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Endocrinology Vol. 138, No. 6 2249-2258
Copyright © 1997 by The Endocrine Society

ARTICLES

Luteinizing Hormone Secretion and Corticotropin-Releasing Factor Gene Expression in the Paraventricular Nucleus of Rhesus Monkeys Following Cortisol Synthesis Inhibition1

Dean A. Van Vugt, Jonathan Piercy, Anne E. Farley, Robert L. Reid and Serge Rivest

Department of Obstetrics and Gynecology (D.A.V.V., J.P., A.E.F., R.L.R.); Department of Physiology (D.A.V.V.), Queen’s University, Kingston, Ontario; and Laboratory of Molecular Endocrinology (S.R.), CHUL Research Center and Laval University, Quebec, Canada

Address all correspondence and requests for reprints to: Dean A. Van Vugt, Department of Obstetrics and Gynecology, 3022 Etherington Hall, Queen’s University, Kingston, Ontario, Canada, K7L 3N6.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Corticotropin-releasing Factor (CRF) is an important inhibitory neuromodulator of GnRH/LH secretion, and mediates in part the inhibitory effects of stress on the hypothalamic-pituitary-gonadal axis. The purpose of the present study was to further investigate CRF’s role in regulating LH secretion in primates. This was accomplished by examining LH secretion in ovariectomized rhesus monkeys (n = 7) following cortisol synthesis inhibition with metyrapone. Infusion of metyrapone (5 mg/kg per h) for 4 h decreased cortisol levels to less than 20% of controls while increasing ACTH approximately 10-fold. LH concentrations were not affected by this acute activation of the hypothalamic-corticotroph axis. In a second experiment, metyrapone was infused for 10 h before collecting serial blood samples every 15 min for 6 h. Although this protocol produced a sustained increase in ACTH, no apparent effect on pulsatile LH secretion compared with saline controls was observed. Mean LH (± SEM) levels calculated for consecutive 2-h increments were 87.6 ± 9.2 (0–2 h) 82.1 ± 5.5 (2–4 h), and 80.7 ± 4.8 (4–6 h) ng/ml in saline pretreated animals compared with 83.6 ± 4.9, 79.8 ± 5.8, and 72.5 ± 6.2 ng/ml, respectively, in metyrapone pretreated monkeys. The same regimen of metyrapone infusion increased CRF messenger RNA levels in the paraventricular nucleus by approximately 33% (P < 0.0002). In a final experiment designed to examine the potential synergy between CRF and cortisol, the LH response to insulin-induced hypoglycemia was contrasted in saline and metyrapone pretreated monkeys. LH concentrations were reduced to approximately 40% of basal levels following insulin in both metyrapone and saline pretreated monkeys. Therefore, even though inhibition of cortisol synthesis leads to an increase in CRF messenger RNA in the paraventricular nucleus and a robust increase in ACTH secretion in rhesus monkeys, presumably due in part to increased neuroendocrine CRF secretion, LH secretion was not inhibited during either the acute or more chronic phase of corticotroph activation. Absence of LH inhibition was not due to low cortisol concentrations resulting from metyrapone because metyrapone did not prevent hypoglycemia-induced suppression of LH secretion. We conclude that increased neuroendocrine CRF secretion following metyrapone does not inhibit LH secretion under these conditions. Several explanations for this result are discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STRESS IS known to disrupt reproductive cycles in the female (1, 2). Stress can produce a state of hypogonadotrophic-hypogonadism as a result of inhibiting GnRH. Several different neuromodulators have been postulated to mediate the inhibitory effect of stress on GnRH/LH secretion (see Ref. 3 for review). In this regard, CRF is recognized as a critical inhibitory neuropeptide for several reasons. In most cases, stresses that inhibit LH secretion also activate the hypothalamic-pituitary-adrenal axis (HPA) (4, 5, 6, 7, 8). CRF administration to rats, monkeys, and humans was reported to reduce LH levels (9, 10, 11), possibly involving an opioid mechanism (11, 12, 13, 14). Because CRF neurons synapse with GnRH-containing neurons (15), and CRF can inhibit both in vivo and in vitro GnRH release (16, 17), LH inhibition most likely results from CRF inhibition of GnRH.

