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Endocrinology Vol. 145, No. 4 1750-1759
Copyright © 2004 by The Endocrine Society

A Hypothalamic-Testicular Neural Pathway Is Influenced by Brain Catecholamines, But Not Testicular Blood Flow

Daniel J. Selvage, Soon Y. Lee, Loren H. Parsons, Dong O. Seo and Catherine L. Rivier

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute (D.J.S., S.Y.L., D.O.S., C.L.R.), La Jolla, California 92037; and Department of Neuropharmacology, The Scripps Research Institute (L.H.P.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D, Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported the existence of a descending multisynaptic, pituitary-independent, neural pathway between the hypothalamus and the testes in the male rat. Stimulation of this pathway by the intracerebroventricular (icv) injection of IL-1ß or corticotropin-releasing factor blunts the testosterone (T) response to human chorionic gonadotropin (hCG). This response is mediated at least in part by catecholamine ß-adrenergic receptor activation. The present work was performed to further investigate the role of brain catecholamines and testicular blood flow in this pathway. The icv injection of 5 µl of 200 proof ethanol (EtOH; 86 µmol) did not result in detectable levels of the drug in the general circulation and did not induce neuronal damage, but rapidly blunted hCG-induced T release while not decreasing LH levels or altering testicular blood flow. EtOH significantly up-regulated transcripts of the immediate-early gene c-fos in the paraventricular nucleus (PVN) of the hypothalamus. Lesions of the PVN blocked the inhibitory effect of IL-1ß on T, but only partially interfered with the influence of EtOH. PVN catecholamine turnover significantly increased after icv injection of IL-1ß, but not EtOH. Brain catecholamine depletion due to the neurotoxin 6-hydroxydopamine did not alter the ability of hCG to induce T release, but significantly reversed the inhibitory effect of icv EtOH or IL-1ß on this response. Collectively, these results indicate that icv-injected IL-1ß or EtOH blunts hCG-induced T secretion through a catecholamine-mediated mechanism that does not depend on either peripherally mediated effects or pituitary LH, and that the PVN plays a role in these effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AN UNRESOLVED issue regarding the control of the hypothalamic-pituitary-gonadal axis pertains to the ability of some stressors to decrease plasma testosterone (T) levels in the absence of detectable changes in mean LH concentrations (1). One possibility is that these stressors release inhibitory substances into the general circulation or generate their synthesis within the testes, thus bypassing perturbations in LH secretion. We have recently proposed that in addition to the well recognized control exerted by pituitary LH (2), T release is regulated by an efferent neural pathway from the hypothalamus to the testes (3, 4) that works independently from the pituitary (5). This pathway, identified by pseudorabies virus studies (3, 6), was retrogradely mapped from the testes, up the spinal cord (T10-L1, L5-S1) to brain stem areas, including the caudal raphe nucleus, nucleus of the solitary tract, and locus coeruleus. Hypothalamic areas identified as possible components of this pathway include the paraventricular nucleus (PVN) and the lateral hypothalamus, whereas telencephalic structures include the preoptic area, central amygdala, and insular cortex. Functionally the identified circuit, which is activated by the intracerebroventricular (icv) injection of IL-1ß, corticotropin-releasing factor (CRF), and ß-adrenergic agonists, exerts an inhibitory influence on Leydig cell responsiveness to gonadotropins such as human chorionic gonadotropin (hCG) (5, 7).

Although these effects are well established, the relative importance of the structures that are part of the anatomical connection between the hypothalamus and the testes, remains poorly understood. Similarly, the role of central (i.e. brain-mediated) vs. peripheral mechanisms, such as altered testicular blood flow, has been controversial. Also, as receptor antagonists used to block the influence of circulating secretagogues often themselves influence T release, dissecting the role of peripheral/intratesticular signals from that of brain-mediated events, such as decreased levels of the hypothalamic peptide LH-releasing hormone (LHRH) and/or activation of the proposed neural pathway (3), has not been possible. We therefore developed an experimental model that would allow us to alter Leydig cell function in the absence of stressor-induced increases in circulating and/or testicular inhibitory signals.

The intragastric or ip injection of ethanol (EtOH) lowers plasma T levels through mechanisms that involve both decreased LH secretion and a direct effect of EtOH on testicular steroidogenic enzymes (8), although the respective roles of these different mechanisms have not been determined. We know that after peripheral EtOH administration, brain levels of this drug rapidly reach pharmacologically meaningful levels (9, 10, 11). We therefore reasoned that if we could inject EtOH directly into the brain ventricles at a dose that modified Leydig cell activity but did not leak to the periphery, we could differentiate between the influence of this drug on the central nervous system (CNS) and on the testes. Results obtained in this model were then compared with those obtained after the icv injection of IL-1ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Harlan Sprague Dawley rats (Harlan Laboratories, Indianapolis, IN, and San Diego, CA), weighing 180–200 g on arrival, were housed individually after surgical treatments and maintained under controlled lighting conditions (12 h of light, 12 h of darkness), with food and water available ad libitum. All protocols were approved by The Salk Institute animal care and use committee.

