO R. OJEDA,
W. LES DEES
AND
Oregon National Primate Research Center (G.A.D., S.R.O.), Oregon Health and Science University, Beaverton, Oregon
97006-3448; and Department of Integrative Biosciences (R.K.D., H.M.S., W.L.D.), College of Veterinary Medicine, Texas
A&M University, College Station, Texas 77843-4458
We determined whether the effect of alcohol (ALC) to suppress
LH secretion in immature female monkeys is due to a hypothalamic or pituitary site of action. Beginning at 20 months of
age, four monkeys received a single intragastric dose of ALC
(2.4 g/kg), and four monkeys received an equal volume of a
saline/sucrose solution daily until they were 36 months old.
For the hypothalamic response test, two basal samples (3.5 ml)
were collected at 15-min intervals via the saphenous vein, and
then N-methyl-D-L-aspartic acid (NMA; 20 mg/kg) was given iv
and four more blood samples collected. Three weeks later, this
protocol was repeated except LH-releasing hormone (LHRH)
(5 ␮g/kg) was used to test pituitary responsiveness. NMA or
LHRH was administered 3 h after the ALC. After the pituitary
challenge, each monkey was ovariectomized and 6 wk later,
I
N RECENT YEARS, there has been an increase in alcohol
(ALC) use and abuse during adolescence, especially by
females (1). The adolescent period is a potentially vulnerable
time for developing individuals who may be more sensitive
to the drug and less tolerant to its detrimental effects than
adults. This concept has been supported by work using the
rat as an animal model (2, 3). The possibility that ALC could
alter neuroendocrine development has been suspected for
years because a history of any drug ingestion is routinely
conducted to identify potential causes of altered endocrine
function or pubertal development. Any detrimental effect of
ALC on puberty-related hormones at this critical time of
growth and development could elevate the risk for developmental deficits.
Research with experimental animals and case reports from
humans further suggest that determining the effects of ALC
on endocrine development warrants serious consideration.
Studies using rats have shown that ALC suppresses the levels of LH and estradiol (E2) and causes delayed female puberty (4 – 6). Even though case studies involving ALC use and
abuse by adolescent and teenage humans are limited in number and scope, they have suggested that the drug can disrupt
endocrine function, stature, and weight distribution in
Abbreviations: ALC, Alcohol; BAC, blood ALC concentration; E2,
estradiol; LHRH, LH-releasing hormone; NMA, N-methyl-d-l-aspartic
acid; NMDA, N-methyl-d-aspartate.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
implanted with an indwelling subclavian vein catheter. Blood
samples were drawn every 10 min for 8 h to assess effects of
ALC on post-ovariectomy LH levels and the profile of LH pulsatile secretion. The hypothalamic challenge showed NMA
stimulated LH release in control monkeys, an action that was
blocked by ALC. The pituitary challenge revealed that LHRH
stimulated LH release equally well in control and ALC-treated
monkeys. A post-ovariectomy rise in LH was observed in both
groups, but levels were 45% lower in ALC-treated monkeys.
This reduction was attributed to an ALC-induced suppression
of both baseline and amplitude of pulses. Results demonstrate
that the ALC-induced suppression of LH in immature female
rhesus monkeys is due to an inhibitory action of the drug at
the hypothalamic level. (Endocrinology 145: 4558 – 4564, 2004)
young people (7–9), as well as place them at risk for nutritional deficiencies (10). Using the developing female rhesus
monkey as an animal model, we have shown previously that
chronic ALC administration inhibits the secretion of pubertyrelated hormones, including LH, and delayed the development of a normal pattern of menstruation (11). These results
lead us to suggest that the suppression in LH contributed to
the suppression observed in circulating E2 and to the lengthened time interval between menstruations. The fact that ALC
causes suppressed serum LH at this critical time of development is important, but a question remains as to whether
this effect is due to a hypothalamic or a pituitary site of
action. In immature rodents there have been several reports
suggesting an ALC-induced deficit at the hypothalamic level
(12–15), but this has not been tested in immature primates.
This is an important question considering the major influence
of E2 directly at the pituitary level in primates (16). Thus, to
determine the site of action for ALC to suppress LH secretion
in immature primates, we assessed hypothalamic and pituitary responsiveness to N-methyl d-l-aspartic acid (NMA)
and LH-releasing hormone (LHRH), respectively. Furthermore, we evaluated the effect of ALC on the post-ovariectomy LH response to gonadal steroid removal and on the
parameters controlling the pulsatile secretion of LH.
Materials and Methods
Animals
Immature female rhesus monkeys (Macaca mulata) were purchased
from Shared Enterprises (Devon, PA). The monkeys were captive born
4558
Dissen et al. • ALC Alters LH Release by Hypothalamic Action
at the Institute of Laboratory Animal Science (Chinese Academy of
Medical Sciences, Beijing, China). At 15 months of age, they were individually caged for 30 d before shipment. Upon arrival at the Oregon
National Primate Research Center (ONPRC), they were quarantined in
individual cages of 90 d indoors with controlled lighting (12-h light, 12-h
dark; lights on, 0700 h) and temperature (22 C). After the quarantine
period, the monkeys were paired socially in the same light and temperature conditions. They were fed monkey chow (Jumbo 537, Ralston
Purina Co., St. Louis, MO) twice daily at 0800 and 1500 h. Food consumption was assessed and recorded daily. All monkeys were weighed
at 2- to 4-wk intervals, and the amount of food provided (4 –5% of body
weight) was adjusted as the animals grew. Their diet was supplemented
daily with fruit and other food additives as part of their environmental
enrichment. The monkeys selected for this study were closely matched
in age, with their mean (⫾sem) beginning age being 20.0 ⫾ 0.2 months.