In addition to the evidence cited above, there are several reports that question the importance of CRF as a regulator of LH secretion, particularly in the primate. The effects of CRF administration on LH secretion in humans or monkeys are inconsistent. CRF was reported to have no effect, and in some instances to stimulate LH secretion (18, 19, 20, 21). Furthermore, although an inhibitory effect of CRF administration on LH secretion supports the hypothesis that CRF is an inhibitory neuromodulator of LH secretion, it is by itself inadequate proof. Intracisternal or iv injection of CRF does not mimic endogenous CRF release. Brain regions not innervated by CRF neurons may be exposed to high CRF levels following exogenous CRF administration. Inadequate amounts of CRF may cross the blood-brain barrier following iv administration, whereas the pituitary and gonads are exposed to nonphysiological concentrations.

We have attempted to address the question of CRF’s relevance as a physiological neuromodulator of LH secretion in the rhesus monkey using metyrapone to stimulate endogenous CRF activity. Metyrapone inhibits 11-deoxycortisol conversion to cortisol resulting in reduced cortisol negative feedback (22). Given the incontrovertible evidence that glucocorticoid negative feedback occurs at both the hypothalamus and pituitary (23, 24, 25), we reasoned that metyrapone would increase CRF metabolism as a result of reduced cortisol negative feedback. Indeed, messenger RNA (mRNA) levels of CRF in parvocellular neurons were increased following metyrapone administration to rats (26, 27). Concentrations of CRF in hypophysial portal blood of rat (28) and sheep (29), and both CRF and vasopressin concentrations in pituitary venous blood of mares were increased by acute metyrapone administration (30). We hypothesized that if CRF originating from the paraventricular nucleus (PVN) is an important physiological inhibitory neuromodulator of LH secretion in the primate, LH concentrations should be reduced by metyrapone administration. In addition to monitoring ACTH, cortisol, and LH in the peripheral circulation, the effect of metyrapone on CRF mRNA in the PVN of monkeys was semiquantified by in situ hybridization to confirm or refute its effect reported in other species. Finally, because cortisol and CRF may synergize to inhibit LH secretion (31, 32), we reasoned that low cortisol levels resulting from metyrapone infusion may prevent or reduce metyrapone-induced inhibition of LH. To address this possibility, the effect of hypoglycemia-induced LH inhibition was compared in saline and metyrapone pretreated monkeys.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal husbandry
All experiments were performed on 10 adult ovariectomized rhesus monkeys. Their age ranged from 6–15 yr and their weights ranged from 6–8.5 kg. They had been ovariectomized for at least 3 months. All animals were housed in individual cages in a light and temperature controlled room (lights on 0600–1800 h; temperature 21–23 C). Their diet consisted of a twice daily ration of monkey chow that was supplemented with fruit and vegetables. Water was available ad libitum. All animal husbandry practices and all experimental procedures conformed to the guidelines of the Canadian Council on Animal Care and were approved by the Queen’s University Animal Use Committee.

Exp 1: acute effects of metyrapone on hormone secretion
The objective of this experiment was 2-fold: determine the onset and duration of hypothalamic-corticotroph axis activation after cortisol synthesis inhibition with metyrapone and determine the acute LH response to this inhibition. Monkeys (n = 7) were lightly sedated with ketamine HCl (5–10 mg/kg; Rogarsetic, Rogar/STB, Montreal, Quebec) and placed in primate chairs between 0800–0900 h. An angiocatheter was inserted into a femoral vein for blood collection and into a saphenous vein for metyrapone or saline infusion. Beginning at 1100 h, monkeys received a 4-h infusion of metyrapone (5 mg/kg per h; Sigma Chemical, St. Louis, MO) or an equivalent volume of saline. Blood samples (2.5 ml) were collected at hourly intervals from 1100–1400 h and at 15-min intervals from 1400–1700 h. Each animal received both treatments in random order. The interval between treatments was at least 2 weeks.

Exp 2: effects of longer term metyrapone infusion on hormone secretion
The effect of a longer period of cortisol synthesis inhibition on the hypothalamic-corticotroph axis and its consequence on LH secretion were examined in a second experiment. Monkeys (n = 7) were prepared as described in the first experiment at approximately 1600 h. Commencing at 2200 h, a 10-h infusion of metyrapone (5 mg/kg per h) or saline was given. On completing the metyrapone infusion at 0800 h, blood samples were collected at 15-min intervals for 6 h. Each animal received both treatments in random order. The interval between treatments was at least 2 weeks.