Surgeries
Intracerebroventricular cannulation.
With the anesthetized animals placed in a stereotaxic surgery apparatus, icv cannulas were implanted in the left lateral ventricle (coordinates: anterior-posterior, -0.4 mm; lateral, ±1.4 mm; dorsoventral, -3.8 mm). Cannula placement was verified at the end of each experiment, and only animals with correct placement were included in the analysis of the data. Castration, when appropriate, was performed under isoflurane anesthesia. After a 7- to 10-d recovery time after any of these surgeries, the animals were fitted with a jugular vein cannula, inserted under isoflurane anesthesia as previously described (4, 5, 7). Rats were allowed to recover for an additional 2–3 d before experimentation.

PVN lesions.
Bilateral, electrolytic lesions of the PVN were made by passing a 0.8-mA current through a tungsten-tipped electrode (A-M Systems, Inc., Carlsborg, WA) for 35 sec, as previously described (4). The coordinates for electrode placement from bregma were: anterior-posterior, -1.65 mm; lateral, ±0.4 mm; dorsoventral, -8.0 mm. Sham lesions were performed using the same coordinates, except the electrode was lowered -5 mm dorsoventral, and no current was passed through it. All animals were also fitted with icv cannula as described above and with an iv cannula 7–8 d later. Brains were perfused in 4% paraformaldehyde, removed, and stored in a solution of 10% sucrose in 4% paraformaldehyde overnight, and coronal sections were cut at 30 µm on a cryostat. Only animals with PVN lesions that were determined to encompass 75% or more of the nucleus were used in statistical analysis of the data.

6-Hydroxydopamine (6-OHDA) lesions.
The catecholamine neurotoxin 6-OHDA was dissolved in sterile apyrogenic water with 0.2% ascorbic acid. Seventy-five micrograms of 6-OHDA were administered into the left brain ventricle (injection rate, 1 µl/10 sec) 4 d before experimentation, and a second 75-µg dose was injected 2 d later. This regimen was chosen because it does not alter behavior (12), and it depletes synaptic norepinephrine (NE) and dopamine (DA) levels by more than 95% (13, 14). As 6-OHDA does not alter serotonin (5-HT) levels (Seo, D. O., and C. L. Rivier, unpublished observations), it is considered a specific neurotoxin for catecholamines. At the end of the experiments, the rats were anesthetized with chloral hydrate and perfused transcardially with saline, followed by 4% paraformaldehyde plus 1% glutaraldehyde and 0.2% Na2S2O5 in 0.1 M NaPO4 buffer. After a 4-h postfix and overnight immersion in cryoprotectant (20% sucrose in PBS), brains were cut into 30-µm coronal sections, and sections containing the PVN were processed for NE and tyrosine hydroxylase immunoreactivity using an NE antibody (ab8887, Abcam, Cambridge, MA) in a 1:4 potassium dilution or a tyrosine hydroxylase antibody (NB300–109, Novus Biologicals, Inc., Littleton, CO) at a 1:5 potassium dilution using previously described immunocytochemistry techniques (15). Sections containing the PVN, which receives significant noradrenergic innervation, were examined using light microscopy, and only rats showing little or no NE immunoreactivity in this area after 6-OHDA lesions were used for statistical analysis of the data. The results of control experiments involving omission of primary antisera or secondary antisera supported the specificity of each antiserum.

Experimental protocol
On the day of experimentation, the animals were removed to a sound-proof room and housed individually in opaque buckets, with their cannulas connected by polyethylene tubings to injection sites outside the buckets so treatments could be administered, and blood withdrawn without disturbing the freely moving animals. All injections were given at least 2 h after rehousing to allow hormone levels to return to normal. Blood samples (0.3 ml) were taken through the iv cannula and immediately replaced with an equivalent volume of apyrogenic isotonic saline.

Fluoro-Jade (FJ) detection of neuronal damage
FJ is an anionic tribasic fluorescein derivative that stains the cell bodies, dendrites, axons, and axon terminals of degenerating neurons, but does not stain healthy neurons, myelin, vascular elements, or neuropil (16, 17). After icv EtOH or vehicle (sterile water) administration, brain sections were examined following FJ staining by light microscopy using a 450- to 490-nm excitation filter.

Measurement of testicular blood flow
We used laser-Doppler flow cytometry because this technique does not require the extensive surgery necessary in other methods and can be performed under mild anesthesia (18). This type of flow cytometry yields measurements proportional to the amount of blood flow, not the rate of flow or blood pressure (19), and has been used to monitor testicular microvascular flow under a variety of experimental manipulations (20, 21). Rats were anesthetized with isoflurane, and core temperature was maintained at 37 C using a circulating heating pad. The level of anesthesia was held constant by keeping the breaths per minute rate between 35–40. After opening the scrotum, the left testicle was exposed, and a laser-Doppler probe (0.85-mm diameter) attached to a blood perfusion monitor (model 403A, TSI, St. Paul, MN) was placed on two areas of the testes where there were no obvious surface blood vessels. During the experiment, the left testes were repeatedly rinsed with warm saline to prevent dehydration of the exposed tissues. Five measurements, in the form of perfusion units (PU), were recorded from each area, then averaged to determine basal flow rates and whether iv administered hCG, followed by icv injections of vehicle, EtOH, or IL-1ß, altered these rates.