None of the monkeys had been exposed previously to ALC or any other
drug. Animal protocols and experiments were approved by the ONPRC
institutional animal care and use committee in accordance with the
National Institutes of Health policy on the use of animals in research and
the Guide for the Care and Use of Laboratory Animals. The health of the
monkeys was monitored by veterinarians in the Division of Animal
Resources at the ONPRC.
ALC dosing procedure
Daily at 1230 –1330 h, four animals received a single intragastric dose
of ALC (2.4 g/kg; ethanol diluted in saline) and four animals received
a saline/sucrose control solution that was isocalorically equivalent to the
ethanol/saline solution. Both solutions were administered in equal volumes (10 ml/kg body weight) to unanesthetized monkeys using a pediatric-grade gastric intubation tube (5-French, Professional Medical
Products, Inc., Greenwood, SC). The dosing continued daily until the
monkeys were 36 –37 months of age when the tests described below were
performed. Monitoring of progesterone levels three times per week after
30 months of age confirmed that none of the monkeys had ovulated at
the time of their testing.
Endocrinology, October 2004, 145(10):4558 – 4564 4559
system that included a two-channel swivel attached to the top of the
cage. Tubing attached to the top of the swivel was passed out of the
animal room to the sampling room. The patency of the catheter was
maintained by continuous infusion of a heparin solution (5 U/ml in 0.9%
saline; 0.6 ml/h). A pharyngostomy tube, for administration of ALC or
saline/sucrose control solution, was passed from the caudal oropharynx
through the skin of the neck into the sc tissues caudal to the mandibular
ramus, and was then tunneled sc to the midscapular region where it was
exteriorized for catheter protection attachment. The pharyngeal portion
of the tubing was inserted into the esophagus using a laryngoscope, and
advanced to the stomach. The pharyngeal tube was used to administer
the ALC solution to the monkeys. Two of the control monkeys removed
the pharyngeal portion of their pharyngostomy tube; thus, it was not
possible to administer the saline/sucrose control solution via the tubing.
Because this solution is viscous and difficult to pass through the small
tubing, the control monkeys were allowed to drink their treatment from
the sipper bottles hung on the outside of their cage. All the monkeys
were individually caged while the catheters were inserted, and seethrough screens separated monkeys that were paired previously. The
monkeys were free to move about their cage, and all eight adapted well
to the catheter protection system. Beginning at 0800 h, a 0.5-ml blood
sample was collected once every 10 min for 3 h, and then the monkeys
received their daily ALC or saline/sucrose solution. After a 90-min
period to allow for ALC absorption, the sampling resumed for another
4 h. Serum samples were stored frozen until assayed to assess the
following characteristics of pulsatile LH release: 1) mean LH, 2) baseline
LH (mean of all troughs recorded between peaks), 3) amplitude (peaktrough difference) of peaks, and 4) frequency of peaks (per hour).
Blood ALC analysis
In each of the three experiments, the blood samples at 3 h post ALC
administration were also used to assess blood ALC concentration (BAC).
The BAC of each monkey was measured in duplicate by an enzymatic
method (Sigma Inc., St. Louis, Mo.) that is both sensitive and reliable (19).
The BAC for each of the above experiments was reported as the mean
(⫾sem) for the four ALC-treated monkeys.
Hypothalamic response test
NMA is known to stimulate LH by acting directly within the hypothalamus to induce LHRH release and does not act at the pituitary level
(17, 18). On the day of the test, the monkeys received either their ALC
or saline/sucrose control solution as they had for approximately 16
months, and 2 h and 45 min later the first basal blood sample (3.5 ml)
was collected from unanesthetized monkeys via the saphenous vein. A
second basal sample was collected 15 min later. Immediately after the
second basal sample (3 h after administration of the ALC or control
solution), a single bolus dose of NMA (20 mg/kg body weight) was
given iv, and four more blood samples were collected as above every 15
min. All blood samples were allowed to clot and then centrifuged, and
the serum frozen for subsequent analysis of LH by the method described
below. None of the monkeys had a menses the month of the experiment,
although three of four in each group had shown signs of menses previously with only two in the ALC group and three in the control group
having menses more than 1 d.
Pituitary response test
Approximately 3 wk after the hypothalamic response test, the same
monkeys were used to assess their pituitary responsiveness. During the
interval between experiments, no menses were noted and the monkeys
continued to receive their daily dose of ALC or the control solution. On
the day of the experiment, we used exactly the same protocol and time
table as above except that LHRH (5 ␮g/kg) was used to stimulate LH
release from the pituitary directly.
Pulsatile LH assessment
After the above response test, the monkeys were ovariectomized
using a laparoscopic technique and allowed to recover for 6 wk. Before
remote blood sampling, an indwelling catheter was surgically inserted
into the subclavian vein of each animal. The catheter was exteriorized
in the mid-scapula region and passed through a catheter protection
LH RIA
Monkey LH was assessed in serum as described previously (11) using
a kit purchased from the National Hormone and Pituitary Program
(National Institute of Diabetes and Digestive and Kidney Diseases). The
sensitivity of the assay was 0.07 ng/ml. Samples for the NMA and LHRH
challenge experiments were assessed in one assay and the intraassay
variation was 3.2%. Likewise, all of the samples from the pulsatility
study were assessed in one assay and the intraassay variation was 3.9%.
