Tonic Support of Luteinizing Hormone Secretion by Adrenal Progesterone in the Ovariectomized Monkey Replaced with Midfollicular Phase Levels of Estradiol
Journal of Clinical Endocrinology and Metabolism
Copyright © 1997 by The Endocrine Society
Tonic Support of Luteinizing Hormone Secretion by Adrenal Progesterone in the Ovariectomized Monkey Replaced with Midfollicular Phase Levels of Estradiol*
ENNIAN XIAO 0
LINNA XIA-ZHANG 0
DAVID SHANEN 0
MICHEL FERIN 0
0 Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons , New York, New York 10032 , USA
Although it is known that progesterone facilitates the estradiolinduced gonadotropin surge at midcycle, its effect on LH secretion at other times of the follicular phase remains to be investigated. In this study, we investigate the role of progesterone on tonic LH secretion in the ovariectomized primate replaced with estradiol at levels representative of the follicular phase. The experiments were performed in nine ovariectomized rhesus monkeys, either unreplaced with estradiol or after a 5-day estradiol therapy to mimic early follicular (10 -36 pg/mL; low dose) and midfollicular (medium dose; 40 -75 pg/mL) concentrations. We used two antiprogesterone compounds, RU-486 (5 mg) and ORG-31806 (1 mg), to antagonize endogenous progesterone activity and studied their acute effects on LH secretion in each group. LH concentrations were measured at 15-min intervals for a 3-h baseline period and during a 5-h period after antagonist administration. LH concentrations remained unchanged after either antiprogesterone compound or diluent (ethanol) administration in the estrogen-unreplaced monkeys or after low dose estradiol replacement. However, both antiprogesterone compounds significantly decreased LH secretion in monkeys pretreated with the medium dose of estradiol; by 5 h, the mean (6SE) areas under the LH curve were 54.8 6 4.1% and 64.0 6 4.2% after RU-486 and ORG-31806, respectively (P , 0.05 vs. unreplaced and low dose estrogen-replaced groups). To exclude the possibility that
TUDIES IN various species have demonstrated a role for
progesterone in the control of LH secretion. Several
reports, for instance, support a facilitative effect of
progesterone on the spontaneous or estradiol-induced LH
). RU-486, a progesterone antagonist (
indeed been shown to delay or block the LH surge when
administered at the end of the follicular phase in humans
) and monkeys (
). Other studies in which RU-486 was
administered earlier in the follicular phase suggest that in
addition to influencing the estradiol- related LH surge,
progesterone may modulate tonic LH secretion during the
follicular phase. Indeed, when administered during the
midfollicular phase, RU-486 produces a delay in
folliculogenesis and a decrease in estradiol secretion (
), both of
which are presumably related to a decline in tonic
gonadotropin secretion (
the LH response reflects an agonist action of the progesterone
antagonist, LH responses to progesterone infusions (at three doses
to reproduce preovulatory, luteal, and pharmacological levels)
were also examined in monkeys pretreated with midfollicular
levels of estradiol. In none of these was there a decrease in LH; rather,
progesterone infusions resulted in an increase in LH secretion in
all three groups (to 115–194% of baseline in seven of eight
monkeys). Finally, we determined that at the dose used in our protocol,
neither of the two progesterone antagonists was able to prevent
dexamethasone-induced cortisol suppression, thus excluding the
possibility that results after progesterone antagonist
administration may reflect a putative antiglucocorticoid activity of these
compounds. When the doses of the antiprogesterone compounds were
increased 6 times, only RU-486 counteracted the effect of
dexamethasone on cortisol.