Reagents
Absolute, reagent grade EtOH (USP, 200 proof) was purchased from Accurate Chemical and Scientific Co. (Shelbyville, KN). It was free of any additives. We conducted preliminary studies to determine the optimum dose, which was chosen to be that producing maximum biological responses but no neuronal damage (Selvage, D. J., and C. L. Rivier, unpublished observations; also see Results). Recombinant human IL-1ß was provided by Otsuka Pharmaceutical Co. (Tokushima, Japan). Rat/human CRF and the GnRH antagonist Azaline B (provided by Dr. Jean Rivier, The Salk Institute, La Jolla, CA) were synthesized by solid phase methodology (22). IL-1ß and CRF were diluted in apyrogenic water, and Azaline B was diluted in phosphate-buffered apyrogenic saline that contained 0.1% crystalline BSA and 0.01% ascorbic acid. hCG was purchased from Sigma-Aldrich Corp. (St. Louis, MO) and diluted in apyrogenic saline. The doses of IL-1ß (80 ng, icv), Azaline B (40 µg/kg, iv; -15 min), and hCG (1 U/kg, iv) were chosen on the basis of our previous experience (4, 5, 7). Vehicle treatments consisted of apryogenic water alone in the EtOH studies. Phenylephrine HCl gel (0.25%) was obtained from Whitehall-Robins (Madison, NJ).

Assays
Blood alcohol levels (BALs) were measured in 5 µl plasma using an AM 1 analyzer (Analox Instruments Ltd., Lunenburg, MA) (23). Plasma T levels were measured in 50 µl unextracted plasma samples with a commercially available, solid phase RIA kit (Diagnostic Products Corp., Los Angeles, CA) (3). Plasma LH levels were measured in 50-µl unextracted plasma samples with RIA reagents provided by the National Pituitary and Hormone Distribution Program, NIDDK (24).

PVN monoamine levels
Animals were decapitated, and the whole brains were quickly removed, quick-frozen with cold isopentane in dry ice, and stored at -70 C until assayed. The hypothalamus was isolated using the landmarks of the optic chiasm on the ventral surface and the fornices for the dorsal and lateral boundaries, and the PVN areas were collected using 2-mm diameter neuropunches (Fine Science Tools, Inc., Foster City, CA). Monoamine measurement by HPLC was based on previously published methodology (25).

Brain preparation for in situ hybridization
Rats were deeply anesthetized with chloral hydrate, a drug that does not increase immediate-early gene/peptide mRNA levels (Lee, S. Y., and C. L. Rivier, unpublished observations). They were then perfused as described above. Brains were removed and postfixed in 4% paraformaldehyde for 4–5 d, then placed overnight in 10% sucrose/4% paraformaldehyde/0.1 M borate buffer. They were cut into 30-µm coronal slices obtained at 120-µm intervals throughout the hypothalamus and stored at -20 C in a cryoprotectant solution (50% 0.1 M PBS, 30% ethylene glycol, and 20% glycerol) until histochemical analysis. Brains from control and experimental animals belonging to the same experiment were always analyzed in the same hybridization experiment. Hybridization histochemical localization of the immediate-early gene c-fos was carried out with {alpha}-35S-labeled cRNA probe prepared as previously described (26, 27). A sense probe was used as a control for nonspecific signal in some adjacent sections for in situ hybridization.

Quantitative analysis of in situ hybridization results
Semiquantitative densitometric analysis of hybridization signals was carried out in nuclear emulsion-dipped slides, as previously described (27). Brain paste standards containing serial dilutions of [35S]UTP, used for quantification of mRNA signal, were prepared concurrently to ensure that OD was found within the linear range of the standard curve (28). Analyses with emulsion-coated slides were carried out with two or three different exposure times to confirm that signals were not saturated. Densitometric analyses of autoradiographic signals were performed over the confines of cells within the PVN using an optical system (Leitz, Deerfield, NJ) coupled to a Macintosh II computer (Apple Computer, Cupertino, CA) and Image software (version 1.61, W. Rasband, NIH, Bethesda, MD). Darkfield measurements for the parvo- and magnocellular divisions of the PVN were obtained separately, as previously reported (29). Gray level measurements (OD) were taken under darkfield illumination of hybridized sections over the medial parvocellular PVN or magnocellular PVN, as defined by redirected sampling from the corresponding Nissl-stained sections under brightfield images. Data were expressed in Gray scale values of 1–256. All Gray level measurements were corrected for background, which corresponded to the areas immediately adjacent to those under study. Signals were measured in both sides of the brain, and mean values for all animals were determined in three or four sections for each rat throughout the PVN.