Statistics
In both the NMA and LHRH challenge experiments, the first two
blood samples from each animal were used to determine the average
basal level of LH for that animal. The sample showing the highest level
of LH after the respective challenge represented the peak response that
was induced. Differences in LH responsiveness between the four control
and the four ALC-treated monkeys were analyzed using the unpaired
Student’s t test. INSTAT and PRISM software (GraphPad, San Diego,
CA) were used to calculate and graph these results.
The algorithms incorporated in the PULSAR program was used as
described previously (20) to assess the characteristics of the pulsatile
release of LH. Cut-off points for peak determination were 3.8, 2.6, 1.9,
1.5, and 1.2 for 1-, 2-, 3-, 4-, and 5-point peaks, respectively. The sensitivity of the assessment was set to 0.07 ng/ml (LH assay sensitivity) and
the sd for the linear term set at 3.9 (intraassay variation). PULSAR was
run separately for the morning (180 min) and afternoon (240 min) periods from each monkey. These assessments were separated by the
designated administration/absorption break.
Because there were multiple measurements recorded per monkey,
dependence among observations within each monkey was expected. To
provide a more conservative estimate of the significance of any differences in mean LH between treatments and across sampling periods
(morning and afternoon), we used a mixed model (PROC MIXED, version SAS 8.2, SAS Institute, Cary, NC) that included a full factorial design
4560
Endocrinology, October 2004, 145(10):4558 – 4564
Dissen et al. • ALC Alters LH Release by Hypothalamic Action
for the fixed effects of treatment and sampling period, and a random
effect (variance components correlation structure) for the effect of each
monkey. Type 111 tests of fixed effects, including main effects and
interaction terms, were assessed. Least-squares means (⫾sem) were
determined for both fixed effects and interactions. The significance of
differences between the means for each of the two main effects (treatment and period) as well as for the morning to afternoon difference
within each treatment was assessed.
The mixed model approach outlined above was also used to assess
the effects of treatment and period on mean peak amplitude and mean
baseline (trough) LH. The differences in mean hourly pulse frequency
were assessed in full factorial ANOVA design using a general linear
model (SPSS 11.0; SSS Inc., Chicago, IL). Probability values of P ⬍ 0.05
were considered statistically significant for all of the evaluations described above.
Results
The 2.4 g/kg dose of ALC administered intragastrically
produced a mean (⫾sem) BAC of 182 ⫾ 7. 3 mg/dl and 179 ⫾
5.7 mg/dl, respectively, for the NMA and LHRH challenge
experiments, and 180 ⫾ 4.2 mg/dl for the LH pulsatility
evaluation study. During the 4 months before these tests,
BACs were assessed eight times and consistently found to be
in this range (178 ⫾ 3.6 mg/dl). The monkeys appeared only
mildly intoxicated at this 3-h time point, and as reported
previously (11), periodic sampling indicated that there was
little to no ALC remaining in blood by the following morning. Throughout the course of the study, appetite and food
consumption were not affected by the ALC. The body
weights were not different (P ⬎ 0.05) between control and
ALC-treated monkeys.
FIG. 1. The effects of chronic ALC exposure on NMA-induced LH
release in intact immature female monkeys. The left panel depicts
that NMA induced a 2-fold increase in serum LH release over basal
LH levels in control animals. The right panel demonstrates that basal
LH levels were depressed in the ALC-treated monkeys and that the
NMA challenge failed to elicit a release in serum LH over basal levels.
Open bars represent the mean (⫾SEM) basal LH levels, and solid bars
represent the mean (⫾SEM) peak LH response induced by NMA. **,
P ⬍ 0.01 vs. its own basal level; ⫹, P ⬍ 0.05 vs. control basal levels
(n ⫽ 4 monkeys in each group).
Hypothalamic responsiveness
In the control animals, NMA induced an increase in serum
LH that was detectable 10 min after the challenge. By comparison, the monkeys that received ALC did not show a
change in LH levels in response to the NMA challenge.
Figure 1 depicts the mean (⫾sem) basal and peak response
for both control and ALC-treated monkeys. The controls
showed a basal level of LH at 6.3 ⫾ 0.8 ng/ml, with a 2-fold
increase (P ⬍ 0.01) to 11.9 ⫾ 0.7 ng/ml after the NMA
challenge. The monkeys that received ALC showed a basal
level of LH at 4.5 ⫾ 0.3 ng/ml, a moderate suppression (P ⬍
0.05) compared with the basal level observed in the control
monkeys. After the NMA challenge, LH levels in the ALCtreated monkeys were 5.5 ⫾ 0.6 ng/ml, indicating that ALC
blocked the NMA-induced release of LH.
FIG. 2. The effects of chronic ALC exposure on LHRH-induced LH
release in intact immature female monkeys. The left panel depicts
that LHRH induced a 2.8-fold increase in serum LH release over basal
LH levels in control animals. The right panel demonstrates that basal
LH levels were depressed in the ALC-treated monkeys, and that the
LHRH challenge induced a 3-fold increase in serum LH release over
their basal levels. Open bars represent the mean (⫾SEM) basal LH
levels, and solid bars represent the mean (⫾SEM) peak LH response
induced by LHRH. **, P ⬍ 0.01 vs. its own basal levels; ⫹, P ⬍ 0.05
vs. control basal levels (n ⫽ 4 monkeys in each group).