In summary, our data indicate support by progesterone of tonic LH
secretion in the nonhuman primate under estrogenic conditions
similar to the midfollicular phase of the menstrual cycle. Significantly,
because the experiments were performed in ovariectomized monkeys,
and endogenous progesterone was most probably of adrenal origin,
the data also demonstrate a role of the
hypothalamo-pituitary-adrenal axis in support of gonadotropin secretion. (J Clin Endocrinol
Metab 82: 2233–2238, 1997)
Although the ovary is not thought to secrete substantial
amounts of progesterone during the follicular phase,
recent data using sensitive assay methods have clearly
demonstrated small, but detectable, amounts of circulating
progesterone in women during that period of the cycle (
The source of the steroid at that time was presumed to be
the adrenal gland (
). Thus, the possibility exists that
progesterone of adrenal origin may play a role in support of
the tonic secretion of gonadotropin. To study this
phenomenon in the absence of ovarian input, we used the
ovariectomized (OVX) monkey as a model and studied the
effects of two different progesterone antagonists, RU486
) and ORG-31806 (
), on pulsatile LH secretion in the
presence of estradiol replacement that reproduces
estrogen amounts observed during the early or midfollicular
phase. To exclude the possibility that the results could be
due to a putative progesterone agonist activity of the
progesterone antagonist in the presence of estradiol, as has
been speculated previously (
), we also report on the
effects of progesterone infusions on LH secretion in the same
animals. An additional experiment was performed to
demonstrate that at the dose used in our protocols, the two
progesterone antagonists do not exert any overt
Materials and Methods
Nine adult female rhesus monkeys (Macaca mulatta), weighing 5–7 kg,
were used in these experiments. The animals had been OVX at least 12
months before these studies. They were kept in individual cages in light
(lights on, 0730 –1930 h)- and temperature-controlled rooms and were
fed once a day with Purina monkey chow (Ralston Purina, St. Louis, MO)
and fresh fruit or vegetables. They had free access to water at all times.
The experimental protocols were approved by the institutional animal
care and use committee of Columbia University.
Exp 1. The effects of two antiprogesterone compounds, RU-486
(mifepristone, provided by Roussel-UCLAF, Romainville, France) and
ORG31806 (provided by NV Organon, Oss, The Netherlands), on LH
secretion were investigated. Each compound was tested in OVX monkeys in
the absence of estradiol replacement and after a 5-day estradiol therapy
at two concentrations. For estradiol replacement, one SILASTIC capsule
(SILASTIC brand tubing, Dow Corning, Midland, MI; id, 3.3 mm; od, 4.6
mm; length, 1 or 3 cm) was inserted sc under ketamine tranquilization.
Each capsule had been filled with 17b-estradiol (Steraloids, Wilton, NH)
and preincubated for at least 24 h before implantation. RU-486 (5 mg)
and ORG-31806 (1 mg) were dissolved in 0.5 mL ethanol and
administered once im after the 3-h baseline control period. Control monkeys
(nonreplaced and medium estrogen replacement) received an ethanol
injection only. On the evening before the experiment, the monkeys were
briefly tranquilized (15–30 min) with ketamine hydrochloride (5–7 mg/
kg; Ketaset, Aveco Co., Fort Dodge, IA), and a catheter was inserted into
the femoral vein for blood collection. They were then seated in a primate
chair, to which they had previously been habituated. The experiment
was initiated the next morning (0730 – 0800 h) and lasted 8 h. Blood
samples (1.2 mL) were obtained at 15-min intervals for a 3-h baseline
control period and a 5-h experimental period, and an equivalent amount
of physiological saline was infused after each sample. The SILASTIC
capsules were removed at the end of the experiment, and animals were
returned to their home cage.
Exp 2. To exclude the possibility that the results of Exp 1 could be related
to a putative progesterone agonist effect of the two antiprogesterone
compounds, we also studied the effects of progesterone infusions on LH
secretion in OVX monkeys pretreated with the higher dose of estradiol.
A protocol similar to that employed in Exp 1 was used, except that a
progesterone infusion replaced that of the progesterone antagonist.
Three doses of progesterone were infused iv to produce serum levels of
0.85 6 0.05 ng/mL (group 1; n 5 3), 4.1 6 0.29 (group 2; n 5 3), and 18.3 6
0.23 (group 3; n 5 2). The progesterone (ICN Biochemical, Cleveland,
OH) stock solution was prepared, as described previously (
diluted with normal saline containing 10% monkey plasma. Infusion of
progesterone was initiated after a 3-h baseline control period and
continued for the 5-h experimental period.
Exp 3. To evaluate whether the results of Exp 1 reflect a putative
antiglucocorticoid activity of either of the two antiprogesterone compounds,
an additional experiment was performed. Dexamethasone (0.4 mg, im;
Lyphomed, Deerfield, IL) alone or together with two doses (equal to that
in Exp 1 and 6 times that dose) of RU-486 or ORG-31806 was
administered at 1900 h to free moving caged monkeys. Blood samples were
obtained by venous puncture at 0800, 0900, and 1000 h on the next day,
and the cortisol response to dexamethasone was measured.