Statistical analysis
Depending on what we estimated to convey the clearest information, data are presented as either the sum of T levels measured in three or more samples taken up to 90 min after hCG or as T released as a function of time. Results were first analyzed using ANOVA with repeated measures, followed by comparison of individual time points employing t test or Bonferroni/Dunn test. In all cases, a difference was considered statistically significant if it reached the level of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose- and time-related effect of icv injection of EtOH on T response to hCG
Preliminary studies conducted with 1–5 µl EtOH (200 proof) as well as 5 µl of a 50–100% final concentration indicated that 5 µl undiluted EtOH (86 µmol) yielded the most consistent data. This dose, which caused a small degree of arousal (i.e. short-lived locomotion and grooming), but no obvious pain behaviors, such as squealing, scratching of the head, or crouching, was therefore chosen for all subsequent experiments. Injection of alcohol treatments 30–90 min before hCG induced significant (P < 0.01) decreases in basal T levels (nanograms of T per milliliter ± SEM; n = 4–5): control, 0.81 ± 0.29; EtOH, -15 min, 0.85 ± 0.25; EtOH, -30 min, 0.70 ± 0.19; EtOH, -60 min, 0.52 ± 0.11; and EtOH, -90 min, 0.27 ± 0.05. As illustrated in Fig. 1Go, significant inhibition of the T response to hCG (calculated by measuring the sum of T levels 20, 45, and 90 min after hCG injection) was also observed when EtOH was administered 15–60 min before gonadotropin. On the basis of this experiment as well as others, hCG was usually injected 20–30 min after EtOH in most subsequent studies.



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  		FIG. 1. Time-related effect of icv injection of EtOH (5 µl of 200 proof) on the T response to hCG (1 U/kg). EtOH was administered 0–90 min before hCG. Data are presented as both the time course of T release (A) and the sum of T levels measured 20, 45, and 90 min after hCG administration (B). {bullet}, Control; {blacksquare}, EtOH (-15 min); {blacktriangleup}, EtOH (-30 min); {square}, EtOH (-60 min); {circ}, EtOH (-90 min). Each point or bar represents the mean ± SEM of five or six rats. *, P < 0.05; **, P < 0.01 (vs. vehicle).

 
Effect of icv injection of EtOH on the integrity of PVN neurons
No FJ staining was observed over the course of the experiments in PVN slices of vehicle- or EtOH-injected rats, indicating that there was no detectable neuronal damage over the time course relevant for alcohol studies (90 min; Fig. 2Go). In contrast, icv injection of 2 µg kainic acid induced significant FJ staining in the brains collected 2 d later.



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  		FIG. 2. Effect of icv injection of alcohol (5 µl; A) or kainic acid (2 µg; B) on FJ staining. A, The PVN, which is the focus of the alcohol studies. B, The thalamus, which is one of the areas that was stained positively for FJ after kainic acid treatment. Data are illustrated for the time course relevant for alcohol studies (90 min; A) or 2 d after treatment in the case of kainic acid (B; magnification, x130).

 
Effect of icv injection of EtOH on BALs
Blood samples were obtained before icv vehicle or EtOH injection as well as 5, 10, 20, 30, 45, and 60 min later. BALs of all samples remained below 1 mg/100 ml.

Effect of icv injection of EtOH or IL-1ß on testicular blood flow
We first performed various studies to ensure that the technique we used was suitable to detect significant changes in blood flow. In particular, we tested the effect of topical application of a vasoconstrictor, phenylephrine, on the testicle, because the intratesticular injection of epinephrine was reported to interfere with microvasculature parameters (20). We also investigated the effect of iv injection of several doses of hCG because the sc administration of 100 U/kg or more of this gonadotropin was shown to increase testicular blood flow (30, 31, 32). Subsequent experiments comprised rats injected with the vehicle, EtOH, or IL-1ß icv, hCG alone, or hCG preceded by the vehicle or EtOH. Finally, the influence of icv injection of IL-1ß, a compound also known for its ability to inhibit the T response to hCG (5), was included for the sake of comparison. The mean blood flow of vehicle-injected rats (n = 30) was 16.53 ± 1.21 PU, which is in excellent agreement with the previously reported value of 16.3 ± 1.3 PU (20). The iv injection of 1 and 10 U hCG/kg rapidly and dose-dependently increased blood flow, whereas topical phenylephrine decreased it (Fig. 3AGo). Despite its reported ability to increase blood pressure in the general circulation (33), icv-injected IL-1ß caused no detectable influence on testicular microvasculature flow (Fig. 3BGo). Compared with the data presented in Fig. 3AGo, icv IL-1ß did not significantly alter the effect of hCG (1 U/kg) on blood flow. The icv injection of EtOH modestly, but significantly (P < 0.01), increased basal blood flow. It also slightly decreased the stimulatory effect of hCG (1 U/kg), although the overall difference did not reach statistical significance.