LH pulsatility assessments
Pituitary responsiveness
LHRH induced an increase in serum LH within 10 min
after the challenge in both control and ALC-treated monkeys.
Figure 2 demonstrates the mean (⫾sem) basal and peak response for both groups. The controls showed a basal level of
LH at 5.3 ⫾ 0.4 ng/ml, then a 2.8-fold increase (P ⬍ 0.01) to
14.9 ng/ml ⫾ 1.3 after the LHRH challenge. Monkeys that
received ALC showed a basal level of 3.7 ⫾ 0.4 ng/ml, which
was suppressed (P ⬍ 0.05) compared with the basal level
observed in the controls. After the LHRH challenge, there
was a 3-fold increase (P ⬍ 0.01) to 11.3 ⫾ 1.9 ng/ml, indicating ALC did not alter pituitary responsiveness.
Representative LH secretory profiles from individual
monkeys that received either sucrose or ALC are presented
in Fig. 3. As shown, LH release was pulsatile, and the frequency of the pulses appears to be equivalent between treatment groups and across the morning and afternoon sampling
periods. However, the overall mean levels of the hormone,
as well as the amplitude of the peaks, were lower in ALCtreated than in control monkeys. In addition, baseline
(trough) LH levels were lower during the afternoon compared with the morning hours in the ALC-treated monkeys.
A detailed evaluation of all animals revealed significant
alterations in the LH secretory profile as a result of chronic
Dissen et al. • ALC Alters LH Release by Hypothalamic Action
Endocrinology, October 2004, 145(10):4558 – 4564 4561
FIG. 3. Representative pulsatile secretory pattern for LH in control and ALC-treated ovariectomized immature monkeys. Note that the frequency of LH pulses were similar between control
(A) and ALC-treated (B) animals; however, the
mean levels of LH, the baseline (trough between
peaks), as well as amplitude of the peaks, were all
lower in the ALC-treated monkey. Additionally,
the baseline LH level in the ALC-treated monkey
was markedly lower in the afternoon than in the
morning. The blood sample points were at 10-min
intervals and represent the pulsatile nature of LH
secretion. Asterisks indicate the pulse peaks as
determined by PULSAR analysis.
ALC exposure. The subclavian catheter of one of the control
monkeys failed the day before the sampling protocol and
thus, this monkey could not be included in the study. Six
weeks after ovariectomy, the control monkeys exhibited
markedly (P ⬍ 0.0001) higher mean (⫾sem) overall daily LH
levels compared with the ALC-treated monkeys which were
45% lower (11.0 ⫾ 0.23 ng/ml vs. 6.1 ⫾ 0.20 ng/ml). Figure
4 depicts LH levels in both groups, but separated into the
morning and afternoon sampling periods. The control monkeys showed a mean (⫾sem) morning LH level of 11.0 ⫾ 0.3
ng/ml, which was markedly (P ⬍ 0.001) higher when compared with the 6.8 ⫾ 0.3 ng/ml observed in the ALC-treated
monkeys. A similar effect was noted in the afternoon, as
controls showed a mean level of 10.9 ⫾ 0.3 ng/ml, which
again was markedly (P ⬍ 0.0001) higher compared with the
5.5 ⫾ 0.2 ng/ml observed in the ALC-treated monkeys. No
differences were observed between the morning and afternoon levels of LH in the control monkeys. Conversely, in the
ALC-treated monkeys, the mean level of LH was suppressed
(P ⬍ 0.05) in the afternoon compared with the morning,
demonstrating an acute effect of the ALC administered just
before the afternoon sampling period. Additionally, by comparing these post-ovariectomy levels of LH (Fig. 4A, morning
values) to the preovariectomy levels (Fig. 2, basal values), we
observed that after the removal of gonadal steroids the expected LH increases occurred in both groups, but that the
mean post-ovariectomy levels of LH in the ALC-treated animals only rose to levels that were similar to the preovariectomy levels observed in the control monkeys.
Figure 4B shows the effect of ALC on baseline LH as
assessed by determining the mean (⫾sem) of the trough
(between peak) levels of the hormone throughout the day.
The control monkeys showed a morning baseline LH level of
7.8 ⫾ 0.7 ng/ml, which was higher (P ⬍ 0.05) compared with
the 5.0 ⫾ 0.3 ng/ml observed in the ALC-treated monkeys.
In the afternoon, the control animals continued to show a
baseline LH level of 7.1 ⫾ 0.6 ng/ml, which was markedly
(P ⬍ 0.01) higher compared with the 3.7 ⫾ 0.3 ng/ml observed in the ALC-treated monkeys. There were no differences observed between the morning and afternoon baseline
levels of LH in the control monkeys. The ALC-treated monkeys, however, showed a suppressed (P ⬍ 0.05) mean base-
4562
Endocrinology, October 2004, 145(10):4558 – 4564
Dissen et al. • ALC Alters LH Release by Hypothalamic Action
FIG. 5. The effect of chronic ALC exposure on mean peak amplitude
of serum LH released in ovariectomized immature monkeys. Blood
samples were collected and data assessed as described in Fig. 4. Data
represent mean (⫾SEM) peak amplitude (trough to peak difference) in
LH levels. Note that compared with controls the mean peak amplitude
was suppressed in the ALC-treated monkeys throughout both the
morning and afternoon periods. No morning and afternoon differences
were noted within treatment groups. *, P ⬍ 0.05 vs. morning and
afternoon control levels.