Assays and statistical analysis
After centrifugation, sera were stored at 220 C until assay. LH was
measured using a homologous RIA method (
progesterone, and cortisol were also measured by RIA (Coat-A-Count, Diagnostic
Products, Los Angeles, CA). Before the progesterone RIA, samples were
extracted with petroleum ether (Aldrich Chemical Co., Milwaukee, WI);
the extraction recovery rate was 94.2 6 2.1%. Intraassay coefficients of
variation were 6.6% (LH), 2.9% (cortisol), 3.0% (estradiol), and 4.8%
(progesterone); interassay coefficients of variation were 13.3% (LH),
6.1% (cortisol), 11.0% (estradiol), and 9.1% (progesterone).
Hourly areas under the LH curve during the 5-h experimental period
were calculated and expressed as a percentage of the 3-h baseline control
period. The posttreatment percent change in different treatment groups
was then analyzed by the Kruskal-Wallis ranking test, followed by
Tukey’s test. Student’s t test was used to compare control morning
baseline cortisol concentrations with those obtained after
dexamethasone alone or in combination with RU-486 or ORG-31806.
LH remained unchanged after the administration of either
progesterone antagonist (RU-486 or ORG-31806) to OVX
monkeys in the absence of estrogen replacement or after the
lower 5-day estradiol replacement therapy (1-cm SILASTIC
capsule; early follicular phase levels; Fig. 1, left and center
panels). In contrast, both antagonists acutely decreased LH
secretion after the medium estradiol replacement therapy
(3-cm SILASTIC capsule; midfollicular phase levels; Fig. 1,
right panels). By 5 h after administration of the progesterone
antagonist, the mean area under the LH curve was 54.8 6
4.1% (6se) of the 3-h baseline period and 64.0 6 4.2% after
RU-486 and ORG-31806, respectively (P , 0.05 vs. ethanol,
unreplaced or low estrogen groups). Table 1 summarizes
mean estradiol concentrations in all groups and overall LH
concentrations before and after progesterone antagonist
administration. Figure 2 illustrates the effects of the
progesterone antagonists in two monkeys. Progesterone
concentrations did not significantly differ between groups. Overall, the
mean morning baseline concentration was 88 6 9.7 (6se)
pg/mL, whereas the posttreatment afternoon concentration
was 67 6 7.3 pg/mL.
In none of the three groups receiving different amounts of
progesterone did we observe a decrease in LH secretion
within the 5-h experimental period (mean estradiol
concentration in all three groups, 58.7 6 3.6 pg/mL). Instead of a LH
decrease, we observed an increase in LH in all eight
monkeys, independent of the progesterone dose. This LH
increase ranged from 115–194% of baseline by 5 h in seven
animals. One monkey showed a larger increase of 430%.
Figure 3 illustrates the mean hourly LH changes over
baseline during the 5-h progesterone infusion in seven of eight
Administration of dexamethasone at 1900 h inhibited
cortisol secretion. At 1000 h the next morning, for instance, the
mean (6 se) cortisol concentration was 4.3 6 0.6 mg/dL after
dexamethasone treatment vs. 21.3 6 2.4 in controls (P , 0.05;
Fig. 4). This inhibitory effect of dexamethasone was still
present when it was coadministered with RU-486 at the 5-mg
dose (4.6 6 1.1 mg/dL) or with ORG-31806 at the 1-mg (5.9 6
1.2) or 6-mg (5.3 6 1.5) dose. At the dose of 30 mg, RU-486
counteracted this inhibitory effect; at 1000 h, the cortisol
concentration was 22.7 6 7.3 (P 5 NS vs. control).
Our data show a significant decrease in tonic pulsatile LH
secretion after the administration of either of two
progesterone antagonists to OVX monkeys pretreated with
estradiol for 5 days to mimic estrogen concentrations typical of the
midfollicular phase of the cycle. The observed decrease in LH
concentrations after RU-486 or ORG-31806 treatment is rapid
and observed by 3 h after injection of the progesterone
antagonist. This inhibitory effect is not present when either
antagonist is given to steroid-unreplaced animals or with
lower estradiol concentrations representative of the early
Several studies in the human and the nonhuman primate
have investigated the effects of RU-486 on the menstrual
cycle, although most of these have focused on the time of the
cycle when progesterone production is maximal, i.e. the
luteal phase (
) or the late follicular phase (
5, 6, 17
). In the
first of two studies in which RU-486 was administered
during the midfollicular phase after the emergence of the
dominant follicle, investigators reported a decline in
serum estradiol concentrations accompanied by a decrease
in the size of the dominant follicle and a delay in the onset
of the gonadotropin surge (7). Although no significant
effects of RU-486 on LH or FSH were observed, this
probably reflected the paucity of blood sampling early in the
experiment. Indeed, a second study in the human clearly
demonstrated that, as in our estradiol-pretreated OVX
monkeys, administration of RU-486 in the midlate
follicular phase produces a substantial reduction in plasma LH
). Significantly, as in our experiment, the reduction was
dependent upon the initial plasma estradiol concentration.