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  		FIG. 3. Effect of icv treatments on testicular blood flow. A, Effect of icv injection of vehicle, followed by iv injection of vehicle ({circ}), 1 U hCG/kg ({square}), or 10 U hCG/kg ({bullet}) or topical application of phenylephrine gel ({blacksquare}). B, Effect of icv injection of IL-1ß (80 ng), followed by vehicle iv ({circ}) or hCG iv (1 U/kg) ({bullet}) or icv injection of EtOH (5 µl of 200 proof), followed by vehicle iv ({square}) or hCG iv (1 U/kg; {blacksquare}). Each point represents the mean ± SEM of three to seven rats. For the sake of clarity, vehicle data already presented in A were not added to B. PU, Perfusion units.

 
Effect of the icv injection of EtOH on LH release
We first determined the ability of icv-injected EtOH to alter LH release in testes-intact rats. These experiments failed to indicate significant changes in LH levels compared with those of rats injected with the vehicle (sum of LH levels measured 15, 30, 60, and 90 min after icv treatments: vehicle, 0.22 ± 0.03 ng/ml; EtOH, 0.21 ± 0.04 ng/ml; P > 0.05). However, we considered the possibility that the low basal LH levels measured in this model, which fall in the far left part of the RIA curve, might have masked significant changes. We therefore repeated this study in castrated rats and found that they similarly did not respond to icv-injected EtOH with significant changes in LH concentrations (sum of LH levels measured 15, 30, 60, and 90 min after icv treatments: vehicle, 28.16 ± 1.93 ng/ml; EtOH, 26.95 ± 1.76 ng/ml; P > 0.05).

Role of LH in the EtOH-induced inhibition of T response
We had previously reported that the ability of IL-1ß and CRF, injected icv, to interfere with hCG-induced T release was not impaired by removal of endogenous LH, which suggested that this pituitary hormone was not involved (5). We used a similar approach in the present work to ensure that even though EtOH did not alter mean LH levels, possible changes in the pulsatile pattern of secretion of this hormone, which would not be reflected by decreased mean concentrations, did not mediate the inhibitory influence of EtOH. We therefore compared the effect of icv injection of EtOH on hCG-induced T secretion in rats that had been pretreated with vehicle or Azaline B 15 min earlier. As in the case of IL-1ß and CRF (5), both groups of animals exhibited a comparable inhibition of T release when administered EtOH, and these responses were not altered by the LHRH antagonist (mean cumulative nanograms of T per milliliter ± SEM measured 20, 45, and 90 min after hCG: vehicle/icv vehicle, 20.3 ± 2.23; vehicle/icv EtOH, 12.1 ± 1.8; Azaline B/icv vehicle, 18.77 ± 2.14; Azaline B/icv EtOH, 11.55 ± 1.65; n = 5–7; P > 0.05 vs. decrease measured in the absence of the GnRH antagonist).

PVN neuronal response to icv injection of EtOH
Compared with vehicle administration, icv injection of EtOH 30 min earlier significantly up-regulated c-fos mRNA transcripts in the parvocellular portion of the PVN (OD, arbitrary units: vehicle, 9.58 ± 2.92; EtOH, 62.62 ± 9.79; P < 0.01; Fig. 4Go).



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  		FIG. 4. Representative darkfield photomicrographs illustrating PVN c-fos mRNA transcripts measured 30 min after icv injection of vehicle or EtOH (5 µl). III, Third ventricle. Magnification, x130.

 
Consequence of PVN lesions (PVNx) on T response to icv-injected EtOH or IL-1ß
As in our previous studies and according to criteria generally accepted in the literature (4, 34, 35), we chose more than 75% destruction of the PVN as the criterion for inclusion of an animal in the analysis of the data. We had previously reported that PVN lesions reversed the inhibitory effect of icv-injected CRF or the ß-adrenergic agonist isoproterenol on the T response to hCG (4). We therefore investigated the ability of these lesions to interfere with the influence of EtOH and compared these results with those of rats injected with IL-1ß. We found that, as we had previously shown (4), these lesions did not in themselves significantly alter the T response to hCG. Thus, we combined results measured in sham-operated and PVN-lesioned rats injected with vehicle icv before hCG. The icv injection of EtOH or IL-1ß significantly (P < 0.01) blunted T levels in sham-operated animals (Fig. 5Go). PVN lesions totally blocked the ability of IL-1ß to inhibit hCG-induced T, but only partially restored this response to icv EtOH.



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  		FIG. 5. Consequence of PVN lesions on the T response to iv hCG (1 U/kg) in rats pretreated with vehicle, EtOH (5 µl of 200 proof), or IL-1ß (80 ng) 20 min previously. A: {blacktriangleup}, Sham/PVNx/vehicle; {circ}, sham/EtOH; {blacksquare}, PVNx/EtOH. B: {blacktriangleup}, Sham/PVNx/vehicle; {circ}, sham/IL-1ß; {blacksquare}, PVNx/IL-1ß. Each point represents the mean ± SEM of 5–12 rats. a, P < 0.01 vs. sham/EtOH; **, P < 0.01 vs. sham and PVNx/vehicle.