FIG. 4. The effect of chronic ALC exposure on mean serum LH and
mean baseline LH levels in ovariectomized immature monkeys. Subclavian vein blood samples were collected every 10 min throughout
the daytime hours both in the morning (pre-ALC or saline/sucrose)
and in the afternoon (post ALC or saline/sucrose) as described in
Materials and Methods. Data represent mean (⫾SEM) serum LH levels. To adjust for the dependence of the response data within each
monkey, treatment (ALC or control) and period (morning or afternoon) were assessed as fixed effects in a full factorial design with
individual monkeys as a random effect in a linear mixed model. Note
that, compared with controls, the mean LH (A) and mean baseline LH
(B) levels were suppressed in the ALC-treated monkeys both in the
morning and in the afternoon. Additionally, in both panels only the
ALC-treated monkeys showed mean LH levels that differed between
the morning and afternoon. These results indicate a suppression of
LH as a result of chronic ALC exposure that is sustained throughout
the day and furthermore, depict a short-term or acute suppression of
LH that occurs in the afternoon period after ALC administration. *,
P ⬍ 0.05; ** or ***, P ⬍ 0.001; ****, P ⬍ 0.0001 vs. morning and
afternoon control levels; ⫹, P ⬍ 0.05 vs. own morning level. Control,
n ⫽ 3; ALC-treated, n ⫽ 4.
line level of LH in the afternoon compared with the morning
levels, further demonstrating an acute effect of ALC in the
afternoon.
Detailed analysis of pulse frequency indicated that there
was no difference between groups. The control monkeys
exhibited mean frequencies of 1.1 ⫾ 0.2 LH pulses per hour,
and the ALC-treated monkeys showed 1.2 ⫾ 0.1 LH pulses
per hour. This pulsatile pattern did not vary in either group
as a result of time of day (not shown). Analysis of pulse
amplitude, however, revealed a significant effect of ALC
exposure. Figure 5 shows the effect of ALC on mean (⫾sem)
peak amplitude (trough to peak difference) of LH throughout the day. The control monkeys showed a morning peak
amplitude of 6.3 ⫾ 0.7 ng/ml, which was higher (P ⬍ 0.05)
compared with the 3.4 ⫾ 0.5 ng/ml observed in the ALC-
treated monkeys. Almost identical results were observed in
the afternoon, again demonstrating the suppression (P ⬍
0.05) in peak amplitude in the ALC-treated monkeys. No
differences were detected within each group between the
morning and afternoon hours.
Discussion
We have shown previously that low-dose ALC administration for 1 yr caused suppressed LH and E2 levels, as well
as a delayed the pattern normal of menstrual development
in immature female rhesus monkeys (11). The onset and
progression of primate puberty is characterized by the increased pulsatile secretion of hypothalamic LHRH and subsequent release of pituitary gonadotropins (21, 22). Therefore, in the present study we questioned whether chronic
ALC exposure alters prepubertal LH release in female primates by acting centrally to suppress the hypothalamic
LHRH releasing system, or whether it acts at the pituitary
level directly. Our results are the first to indicate that chronic
ALC exposure suppresses LH release in immature female
monkeys by an action at the hypothalamic level. Several lines
of evidence as discussed below support this conclusion.
The suppressed levels of LH observed in the present study
are consistent with our earlier findings in immature female
rhesus monkeys (11). Chronic ALC exposure also caused
suppressed levels of LH in mid-pubertal boys (7), adult female rhesus monkeys (23), alcoholic women (24, 25), and in
immature and mature rats of both sexes (26, 27). This study
is the first to assess hypothalamic and pituitary function in
immature female monkeys that were exposed chronically to
ALC. The BACs attained are associated with a level of mild
intoxication in humans. Excitatory amino acid analogs of
aspartate, such as NMA and N-methyl-d-aspartate (NMDA),
are known to stimulate the release of LH in both primates (17,
28) and rats (29 –31) via a direct action at the hypothalamic
Dissen et al. • ALC Alters LH Release by Hypothalamic Action
level to induce LHRH release (17, 28 –31). Thus, we used
NMA to assess hypothalamic function and observed that
ALC exposure blocked the NMA-induced release of LH;
hence, providing clear evidence for a hypothalamic effect of
the drug in young female primates. This is supported by our
previous studies using immature female rats, which indicate
that ALC blocks NMA-induced LHRH release in vitro (13), as
well as blocks NMA-induced LH release and attenuates the
ability of NMA to advance female puberty (13, 32). Thus, it
is likely that ALC alters NMDA receptors in the reproductive
hypothalamus, as shown previously in other brain regions
such as the hippocampus (33), cerebral cortex (34), and medial septum (35). It is also possible that ALC may alter the
synthesis of new receptors. These possibilities are important
in view of the fact that NMDA receptors are markedly activated during puberty (36). We suggest, therefore, that the
detrimental effects of ALC on puberty are due, at least in
part, to altered LHRH neuronal responsiveness to excitatory
amino acids. Results of the pituitary function test conducted
in the present study revealed that ALC exposure did not
diminish the ability of the pituitary to release LH in response
to LHRH, further demonstrating that the principal effect of
the drug on LH secretion in immature female monkeys is at
the hypothalamic and not the pituitary level. We are unaware
of any previous studies involving LHRH stimulation of LH
after chronic ALC exposure in immature or mature monkeys
of both sexes, or in ALC-dependent women. However,
LHRH-induced LH release was shown to be unaffected in
alcoholic men (37), and in alcoholic women during abstinence when compared with nonalcoholic controls (24, 38).