The arrest in the growth of the dominant follicle and the
decreased estradiol secretion observed in the first study (
may then have reflected the dependence of the follicle on
gonadotropin support at this stage of development. This
conclusion is supported by the demonstration that a short
term blockade of pulsatile LH secretion by a GnRH
antagonist during the follicular phase results in a transient
arrest in the ongoing process of follicular maturation and,
hence, a decrease in estradiol secretion (
RU486 administration during the follicular phase also
effectively suppresses folliculogenesis (
). In accord with the
above data, our study in the nonhuman primate indicates
a role for progesterone in the maintenance of tonic
pulsatile LH secretion during the normal follicular phase.
FIG. 3. Mean (6SE) LH response (illustrated as the hourly area under
the LH curve, expressed as a percentage of the 3-h baseline control
value) to 5-h iv progesterone infusions in seven estrogen-replaced (E;
58.7 6 3.6 pg/mL) monkeys. As an increase in LH occurred in all
animals independent of the progesterone dose, results were pooled.
The eighth animal was excluded because, although it also showed a
LH increase, this increase (430%) was well outside the range of
response in the other seven animals. In none of the animals was there
a LH decrease such as that observed with the progesterone
antagonist, thus excluding the possibility that the inhibition of LH observed
in Figs. 1 and 2 is related to an agonist effect of the progesterone
The mechanism by which progesterone may act to modify
tonic LH concentrations remains to be investigated. Because
of the short term observation period, we could not ascertain
whether the decrease in LH concentrations relates to a
FIG. 4. Mean (6SE) cortisol concentrations after dexamethasone
(DEX; 0.4 mg administered at 1900 h on the evening of the previous
day) alone or in combination with the progesterone antagonist
RU486 (RU; 5 or 30 mg/animal administered at the same time as DEX;
left panel) or ORG-31806 (ORG; 1 or 6 mg administered at the same
time as DEX; right panel). Closed circles represent control morning
cortisol concentrations in untreated OVX monkeys. As expected, DEX
inhibits cortisol secretion; this inhibition is unaffected by RU (5 mg)
or ORG (1 or 6 mg). However, RU at the dose of 30 mg significantly
prevents this effect. *, P , 0.05 vs. controls.
change in LH pulse frequency or pulse amplitude. Animal
studies have demonstrated a stimulatory effect of
progesterone on hypothalamic GnRH release (
) and GnRH
messenger ribonucleic acid levels (22), suggesting a possible
hypothalamic site of action. However, a pituitary site
cannot be discounted. What most studies have clearly
indicated is that estrogen is an obligatory requirement for
progesterone-stimulated GnRH release (
). This is
also clear from our data and from reports in the human (8),
in that the effectiveness of RU-486 to decrease tonic LH
release depends upon the presence of estradiol
concentrations above a critical level. Indeed, there was no change
in LH after RU-486 treatment in the steroid-unreplaced
OVX animal or in the presence of early follicular phase-like
concentrations. Although there are some reports that
hypothalamic areas of spayed monkeys or hamsters still
contain some progesterone receptor-positive cells (
most species hypothalamic progesterone receptor activity
is lacking in OVX animals, but increases dramatically after
estradiol treatment (
24, 26 –28
). Thus, it may be speculated
that the absence of an effect by the antiprogesterone in
individuals lacking the critical estradiol concentrations
may reflect suboptimal activity of the progesterone
receptor. Unfortunately, there is no report in the literature
quantifying hypothalamic or pituitary progesterone receptor
activity throughout the menstrual cycle. Alternatively, it
is also possible that critical levels of estradiol are required
to induce the formation of intermediary substances
necessary for progesterone’s action on GnRH and/or LH
Of specific relevance is the fact that our experiments were
performed in OVX estrogen-replaced animals, suggesting
that the progesterone antagonist must block the effect of
progesterone from a source other than the ovary, probably
progesterone of adrenal origin. Thus, in this particular
nonhuman primate model, the data may well suggest a role for
adrenal progesterone in support of tonic LH secretion. What
relevance this conclusion may have to events of the follicular
phase is highlighted by observations in women that the
source of small, but significant, amounts of progesterone
during the follicular phase is likely to be the adrenal rather
than the ovary (
). Indeed, in contrast to the luteal phase (
during the follicular phase there is no relationship between
LH and progesterone pulses, and dexamethasone totally
suppresses serum progesterone as well as cortisol,
suggesting that ACTH is the stimulus for pulsatile
progesterone release. The data for progesterone antagonists in
intact women together with our results in the OVX
monkey may then indicate a putative role of the
hypothalamicpituitary-adrenal axis in support of the
hypothalamic-pituitary-gonadal axis and of tonic gonadotropin secretion
during a critical period of folliculogenesis. One important
caveat remains, however, in that it cannot a priori be
concluded that a 5-day estrogen replacement therapy in a long
term OVX monkey entirely mimics the follicular phase.