 
Effect of icv injection of EtOH on PVN monoamine levels
The icv injection of EtOH did not significantly alter PVN levels of any of the monoamines or their metabolites measured 30 min later (Table 1Go). IL-1ß and CRF were studied for the sake of comparison. CRF significantly increased DOPAC levels vs. vehicle, and both CRF and IL-1ß increased catecholamine turnover, as measured by the DOPAC/DA ratio. Finally, IL-1ß significantly decreased PVN 5-HT vs. EtOH or CRF, and CRF increased 5-HT compared with the effect of vehicle.


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  		TABLE 1. PVN monoamine levels

 
Consequence of 6-OHDA lesions on T response to icv-injected EtOH
As we had previously shown that ß-adrenergic agonists stimulated the brain-testes pathway under investigation (4, 7), we tested the ability of both icv EtOH and IL-1ß to decrease hCG-induced T in rats injected with the catecholamine neurotoxin 6-OHDA. Immunohistochemistry indicated a significant decrease (>90%) in amine levels in the hypothalamus of rats with 6-OHDA lesions (not shown), which corresponds to data based on microdialysis measurement of these secretagogues (14). Because depletion of noradrenergic stores in testicular nerve endings decreases LH receptors (36), and because icv-injected compounds can leak to the periphery (37), we first verified that 6-OHDA administration into the brain ventricles was compatible with normal Leydig cell activity. As shown in Fig. 6Go, lesioned rats exhibited dose-related increases in T levels, and the effect of 1 U/kg hCG (the dose we routinely use in all our experiments) was virtually identical to that in sham-operated animals. We then examined the influence of 6-OHDA lesions on T release after icv injection of EtOH and IL-1ß. Both EtOH and IL-1ß blunted the stimulatory effect of hCG, and 6-OHDA lesions significantly reversed the influence of both in sham vs. lesioned animals receiving the same icv treatment (Fig. 7Go). In the case of EtOH, the reversal was such that T levels returned to the same level as in sham animals receiving vehicle, but in animals receiving IL-1ß, T levels were significantly reversed in those with 6-OHDA lesions, but remained well below sham/vehicle levels.



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  		FIG. 6. Effect of depletion of brain catecholamines by 6-OHDA on the T response to hCG (0.3, 1, or 3 U/kg). {blacktriangleup}, Lesions/0.3 U hCG/kg; {bullet}, lesions/1 U hCG/kg; {square}, lesions/3 U hCG/kg; {circ}, sham/1 U hCG/kg. Each point represents the mean ± SEM of five or six rats.

 


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  		FIG. 7. Effect of depletion of brain catecholamines by 6-OHDA on the T response to hCG (1 U/kg) in the presence or absence of EtOH or IL-1ß. EtOH (5 µl of 200 proof) or IL-1ß (80 ng) was injected icv 20 min before hCG. Data are presented as both the time-course release of T (A) and the sum of T levels measured 20, 45, and 90 min after hCG (B). As values for sham and lesioned rats injected with vehicle were comparable, they were collapsed for the time-course graph and presented as a single line. {bullet}, Sham/lesioned- vehicle; {circ}, sham-IL-1ß; {triangleup}, lesioned-IL-1ß; {square}, sham-EtOH; {blacksquare}, lesioned-EtOH. Each point or bar represents the mean ± SEM of five to seven rats. **, P < 0.01 vs. sham/lesioned-vehicle; a, P < 0.05 vs. sham/IL-1ß; b, P > 0.05 vs. sham/EtOH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well known that agents such as EtOH and cytokines inhibit testicular activity through mechanisms that, depending on the route of administration and doses injected, involve blockade of LHRH/LH secretion (38), a direct influence on the gonad (39), and/or stimulation of a catecholaminergic-dependent neural pathway (40). As discussed previously (1, 41, 42), many stressors induce the release of factors into the circulation, such as endogenous opiate-like substances and proinflammatory cytokines, which directly inhibit Leydig cell function. Consequently, these models cannot be used to test the modulating influence of a neural hypothalamic- testicular pathway because at least part of their ability to decrease T levels is expected to depend on a direct testicular influence of blood-borne signals. In contrast to many peripherally administered compounds, EtOH readily diffuses to areas protected by the blood-brain barrier, where it reaches levels in equilibrium with those measured in the periphery (9, 10, 11). Thus, we reasoned that injecting EtOH into the brain ventricles should mimic the CNS effects of its systemic administration while avoiding consequences due to circulating levels of the drug. We show here that the icv injection of EtOH significantly interfered with hCG-induced T secretion. Importantly, this was not accompanied by neuronal damage and did not involve the LHRH-LH axis.