Further evidence for an unimpaired LH response to LHRH
comes from studies using adult female rats (39, 40), adult
female rhesus monkeys (41), and men (42) after acute ALC
exposure.
In addition to data gained from the hypothalamic and
pituitary response tests, we also noted other alterations that
further demonstrate the detrimental effect of ALC on prepubertal LH secretion. Six weeks after ovariectomy, the LH
levels were 45% lower in the monkeys that received the ALC.
It has been shown previously that ovariectomy of immature
monkeys causes a gradual but not an immediate, robust
increase in LH (43– 45). Our results are in agreement with this
observation because relatively modest increases in LH were
observed in both groups at 6 wk post ovariectomy; however,
the fact that LH levels in the ALC-treated monkeys at that
time were similar to levels observed 6 wk earlier in the intact
control monkeys further demonstrates an ALC effect. Detailed analysis of the pulsatile release pattern of LH in these
ovariectomized monkeys revealed differences between the
control animals and those that were treated chronically with
ALC. Both in the morning and in the afternoon, the ALCtreated monkeys consistently showed suppressed overall
mean LH levels. Our results indicate that this is not a result
of any ALC action to alter pulse frequency because they were
the same in both groups, but was due to the action of ALC
throughout the day to drive the mean baseline or trough
levels of LH downward, as well as to lower the amplitude of
the pulses. In addition, there appears to be an acute effect of
ALC superimposed upon the overall chronic effect of the
drug. Mean serum LH levels were suppressed in the after-
Endocrinology, October 2004, 145(10):4558 – 4564 4563
noon after the administration of the daily dose of ALC during
the noon hour. The most likely cause of this effect is the
ability of ALC to suppress the trough or baseline levels of LH
because pulse amplitude was not further suppressed in the
afternoon over the morning hours.
An increase in pulsatile LH secretion has been shown to be
a hallmark of puberty (46 – 48). In female rhesus monkeys, the
amplitude and frequency of LHRH and LH release increase
around 24 months of age. Although the early increases in
frequency are maintained throughout puberty, the amplitude of LH pulses continues to increase as puberty
progresses (46). Evidence that LHRH drives the amplitude of
LH pulses has been shown in LHRH-deficient men, and by
the dose-response profile of LH released from dispersed rat
pituitary cells in vitro in response to LHRH (49). The importance of LHRH in driving LH pulse amplitude is emphasized
by the results of treating women suffering from hypothalamic amenorrhea with low-dose LHRH pulsatile administration. These studies demonstrated a threshold pulsatile
dose of LHRH, above which, these women will exhibit normal cyclicity and will achieve pregnancy. Below this threshold dose, there is increased incidence of anovulation and
reduced conception rate (50, 51). Thus, it would appear that
the continued enhancement of pulse amplitude during puberty is critical for the progression of pubertal development,
and our results indicate that ALC suppresses this increase.
In conclusion, our results are the first to demonstrate that
chronic ALC exposure suppresses LH release in immature
female rhesus monkeys by an action at the hypothalamic
level. We showed that ALC blocked the LH response to
hypothalamic NMA stimulation but did not alter the responsiveness of the pituitary to stimulation with LHRH. Furthermore, the ALC-treated monkeys showed suppressed LH levels after ovariectomy compared with controls, and altered
specific parameters associated with the pulsatile release pattern of LH, the most important of which was altered pulse
amplitude. We suggest that these ALC-induced deficits contribute to the depressed levels of LH and the delay in the
development of a normal pattern of menstruation in rhesus
monkeys (11).
Acknowledgments
Received April 22, 2004. Accepted June 15, 2004.
Address all correspondence and requests for reprints to: W. Les Dees,
Ph.D., Department of Integrative Biosciences, College of Veterinary
Medicine, Texas A&M University, College Station, Texas 77843-4458.
E-mail: [email protected].
This work was supported by National Institutes of Health (NIH)
Grant AA07216 (to W.L.D.) and included a subcontract (to G.A.D.), and
by NIH Grant ES0-1906 to the TAMU Center for Environmental and
Rural Health, by NIH Grant HD25123 (to S.R.O.), and by NIH Grant
RR00163 for operation of the Oregon National Primate Research Center.
References
1. Johnson LD, O’Malley PM, Bachman JG 1996 National survey results on drug
abuse from the monitoring the future study. Vol 1. NIH publication 96-4139.