Exp 3: effect of metyrapone on hypoglycemia-induced suppression of LH secretion
Six monkeys were prepared as in Exp 2 and infused with either metyrapone (5 mg/kg; n = 4) or saline (n = 5) from 2200–0800 h. Beginning at 0800 h, blood samples were collected at 15-min intervals for 1 h to establish baseline LH and cortisol levels. An iv bolus of insulin (1.0 U/kg; Humulin, Eli Lilly Co., Toronto, Ontario, Canada) was given, and blood sampling was continued at 15-min intervals for 5 h to monitor the LH, cortisol, and glucose responses. A second bolus of insulin (0.2 U/kg) was given if glucose levels began to recover 2–3 h after the initial insulin bolus. Three of the six monkeys received both metyrapone and saline before insulin, whereas one received metyrapone only and two received saline only. Treatments were separated by an interval of at least 2 weeks.

Exp 4: effect of metyrapone on CRF mRNA
Four of the seven monkeys used in Exp 1–3, together with three additional ovariectomized animals were prepared as described in Exp 1 and were infused with saline (n = 4) or metyrapone (5 mg/kg per h; n = 3) from 2200–0800 h as described in Exp 2. Immediately following infusion, monkeys were deeply anesthetized with saffan (Pittman-Moore, Middlesex, UK; 1.2 mg/kg). Perfusion of the brain with 4% paraformaldehyde with 3.8% borax (pH = 9) at a rate of 50 ml/min for 20 min via the carotid arteries was begun within 10 min of anesthesia.

In situ hybridization histochemistry.
The brains were removed from the skull, postfixed for 5–8 days, and placed in 10% sucrose in a solution of 4% paraformaldehyde-borax buffer for 48 h at 4 C. The brains were mounted onto a microtome, frozen with dry ice, and cut into 30-µm coronal sections from the olfactory bulb to the caudal medulla. The slices were collected in a cold cryoprotectant solution and stored at -20 C. Hybridization histochemistry using 35S-labeled complementary RNA (cRNA) probes was used to localize CRF transcript for one out of six series (every sixth section) of brain slices from the olfactory bulb to the end of the medulla. Protocols for riboprobe synthesis, hybridization, and autoradiographic localization of the mRNA signals were adapted from Simmons et al. (33). All solutions were treated with diethylpyrocarbonate (depc) and sterilized to prevent RNA degradation. Tissue sections were mounted onto gelatin and poly-L-lysine-coated slides, vacuum dried, fixed in 4% paraformaldehyde for 20 min, and digested by proteinase K (10 µg/ml in 100 mM Tris-HCl, pH 8.0, and 50 mM EDTA, pH 8.0) at 37 C for 25 min. The brain sections were then rinsed in sterile depc water followed by a solution of 100 mM triethanolamine (pH 8.0), acetylated in 0.25% acetic anhydride in 100 mM triethanolamine, and dehydrated through graded concentrations of alcohol (50, 70, 95, and 100%). After vacuum drying, 130 µl hybridization mixture (107 cpm/ml) was spotted on each slide, sealed under a coverslip, and incubated at 60 C for 17–22 h in a slide warmer. The coverslips were then removed, and the slides rinsed four times in 4x standard saline citrate (SSC) at room temperature. The sections were digested by RNase A (20 µg/ml in a solution of 500 mM NaCL, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, pH 8.0) at 37 C for 30 min, rinsed in descending concentrations of SSC (2, 1, 0.5x), washed in 0.1x SSC for 30 min at 60 C, and dehydrated through graded concentrations of alcohol. After being vacuum dried, the sections were exposed at 4 C on x-ray film (Eastman Kodak, Rochester, NY) for 17–48 h, defatted in xylene, and dipped into NTB2 nuclear emulsion (Kodak; diluted 1:1 with distilled water). Slides were exposed for 7 days, developed in D19 developer (Kodak) for 3.5 min at 14–16 C, washed for 15 sec in water, and fixed in rapid fixer (Kodak) for 5 min. Thereafter, the tissues were rinsed under running distilled water for 1 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX (BDH, Laboratory Supplies, Poole, UK).