Our previous studies had indicated that the PVN was an important component of the proposed efferent neural pathway between the brain and testes (3, 4). In the present work, we used c-fos expression to determine whether the icv injection of EtOH up-regulated neuronal activity in this nucleus, and showed that this was the case. These results are in keeping with the known stimulatory effect of peripherally injected alcohol on CRF and vasopressin cell bodies in the PVN (43). However, many signals exert a similar influence, and this does not necessarily reflect a functional role of the PVN in mediating their biological effects. We therefore examined the consequence of PVN lesions in rats injected with EtOH or IL-1ß icv. In contrast to the obligatory role of this nucleus in modulating the ability of icv CRF or isoproterenol to blunt hCG-induced T release (4), destruction of the PVN only partially restored normal Leydig cell activity in rats administered EtOH into the brain ventricles. On the other hand, it completely abolished the effect of icv IL-1ß on hCG-induced T. As we showed that EtOH did not leak to the periphery, it is highly unlikely that a direct influence of the drug on the testes, which would obviously not involve the PVN, may have played a role. Combined with the HPLC data presented here, which showed that IL-1ß and CRF, but not EtOH, significantly increased PVN DA turnover, our results suggest that other, presumably downstream, brain regions may also participate in the influence of EtOH, whereas IL-1ß and CRF appear to work at or upstream of the PVN in this pathway.

As it is well known that the systemic administration of EtOH increases brain levels of these amines (44), we considered the possibility that icv injected EtOH might act through a mechanism that involved catecholamines present within and outside the PVN. We therefore considered the possibility that one of the mechanisms through which this drug interfered with testicular function included stimulation of the catecholamine-dependent, brain testicular pathway. Although significant changes in the levels of hypothalamic NE, epinephrine, DA, 5-HT, or their metabolites over the time course (30 min) separating the injection of EtOH were not detected here, it must be noted that this represents a shorter period than that usually reported to be necessary for the effect of EtOH itself (45, 46) or other signals, such as cytokines, on catecholamine production (47, 48). Thus, the data shown here correspond to the lack of effect of peripherally injected EtOH on brain levels of DOPAC and 5-HT over the short time frame that was necessary for the effect of alcohol. On the other hand, we observed small, but significant, changes in the IL-1ß- or CRF-induced DOPAC/DA ratio in the PVN, an indicator of DA turnover. It remains possible, of course, that by removing tissues 30 min after icv treatment, we missed physiologically relevant changes in catecholamine levels that might have been present at other times. In this context, we therefore need to emphasize two points. First, the time point we chose was based on preliminary data indicating that it represented peak responses in terms of IL-1ß and CRF. Second, the time course over which changes in catecholamine levels are relevant is the one that corresponds to significant effects of icv-injected treatments on T release. Consequently, if such changes occurred at later times, i.e. those more closely related to the data available in the published literature (47, 48), it is difficult to understand how they could be functionally relevant in our model. The only other potentially interesting changes would therefore be those taking place before the 30 min we used in our studies. A final possibility is that catecholamine levels were altered in brain regions that we did not investigate. Of relevance for this hypothesis is our recent finding that, indeed, the icv injection of alcohol induces significant changes in catecholamine levels in the locus coeruleus, and that these changes may be functionally relevant for the ability of the drug to alter Leydig cell activity (Selvage, D. J., and C. L. Rivier, in preparation). While these hypotheses are presently under study, we decided to use another approach to probe the role of catecholamines in rats injected icv with EtOH or IL-1ß. Specifically, we injected the neurotoxin 6-OHDA to deplete amine levels in the hypothalamus. In view of the importance of the splanchnic innervation of the testes in the integrity of LH receptors (7, 49), it was important to first ascertain that this experimental manipulation did not alter the T response to hCG. We show here that depletion of brain catecholamine content was compatible with normal Leydig cell activity. In contrast, 6-OHDA lesions significantly reversed the inhibitory effect of both icv EtOH and IL-1ß when comparing sham- vs. 6-OHDA-lesioned animals receiving these treatments. This observation corresponds to our previous report that the icv injection of ß-adrenergic antagonists significantly, but not completely, reversed the influence of IL-1ß and CRF on Leydig cell function (7). In view of the results illustrated in Table 1Go and Fig. 7Go, future experiments will be necessary to examine the respective roles of DA, epinephrine, and NE in mediating the influence of icv-injected EtOH.