Rockville, MD: NIDA
2. White AM, Ghia A J, Levin, ED, Swartzwelder HS 2000 Binge pattern ethanol
exposure in adolescent and adult rats: differential impact on subsequent responsiveness to ethanol. Alcohol Clin Exp Res 24:1251–1256
3. Crews FT, Braun CJ, Hoplight B, Switzer RC, Knapp DJ 2000 Binge ethanol
4564
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Endocrinology, October 2004, 145(10):4558 – 4564
consumption causes differential brain damage in young adolescent rats compared with adults. Alcohol Clin Exp Res 24:1712–1723
Bo WJ, Krueger WA, Rudeen PK, Symmes SK 1982 Ethanol-induced alterations in the morphology and function of the rat ovary. Anat Rec 202:255–260
Dees WL, Skelley CW 1990 Effects of ethanol during the onset of female
puberty. Neuroendocrinology 51:64 – 69
Dees WL, Nyberg CL, Hiney JK 1995 Neuroendocrine responses of alcohol in
the prepubertal female rat. In: Watson RR, ed. Alcohol and hormones. Totowa,
NJ: Humana Press; 49 –74
Diamond F, Ringenberg L, MacDonald D, Barnes J, Shi HC, Duckett G
Sweetland M, Root A 1986 Effects of drug and alcohol abuse upon pituitarytesticular function in adolescent males. J Adolesc Health Care 7:28 –33
Block GD, Yamamoto ME, Ishii E 1991 Association of adolescent alcohol
consumption of growth and body composition. Alcohol Clin Exp Res 15:361
Block GD, Yamamoto ME, Mallick A, Styche AJ 1993 Effects of pubertal
hormones by ethanol in adolescents. Alcohol Clin Exp Res 17:505
Block GD, Ishii E 1991 Food patterns among adolescents: influence of alcohol
consumption. Alcohol Clin Exp Res 15:359
Dees WL, Dissen GA, Hiney JK, Lara F, Ojeda SR 2000 Alcohol ingestion
inhibits the increased secretion of puberty-related hormones in the developing
female rhesus monkey. Endocrinology 141:1325–1331
Dees WL, Skelley CW, Hiney JK, Johnston CA 1990 Actions of ethanol on
hypothalamic and pituitary hormones in prepubertal female rats. Alcohol
7:21–25
Nyberg CL, Hiney JK, Minks JB, Dees WL 1993 Ethanol alters NMDAinduced LHRH release and the onset of puberty in the female rat. Neuroendocrinology 57:863– 868
Hiney JK, Srivastava VK, Lara F, Dees WL 1998 Ethanol blocks the central
action of IGF-1 to induce luteinizing hormone secretion in the prepubertal
female rat. Life Sci 62:301–308
Hiney JK, Dearth RK, Lara F, Wood S, Srivastava VK, Dees WL 1999 Effects
of ethanol on leptin secretion and the leptin induced LH release from juvenile
female rats. Alcohol Clin Exp Res 23:1785–1792
Chappel SC, Resko JA, Norman RL, Spies HG 1981 Studies in rhesus monkeys on the site where estrogen inhibits gonadotropins: delivery of 17␤ estradiol to the hypothalamus and pituitary gland. J Clin Endocrinol Metab
52:1– 8
Gay V, Plant TM 1987 N-methyl-dl-aspartate elicits hypothalamic gonadotropin-releasing hormone release in prepubertal male rhesus monkeys. Endocrinology 120:2289 –2296
Urbanski HF, Ojeda SR 1990 A role for N-methyl-d-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty. Endocrinology 126:1774 –1776
Redetzki HM, Dees WL 1976 Comparison of four kits for enzymatic determination of ethanol in blood. Clin Chem 22:83– 86
Merriam GR, Wachter KW 1982 Algorithms for the study of episodic hormone
secretion. Am J Physiol 243:E310 –E318
Wildt L, Marshall G, Knobil E 1980 Experimental induction of puberty in the
infantile female rhesus monkey. Science 207:1373–1375
Watanabe G, Terasawa E 1989 In vivo release of luteinizing hormone releasing
hormone increases with puberty in the female rhesus monkey. Endocrinology
125:92–99
Mello NK, Bree MP, Mendelson JH, Elingboe J, King NW, Sehgal PK 1983
Alcohol-self administration disrupts reproductive function in female macaque
monkeys. Science 221:667– 679
Hugues JN, Coste T, Perret G, Jayle MS, Sebaoun J, Modigliani E 1980
Hypothalamo-pituitary ovarian function in thirty-one women with chronic
alcoholism. Clin Endocrinol (Oxf) 12:543–551
Valimaki M, Ylikahri R 1981 Alcohol and sex hormones. Scand J Clin Lab
Invest 41:99 –105
Dees WL, Hiney JK, Nyberg CL 1995 Effects of ethanol on the reproductive
neuroendocrine axis of prepubertal and adult female rats. In: Barnes CD,
Sarkar DK, eds. Reproductive neuroendocrinology of aging and drug abuse.
Boca Raton, FL: CRC Press; 301–334
Emanuele MA, Halloran MM, Uddin S, Tentler TT, Lawrence AM, Emanuele
NV, Kelley MR 1995 Alcohol and cocaine abuse effects on the male reproductive neuroendocrine axis. In: Barnes CD, Sarkar DK, eds. Reproductive
neuroendocrinology of aging and drug abuse. Boca Raton, FL: CRC Press;
271–300
Wilson RC, Knobil E 1982 Acute effects of N-methyl-dl-aspartate on the
release of pituitary gonadotropins and prolactin in the adult female rhesus
monkey. Brain Res 248:177–179
Dissen et al. • ALC Alters LH Release by Hypothalamic Action
29. Ondo JG, Wheeler DD, Dom RM 1988 Hypothalamic site of action for Nmethyl-d-aspartate (NMDA) on LH secretion. Life Sci 43:2283–2286
30. Price MT, Olney JW, Cicero TJ 1978 Acute elevations on serum luteinizing
hormone induced by kainic acid, N-methyl aspartic acid or homocysteic acid.