cRNA probe synthesis and preparation.
The CRF antisense riboprobe was generated from the EcoRI fragment of CRF cDNA (Dr. K. Mayo, Northwestern University, IL), subcloned into pGEM4 plasmid, and linearized with HindIII. Radioactive cRNA copies were synthesized by incubating 250 ng linearized plasmid in 6 mM MgCl2, 40 mM Tris, pH 7.9, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 0.2 mM ATP/GTP/cytidine 5'-triphosphate, [{alpha}-35S]uridine triphosphate, 40 U RNAsin (Promega, Madison, WI), and 20 U SP6 RNA polymerase for 60 min at 37 C. Unincorporated nucleotides were removed by adding 100 µl DNase solution (1 µl DNase, 5 µl 5 mg/ml transfer RNA, 94 µl 10 mM Tris/10 mM MgCl2) for 10 min followed by a phenol-chloroform extraction. The probes were precipitated with 80 µl 5 M ammonium acetate and 500 µl 95% ethanol for 20 min on dry ice. After centrifugation, the pellet was washed with 500 µl 70% ethanol, dried, and resuspended in 100 µl 10 mM Tris/1 mM EDTA. A concentration of 107 cpm probe was mixed into 1 ml hybridization solution \[500 µl formamide, 60 µl 5 M NaCl, 10 µl 1 M Tris, pH 8.0, 2 µl 0.5 M EDTA, pH 8.0, 50 µl 20x Denhart’s solution, 200 µl 50% dextran sulfate, 50 µl 10 mg/ml transfer RNA, 10 µl 1 M dithiothreitol, (118 µl DEPC water-volume of probe used)\]. This solution was mixed and heated for 5 min at 65 C before being spotted on the slides (130 µl/slide).

Semiquantitative analysis.
Semiquantitative analysis of hybridization signal for CRF mRNA was carried out on x-ray films over three bilateral hypothalamic PVN nuclei for each animal expressing a clear positive signal for the transcript. OD of the hybridization signals was measured under a Northern Light Desktop Illuminator (Imaging Research Inc., St. Catharine’s, Ontario, Canada) using a Sony Camera Video System attached to a Micro-Nikkor 55 mm-Vivitar extension tube set for Nikon lens and coupled to a Macintosh computer (Power Macintosh 7100/66, Apple Computer, Cupertino, CA) and NIH Image software version 1.57/ppc (written by W. Rasband at the U.S. NIH and available from the Internet by anonymous ftp from zippy.nih.gov). Sections from experimental and control animals were digitized and subjected to densitometric analysis, yielding measurements of integrated OD (area of nucleus x average OD). The OD of each side of the PVN (bilateral) was then corrected for the average background signal, which was determined by sampling areas immediately outside the PVN (34). The data are expressed as means ± SEM and were analyzed by a one-way ANOVA via the Statview program (version 4.01, Macintosh).

Blood collection.
Blood for ACTH was collected in chilled plastic tubes containing 750 µg EDTA/ml blood. Blood was centrifuged at 6000 x g for 15 min. Plasma was separated and centrifuged a second time. Plasma was stored at -70 C until assayed for ACTH. Blood for LH and cortisol was collected on chilled glass tubes and allowed to clot overnight. Following centrifugation for 15 min at 1500 x g, serum was collected and stored at -20 C.

Assays
LH concentrations were measured in duplicate aliquots of 100 µl using reagents provided by the National Hormone and Pituitary Program (Bethesda, MD). The antiserum used was WP-R13 (pool D). A sheep antirabbit {gamma}-globulin (Prince Laboratories, Toronto, ON) was used to precipitate the antigen-antibody complex. Assay sensitivity, defined as the concentration of reference preparation (NICHD-rhLH) that produced binding that was two standard deviations lower than the maximum binding, was 15 ng/ml. The intraassay and interassay coefficients of variation determined from low, medium, and high serum pools assayed in triplicate at three volumes each were 4.6% and 11.1%, respectively (mean of six assays). Due to depletion of these reagents, some samples in Exp 3 were assayed using a new homologous LH RIA provided by Dr. A.F. Parlow (Pituitary Hormones and Antisera Ctr., Torrance, CA) and the National Hormone and Pituitary Program. Recombinant cynomolgus monkey LH (AFP-6936a) was used as both the iodinated ligand (chloramine-T method) and the reference preparation. Rabbit antiserum (AFP342994) against recombinant cynomolgus LH was used at a final concentration of 1:800,000 and precipitated with a sheep antirabbit gamma globulin (Prince Laboratories). Serum samples (50 µl) were assayed in triplicate. The sensitivity (as calculated for the previous LH assay) was 0.6 ng/ml. The intraassay coefficient of variation calculated from serum pools that spanned the entire standard curve was 6.3%. The ratio of LH values from multiple samples measured by both the old and new LH assays was 18.66 (old/new). Because LH measurements in Exp 3 were made using both the old and new LH assays, the results from each animal were expressed as a percent of its basal LH level by first calculating hourly LH values (mean of four consecutive samples) for each animal. This value was divided by the animal’s basal LH level (mean of the LH level in the five blood samples before insulin) to express individual responses as a percent of basal before a mean response (n = 4 or 5) was calculated.