Although both RU-486 and ORG-31806 have been used
successfully in a variety of experiments to antagonize
progesterone action (
), there are some reports that, for
example, RU-486 may under specific estrogen conditions
exert weak progestational effects on proliferative
6, 30, 31
). Our data showing a lack of inhibitory
effect on LH of progesterone infusions (at levels covering
concentrations representative of the preovulatory period,
the luteal phase, and the pharmacological range) exclude
the possibility that the decrease in LH after the
administration of either progesterone antagonist is the result of a
putative agonist effect, at least within the 5-h experimental
Besides its antiprogesterone properties, RU-486 is
known for its antiglucocorticoid action (
). For instance,
it has been found to inhibit the negative feedback effect of
) and at a dose of 3 mg/kg has been
observed to increase cortisol secretion (
). However, we
do not believe that the inhibitory effects of the
antiprogesterone compounds on tonic LH secretion observed in
this report are due to their antiglucocorticoid action.
Indeed, at the dose used in our experiment (5 mg/monkey),
RU-486 is unable to prevent dexamethasone-induced
cortisol suppression. Further, a similar inhibition of tonic LH
is obtained with ORG-31806, another progesterone
antagonist, which at the dose used (about one fifth the dose of
RU-486) is equally unable to prevent the inhibitory action
of dexamethasone on the hypothalamic-pituitary-adrenal
In conclusion, our data suggest a putative role of the
hypothalamic-pituitary-adrenal axis, through its secretion of
progesterone, in maintaining tonic LH secretion in the OVX
monkey replaced with mid- to late follicular phase levels of
estradiol. Whether a similar effect of the
hypothalamic-pituitary-adrenal axis occurs during the follicular phase
remains to be investigated.
The authors thank Roussel-UCLAF (Romainville, France) for their
generous gift of RU-486, and NV Organon (Oss, The Netherlands) for
their generous gift of ORG-31806. They also thank Dr. A. F. Parlow
(Harbor University-University of California-Los Angeles Medical
Center) and the National Hormone and Pituitary Program, the NIDDK, the
NICHHD, and the USDA for providing the reagents for the homologous
monkey LH RIA.
In the article “The Effect of Growth Hormone (GH) on Histomorphometric Indices of Bone Structure and
Bone Turnover in GH-Deficient Men” by Nathalie Bravenboer, Pauylien Holzmann, Hans de Boer, Jan C.
Roos, Eduard A. Van der Veen and Paul Lips (Journal of Clinical Endocrinology and Metabolism 82:1818 –1822,
1997), an important error has been discovered.
The conversion from 1, 2, 3 IU/m2/day is not 2.9, 5.8, and 8.7 mg/m2/day, as shown in the Abstract (p. 1818),
the “Patients” section of Materials and Methods (p. 1819, column A), and the “Statistical evaluations” section
of Materials and Methods (p. 1819, column B).
The correct conversion values should read: 0.35, 0.69, and 1.3 mg/m2/day.
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In the Discussion section, the conversion from IU GH to mg GH should also be corrected to: 0 . 34 , 0 .68, and 1.2 mg/m2/day.