Neuroendocrinology 26:352–358
31. Urbanski HF, Ojeda SR 1987 Activation of luteinizing hormone-releasing
hormone release advances the onset of female puberty. Neuroendocrinology
46:273–276
32. Nyberg CL, Srivastava VK, Hiney JK, Lara F, Dees WL 1995 N-methyl-daspartic acid receptor (NMDA-R) synthesis and luteinizing hormone release
in immature female rats: effects of stage of pubertal development and exposure
to ethanol. Endocrinology 136:2874 –2880
33. Lovinger DM, White G, Weight FF 1989 Ethanol inhibits NMDA-activated ion
current in hippocampal neurons. Science 243:1721–1724
34. Leslie W, Brown LM, Dildy JE, Sims JS 1990 Ethanol and neuronal calcium
channels. Alcohol 7:233–236
35. Simson PE, Criswell HE, Johnson JB, Hicks RE, Breese GR 1991 Ethanol
inhibits NMDA-evoked electrophysiological activity in vivo. J Pharmacol Exp
Ther 257:225–231
36. Bourguignon JP, Gerard A, Mathieu J, Mathieu A, Franchimount P 1990
Maturation of the hypothalamic control of pulsatile gonadotropin-releasing
hormone secretion at the onset of puberty: increased activation of N-methyld-aspartate receptors. Endocrinology 127:873– 881
37. Bertello P, Gurioli L, Faggiuolo R, Vegilo F, Tamagnone C, Angeli A 1983
Effect of ethanol infusion on the pituitary-testicular responsiveness to gonadotropin releasing hormone and thyrotropin releasing hormone in normal
males and in chronic alcoholics presenting with hypogonadism. J Endocrinol
Invest 6:413– 420
38. Vilimaki M, Pelkonen R, Salasporo M, Harkonen M, Hirvonen E, Ylikahri
R 1984 Sex hormones in amenorrheic women with alcoholic liver disease. J Clin
Endocrinol Metab 59:133–138
39. Dees WL, Rettori V, Kozlowski GP, McCann SM 1985 Ethanol and the
pulsatile release of luteinizing hormone, follicle stimulating hormone and
prolactin in ovariectomized rats. Alcohol 2:641– 646
40. Ogilvie KM, Rivier C 1997 Effect of alcohol on proestrous surge of luteinizing
hormone and the activation of LH-releasing hormone (LHRH) neurons in the
female rat. J Neurosci 17:2595–2604
41. Mello NK, Mendelson JH, Bree MP, Skupny A 1986 Alcohol effects on
luteinizing hormone-releasing hormone-stimulated luteinizing hormone and
follicle stimulating hormone in female rhesus monkeys. J Pharmacol Exp Ther
236:590 –595
42. Ylikahri RH, Huttunen MO, Harkonen M, Leino T, Helenius T, Liewendahl
K, Karonen SL 1978 Acute effects of alcohol on anterior pituitary secretion of
the tropic hormones. J Clin Endocrinol Metab 46:715–720
43. Terasawa E, Bridson WE, Nass TE, Noonan JJ, Dierschke DJ 1984 Developmental changes in luteinizing hormone secretory pattern in peripubertal female rhesus monkeys: relation to menarche and skeletal maturation. Endocrinology 115:2233–2240
44. Wilson ME, Gordon TP, Collins DC 1986 Ontogeny of luteinizing hormone
secretion and first ovulation in seasonal breeding rhesus monkeys. Endocrinology 118:293–301
45. Wilson ME 1989 Relationship between growth and puberty in the rhesus
monkey. In: Delemarre-van de Waal HA, Plant TM, van Rees GP, Shoemaker
J, eds. Control of onset of puberty 111. Amsterdam: Excerpta Medica; 137–149
46. Terasawa E, Fernandez DL 2001 Neurobiological mechanisms of the onset of
puberty in primates. Endocr Rev 22:111–151
47. Richter TA, Terasawa E 2001 Neural mechanisms underlying the pubertal
increase in LHRH release in the rhesus monkey. Trends Endocrinol Metab
12:353–359
48. Ojeda SR, Terasawa E 2002 Neuroendocrine regulation of puberty. In: Pfaff
D, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, eds. Hormones and behavior. Vol. 4. New York: Academic Press, Inc.; 589 – 659
49. Crowley WF, Whitcomb RW, Jameson JL, Weiss J, Finklstein JS, O’Dea LS
1991 Neuroendocrine control of human reproduction in the male. Rec Prog
Hormone Res 47:27– 67
50. Santoro N, Wierman ME, Filicori M, Waldstreicher J, Crowley WF 1986
Intravenous administration of pulsatile gonadotropin-releasing hormone in
hypothalamic amenorrhea: effects of dosage. J Clin Endocrinol Metab
62:109 –116
51. Braat DD, Schoemaker J 1991 Endocrinology of gonadotropin-releasing hormone induced cycles in hypothalamic amenorrhea: the role of the pulse dose.
Fertil Steril 56:1054 –1059
Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.