ACTH concentrations were assayed following the procedure of Nicholson et al. (35). Unextracted plasma aliquots of 50 µl were assayed in duplicate using anti-ACTH serum (IgG-ACTH-1) from IGg Corp (Nashville, TN), labeled h-ACTH from Diagnostic Products (Los Angeles, CA), and sheep antirabbit gamma globulin (Daymar Laboratories, Toronto, ON). Assay sensitivity defined as above was 12.5 pg/ml using h-ACTH AFP-2938C as the reference preparation. The intraassay coefficient of variation was 6.9%.

Cortisol concentrations were measured in duplicate aliquots of unextracted plasma (12.5 µl) using a double antibody kit from Diagnostic Products. Assay sensitivity defined as above was 0.3 µg/dl. Cross-reactivity with 11-deoxycortisol was 0.17%. Intraassay and interassay coefficients of variation were 3.5% and 5.8%, respectively.

Glucose levels were measured using a glucometer (Accuchek Glucometer IIM, Boehringer Mannheim, Laval, Quebec, Canada) and expressed as millimoles per liter.

Statistical analysis
ACTH, cortisol, and LH are expressed as group means ± SEM. A repeated measures ANOVA was used to test for statistical differences in mean levels between metyrapone and saline infused groups followed by Scheffe’s post hoc test. An analysis of pulsatile LH secretion was not attempted because of the inconsistency with which LH pulses occurred in the control group. Although some animals exhibited a definite pulsatile secretory pattern with the expected frequency, other animals displayed more random fluctuations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: acute activation of the hypothalamic-corticotroph axis by metyrapone
The effects of a 4-h infusion of metyrapone on cortisol and ACTH are shown in Fig. 1Go. Cortisol levels were reduced to approximately 50% of controls 2 h after initiating metyrapone administration. The cortisol concentration continued to decline throughout the infusion and reached a level that was approximately 20% of basal levels at 4–6 h after initiating metyrapone infusion. In contrast, ACTH concentrations were increased by metyrapone administration. ACTH levels began to increase 2 h after starting metyrapone infusion and coincided with a significant decrease in cortisol. Peak ACTH levels were achieved 5 h after initiating metyrapone injections. LH concentrations in metyrapone and saline treated animals were comparable both during and immediately following a 4-h metyrapone infusion (Fig. 2Go).



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  		Figure 1. Effect of acute metyrapone infusion on ACTH ({blacksquare}) and cortisol (•) secretion. Time and duration of metyrapone infusion is represented by box. Also shown are cortisol levels in saline infused animals ({circ}). SEM are indicated by vertical lines (n = 7).

 


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  		Figure 2. Effect of acute metyrapone infusion on LH secretion. Mean LH concentrations during and following metyrapone infusion (•) are contrasted to saline controls ({circ}). Time and duration of metyrapone or saline infusion are represented by box. SEM are indicated by vertical lines (n = 7).

 
Exp 2: longer-term activation of the hypothalamic-corticotroph axis
Activation of the hypothalamic-corticotroph axis of a longer duration was achieved with a 10-h infusion of metyrapone. Cortisol levels were maximally suppressed by this regimen of metyrapone infusion (Fig. 3Go). Cortisol levels gradually increased after terminating the metyrapone infusion, but remained below 50% of control levels at the completion of the experiment. In contrast, ACTH levels were increased more than 10-fold by metyrapone and did not decline during the blood sampling period.



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  		Figure 3. ACTH and cortisol concentrations following a 10-h infusion of metyrapone. Mean ACTH ({blacksquare}) and cortisol (•) concentrations following a 10-h infusion of metyrapone or saline ({circ}; cortisol only) are shown. SEM are indicated by vertical lines (n = 7).