One argument that has been repeatedly raised to explain at least part of the inhibitory influence of icv-injected IL-1ß and CRF on Leydig cell activity is that these treatments might decrease blood flow to and/or from the testes. If present, this phenomenon would decrease the amount of hCG delivered to Leydig cells and/or impair T release from the testes. In the past, we presented much indirect evidence that argued against this hypothesis. First, it is well known that the icv injection of CRF increases blood pressure in the general circulation (50), and more recent evidence has indicated a similar phenomenon in rats receiving IL-1ß icv (33). These responses seemed difficult to reconcile with decreased blood flow at the testicular level. Second, various models of immune stimulation, including those accompanied by elevated levels of proinflammatory cytokines such as IL-1ß, are also accompanied by up-regulated blood pressure (51). Third, the finding of specific inhibition of the steroidogenic acute regulatory protein, but not other testicular enzymes, the absence of histological evidence for impaired testicular vascularization, and the persistence of a diminished T response to hCG ex vivo (52), provided what we considered solid support against impaired testicular blood flow as a mechanism responsible for the inhibitory effect of IL-1ß and CRF on Leydig cell activity. Finally, the concept of a functional relationship between decreased T release and altered blood delivery to the testes is not supported by the fact that low T levels, induced, for example, by sepsis, are accompanied by a significant increase in testicular blood flow (51). Nevertheless, repeated encounters with the argument that icv treatments impaired Leydig cell activity by acting on the testicular microvasculature, prompted us to specifically monitor its parameters by Laser-Doppler flow cytometry under a variety of conditions. We first found that, as suggested by the work listed above, changes in peripheral blood pressure are not necessarily valid predictors of altered testicular blood flow. For example, we observed that the peripheral administration of CRF, which is known to lower blood pressure in the general circulation (53), did not significantly influence testicular capillary flow (Selvage, D. J., unpublished observations). However, it was found that the topical administration of phenylephrine gel rapidly and significantly decreased testicular blood flow, an observation in agreement with results obtained after the intratesticular injection of epinephrine (20). We then turned to hCG, which had been reported to cause delayed and prolonged effects on the testicular microvasculature after sc administration of very large doses (30, 31, 32, 54). We report here that even a small amount of this gonadotropin was able to increase blood flow, and that this effect, while transient, was very rapid. In contrast, neither EtOH nor IL-1ß had a marked influence. Collectively, these results suggest that the ability of icv treatments to interfere with T release in response to hCG is unlikely to be mediated by impaired activity of the testicular microvasculature.

In conclusion, we have developed a unique model of stress that avoids the confounding influence of peripheral effects due to blood-borne secretagogues. Specifically, we have shown that the direct injection of a small amount of EtOH into the brain ventricles was capable of rapidly blunting Leydig cell activity independently of altered LH secretion and decreased testicular blood flow. Even though the hypothalamic PVN participates in this antireproductive effect, other brain regions and/or mechanisms are likely to play a role, as indicated by the only partial ability of PVN lesions to restore full T secretion after icv EtOH, but fully restore the ability of IL-1ß to do so. Moreover, we show the importance of an intact catecholaminergic system for the effects of icv IL-1ß and EtOH on T synthesis and secretion, and further delineate the agents (i.e. IL-1ß and CRF) that act at or upstream of the PVN in the pathway under investigation vs. those (EtOH) that act both at and downstream from the PVN. Also, although the inhibitory effect of systemic EtOH on T release is well documented, this is the first demonstration that this drug can modify Leydig cell activity strictly through a CNS site of action. These results suggest that the early phase of the T response to peripheral EtOH administration, which is often not accompanied by decreased LH release and probably precedes steroidogenic defects resulting from circulating levels of the drug, may involve this neural circuit. Until now, the difficulty in isolating the sites of action of EtOH, which include the LHRH-LH axis, a direct influence on testicular steroidogenesis due to circulating amounts of the drug, and a neural hypothalamic-testicular pathway, has hindered the development of treatments aimed at preventing or reversing its detrimental effects on sex steroid production. A better understanding of the importance of a neural pathway mediating the inhibitory influence of EtOH, IL-1ß, and other stress-linked agents on androgen secretion may provide such palliative or restorative therapies for pathologies associated with impaired testicular function that do not depend on abnormal pituitary activity and therefore cannot be addressed with supplemental gonadotropin.


   Acknowledgments
 
The generous gifts of Azaline B from Dr. Jean Rivier (The Salk Institute, La Jolla, CA) and of recombinant human IL-1ß from Otsuka Pharmaceutical Co. (Tokushima, Japan) are gratefully acknowledged. Reagents for the LH RIA were kindly provided by the National Hormone and Peptide Program, NIDDK. Work on testicular blood flow was made possible through the help and guidance generously provided by Dr. Nigel Calcutt (Department of Pathology, University of California, San Diego, CA). We are also indebted to Elaine Law, Melissa Herman, Yaira Haas, Anh-Khoi Nguyen, Keith Hansen, and Richard Schroeder for excellent technical assistance, and to Debbie Doan for manuscript preparation.


   Footnotes
 
This work was supported by NIH Grants AA-12810 (to C.R.) and AA-12294 (to L.P.).

Abbreviations: BAL, Blood alcohol level; CNS, central nervous system; CRF, corticotropin-releasing factor; DA, dopamine; EtOH, ethanol; FJ, Fluoro-Jade; hCG, human chorionic gonadotropin; 5-HT, serotonin; icv, intracerebroventricular; LHRH, LH-releasing hormone; NE, norepinephrine; 6-OHDA, 6-hydroxydopamine; PU, perfusion unit; PVN, paraventricular nucleus; PVNx, PVN lesion; T, testosterone.

Received October 27, 2003.

Accepted for publication December 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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