 
Figure 4Go compares both representative and mean LH and cortisol profiles during the 6-h immediately following a 10-h infusion of metyrapone or saline. In contrast to cortisol, LH concentrations in metyrapone treated animals were no different from controls. LH pulses were evident in some but not all animals. These results are summarized in Fig. 5Go in which mean LH and cortisol levels at 2-h increments are compared. Whereas mean cortisol levels were significantly reduced by metyrapone, no effect on mean LH concentration was seen.



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  		Figure 4. Representative LH and cortisol responses following a 10-h metyrapone infusion. LH (•) and cortisol ({circ}) response to either saline (left) or metyrapone (right) are shown for three monkeys. Mean LH and cortisol responses to saline or metyrapone are shown at the bottom (n = 7). SE bars are eliminated for clarity.

 


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  		Figure 5. Mean LH and cortisol concentrations following a 10-h metyrapone infusion. Mean LH (A) and cortisol (B) responses at 2-h increments were calculated for each animal before calculating group means. Vertical bars represent SEM (n = 7). *, P < 0.05; **, P < 0.01.

 
Exp 3: effect of metyrapone on hypoglycemia-induced suppression of LH
The effect of metyrapone on hypoglycemia-induced suppression of LH secretion and stimulation of cortisol is shown in Fig. 6Go. Insulin significantly reduced LH concentrations by hour 2 in both metyrapone and saline pretreated monkeys (Fig. 6AGo). LH levels declined to less than 50% of basal levels by hour 3. This level of suppression was similar in both groups and was maintained for the duration of the experiment. The cortisol response to insulin alone or metyrapone plus insulin is shown in Fig. 6BGo. Baseline cortisol levels in the metyrapone pretreated animals were significantly decreased compared with saline pretreated monkeys. This response was similar to that observed in Exp 2. Cortisol levels were significantly increased by insulin at 2 h and remained elevated for the duration of the experiment in the saline pretreated group. In contrast, cortisol did not increase following insulin in the metyrapone pretreated monkeys. The glucose response to insulin was identical in both groups (Table 1Go).



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  		Figure 6. Effect of metyrapone on hypoglycemia induced suppression of LH. Mean LH (A) and cortisol (B) concentrations following insulin-induced hypoglycemia were calculated at hourly increments. LH concentrations are expressed as a percent of basal. LH concentrations were significantly decreased at 2 h following insulin and remained suppressed for duration of experiment in both saline pretreated (n = 5) and metyrapone pretreated monkeys (n = 4; P < 0.01 vs. basal). Cortisol concentrations were significantly decreased in metyrapone pretreated monkeys and did not increase in response to insulin. In contrast, cortisol levels in saline pretreated monkeys were significantly increased at 2 h following insulin and remained elevated for duration of experiment. P values are for comparisons with basal cortisol concentration.

 

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  		Table 1. Glucose concentrations in response to insulin in saline and metyrapone pretreated monkeys

 
Exp 4: effect of metyrapone on CRF gene expression
As reported for rodents, mRNA encoding CRF was widely distributed throughout the monkey brain. Figure 7aGo shows representative examples of coronal sections (30 µm) at the level of the medial PVN. Positive hybridization signal for CRF transcript was present in the PVN as well as in various subdivisions of the thalamus (the area just above the PVN), including the anteromedial, anteroventral, and the ventral anterior nuclei. Metyrapone infusion significantly increased CRF gene expression in the PVN nucleus. An example of the increased hybridization signal for CRF in the PVN of a monkey killed immediately after a 10-h infusion of metyrapone is depicted in Fig. 7bGo. The mean OD measured in three bilateral medial sections of the PVN for each monkey indicated that the relative levels of CRF transcript were significantly higher (P < 0.0002) in the metyrapone treated group (Fig. 7cGo). The wide distribution of gene encoding CRF throughout the monkey brain prevented us, at this time, from evaluating the effect of metyrapone on CRF message density in all CRF sensitive regions.



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  		Figure 7. Expression of mRNA encoding CRF in PVN of hypothalamus of ovariectomized female monkeys treated with either metyrapone or vehicle solution. a, Examples of hybridization signal for CRF mRNA in whole 30 µm coronal sections on x-ray film. Positive hybridization signal can be observed in specific subdivisions of thalamus (especially in anteroventral and anteromedial nuclei) and in PVN. b, Darkfield photomicrographs of CRF mRNA positive signal (revealed with nuclear NTB-2 emulsion) of same level of PVN of a vehicle and metyrapone infused animal (magnification x10). c, Average OD of hybridization signal for CRF transcript in PVN. Results are expressed as means ± SEM for vehicle (n = 4) and metyrapone (n = 3) infused monkeys. For more information on image analysis, see Materials and Methods. *, Significantly different (P < 0.0002) from vehicle treated animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We examined the effects of cortisol synthesis inhibition on basal LH secretion and LH secretion in response to a glucopenic stimulus. Based on ACTH and cortisol measurements, the activity of the hypothalamic-corticotroph axis was increased within 2 h of metyrapone infusion and remained elevated for at least 12 h when metyrapone was infused for 10 h. A significant increase in CRF mRNA levels in the PVN occurred following metyrapone infusion, and confirms that this regimen of metyrapone stimulated the CNS component of the HPA axis. Despite activation of the hypothalamic-corticotroph axis, LH secretion was not inhibited during either the acute or more chronic phase of the study. Inhibition of cortisol synthesis and subsequent reduction of cortisol levels did not prevent hypoglycemia-induced suppression of LH. These results indicate that inhibition of the hypothalamic-gonadotroph axis and stimulation of the HPA axis can be dissociated from each other.

This is the first report showing that inhibition of cortisol synthesis in the primate significantly increases CRF mRNA levels in the PVN and is in agreement with observations in several other species. Levels of CRF mRNA were increased in parvocellular neurons following metyrapone administration to rats (26, 27). Herman and colleagues (27) also reported that metyrapone caused expression of CRF primary transcript (heteronuclear), suggesting transcriptional activation of the neuropeptide in the PVN. Concentrations of CRF in hypophysial portal blood of rats (28) and CRF and vasopressin concentrations in both portal blood of sheep and pituitary venous blood of mares were increased by acute metyrapone administration (29, 30).

In the present study, potential effects on LH secretion produced by either acute or more chronic activation of CRF neurons were considered. The first study examined LH secretion before and during the onset of the ACTH response. The time course of acute activation of CRF secretion in this paradigm might resemble increases in CRF achieved by either acute stressors or exogenous CRF infusions. The second study examined LH secretion after corticotrophs had been stimulated for 8–14 h. This protocol was designed to achieve a more prolonged increase in CRF secretion, such as may occur during stressors of intermediate duration. The present study demonstrated that CRF gene expression was significantly increased 10 h after initiating metyrapone infusion. CRF gene expression in the rat was significantly increased 30 min after metyrapone administration (27). Assuming a similarly rapid augmentation of CRF gene transcription in the monkey following metyrapone (an assumption that is supported by cortisol and ACTH measurements), then LH secretion was successfully monitored during both the acute and more chronic phase of increased neuroendocrine CRF secretion. Therefore, we can exclude the possibility that LH inhibition was not observed because CRF activity was increased only acutely, or alternatively, because the hypothalamic-gonadotroph axis had recovered from suppression by CRF.

There are several possible reasons that may explain why activation of CRF by metyrapone did not inhibit LH secretion. Metyrapone may produce a perturbation that is limited to the neuroendocrine component, i.e CRF and possibly vasopressin neurons originating in the PVN that specifically regulate the HPA axis, but have no regulatory influence on GnRH/LH secretion. This hypothesis is supported by studies in which lesions of the PVN blocked ACTH and corticosterone secretion in response to either stress or cytokine injection, but did not prevent either challenge from inhibiting LH secretion (36). These results suggest that CRF neurons in the PVN are indispensable for stress-induced activation of the HPA axis, whereas PVN CRF neurons are not required for inhibition of the hypothalamic-gonadotroph axis. It is possible that CRF neurons outside the PVN influence the hypothalamic-gonadotroph axis but are unaffected by metyrapone. The effect of metyrapone on CRF gene expression in other brain regions of the monkey is yet to be studied. If increased CRF mRNA following metyrapone is restricted to the PVN, it would be revealing to compare this response with CRF mRNA following a stressor, such as hypoglycemia, which we recently showed inhibits LH secretion in the monkey through a CRF mechanism (37). Another possibility is that metyrapone selectively stimulates the activity of a specific subset of parvocellular neurons projecting to the infundibular system without affecting the nonneuroendocrine CRF-containing cells of the PVN. It is known that approximately 50% of the parvocellular CRF neurons of the PVN project to the median eminence and therefore regulate the corticotroph axis, whereas the remaining CRF cells innervate various hypothalamic and extrahypothalamic regions (38). Whether these latter neurons are affected following cortisol synthesis inhibition is technically difficult to answer, especially in monkeys.

The present study also considered the possibility that cortisol and CRF synergize to inhibit LH secretion, and that CRF-induced inhibition of LH is compromised by low cortisol levels following metyrapone. However, our observation that hypoglycemia inhibited LH levels to the same extent in metyrapone and saline pretreated monkeys, even though cortisol levels remained suppressed following insulin in the metyrapone pretreated animals, does not support this explanation. Our results are consistent with the observation that CRF administration inhibited LH concentrations in rats and monkeys that were adrenalectomized or treated with metyrapone (9, 39, 40). Because inhibition of LH by exogenous CRF or hypoglycemia was not prevented by hypocortisolemia, it is unlikely that hypocortisolemia prevented metyrapone-induced LH inhibition. However, because we cannot exclude the possibility that other hypothalamic factors are involved in hypoglycemia-induced LH suppression, we must temper this conclusion at this time.

The present experiments were conducted in ovariectomized monkeys that were seated in primate chairs. We have considered the impact of these two variables (restraint and ovarian status) in our interpretation of why metyrapone did not inhibit LH secretion. Although the animals are habituated to chair restraint by repeated and frequent exposure to this condition, cortisol measurements indicate that the activity of the HPA axis remains elevated following acclimatization. We do not believe that increased HPA axis activity inherent to restraint explains why metyrapone did not inhibit LH secretion. Previous studies demonstrating that CRF administration inhibited LH secretion in the monkey employed chair restraint (10, 14, 40). Secondly, we demonstrated that hypoglycemia inhibited LH secretion in chair restrained ovariectomized monkeys, whereas hypoglycemia did not inhibit LH secretion in ovariectomized monkeys that remained unrestrained in their cage (41). In an attempt to replicate conditions in previous studies, and considering the possibility that metyrapone infusion (as with hypoglycemia) might inhibit LH secretion only when combined with chair restraint, we decided to use chair restraint in these experiments. Furthermore, because cortisol levels are elevated in chaired monkeys, the absolute reduction in cortisol caused by metyrapone is greater than would have occurred in the absence of restraint. This large decline in cortisol negative feedback, may constitute a greater stimulus to CRF neurons than a more complete (but smaller absolute decline) suppression of basal cortisol levels.

It is more likely that our results were influenced by gonadal status. The hypothalamic-gonadotroph axis of ovariectomized monkeys is less susceptible to stress-induced inhibition compared with ovary intact monkeys based on multiunit recordings and LH measurements (42). A similar conclusion was made based on LH measurements following food deprivation in ovariectomized rats (43). We have considered the possibility that in the absence of estrogen, CRF neurons or other peptidergic neurons that mediate CRF’s effect on the hypothalamic-gonadotroph axis are resistant to activation following metyrapone, just as they may be resistant to activation by stress. Although metyrapone increased CRF mRNA levels in the PVN of ovariectomized monkeys, it is possible that a more robust response to metyrapone would occur in ovary intact monkeys. Similar experiments will have to be performed in ovary intact monkeys to determine whether ovarian steroids influence the magnitude of the CRF response to metyrapone. These experiment are in progress.

In summary, we have shown that metyrapone-induced inhibition of cortisol synthesis leads to a significant increase in CRF mRNA levels in the PVN and a robust increase in ACTH secretion in ovariectomized rhesus monkeys. LH secretion was not inhibited during the acute or more chronic phase of corticotroph activation. Furthermore, metyrapone treatment did not alter LH inhibition in response to hypoglycemia. It remains to be determined why hypoglycemia and cortisol synthesis inhibition with metyrapone have different effects on LH secretion, even though they both stimulate neuroendocrine CRF. If it can be demonstrated that ovarian status influences the LH response to metyrapone, metyrapone would be a useful tool for further exploring hypothalamic neuromodulatory systems inhibitory to reproduction.


   Acknowledgments
 
We acknowledge the donation of assay reagents from the National Hormone and Pituitary Program (Rockville, MD) for measuring LH. The technical assistance of Ms. Brenda Roy, Dr. A. Krzemien, Ms. Nathalie Laflamme, and Dr. Rossella E. Nappi is appreciated.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada and was presented in part at the 25th Annual Meeting of the Society for Neuroscience, November 11–16, 1995, San Diego, California. Back

Received December 24, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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